4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
KEYNOTE PAPER
INVESTMENT IN MALAYSIAN BIODIVERSITY: IT’S IMPORTANCE IN SUSTAINING TROPICAL
NATURAL RESOURCES
Prof. Emer. Dato' Dr. Abdul Latiff
Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Malaysia
It is now about 14 years after the launching of the National Biodiversity Policy which had taken a centre
stage in εalaysian environmental management and policy agenda. The government’s agencies together
with non-government organizations have been demanding that biodiversity be conserved and utilized for
future socio- economic development. The country’s tropical forest and the marine ecosystems are
endowed with one of the richest biodiversity assets in the region and efforts to conserve and utilize the
rich flora and fauna have been carried out in the country since the last decade through the establishment
of National Biotechnology Policy and other commercial government and private initiatives. To-day about
7.6% of the forest of all types had been set aside for the conservation of biodiversity but their inventories
are yet to be carried out fully. The country has claimed that these protected areas have captured most of
the diverse ecosystems and species of plants and animals found. Plants and animals including
microorganisms biodiversity is represented by numerous species with flowering plants constituting about
80% whilst the diversity of fauna species is represented by more than 5,000 species excluding
invertebrates. The investment in biodiversity objective should be to achieve a long term capital growth for
subsequent investment of biodiversity asset in biotechnology especially medical and agricultural
biotechnology, health care and ecotourism. To this effect Malaysia has just formulated the National
Biotechnology Policy that envisaged the sustainable use of biodiversity. The genetic resources especially
the seeds, DNA manipulation and microorganism cultures may enhance both the medical and agricultural
products and by-products and some salient features in potential products of biodiversity for commercial
use and management would be discussed.
1
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
NEW LIMONOIDS FROM Chisocheton ceramicus
1*
1
A.Hamid, A.H. , Najmuldeen, I.A. and Morita, H.
2
1
Department of Chemistry, Faculty of Science, University Malaya, 50603 Kuala Lumpur, Malaysia
2
Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa, Tokyo 142-8501, Japan
E-mail: ahamid@um.edu.my
Abstract
An investigation of the bark of Chisocheton ceramicus has yielded five limonoids, three new limonoids,
chisomicine A 1, chisomicine B 2, and chisomicine C 3, with two known mexicanolides, limonoid 4 and
limonoid 5. The absolute structures were determined by 2D-NMR, CD, and computational methods.
Chisomicine A 1 exhibited no production inhibitory activity in J774.1 stimulated by LPS dose-dependent at
high cell viability.
Keywords: Limonoids, chisocheton ceramicus, chisomicine A-C
1. INTRODUCTION
Limonoids from Meliaceae have been the subject of various studies due to their significant biological
activities such as antifeedants, insecticides, antitumor, and antimalarial activities [1, 2]. In addition their
diverse structures with the oxidized backbone and the side chain moiety bonded to ring D in the intact
tetranortriterpenoid nucleus have attracted great interest. [3] In continuation of our research on Meliaceae
family, [4] we have found the alcoholic bark extracts of Chisocheton ceramicus are rich sources in
interesting limonoids [5]. We have isolated five limonoids, ceramicine E 1 with A 2, B-seco limonoid ring,
ceramicine F 2 with phragmalin-type limonoid, and the other one was ceramicine G3 with oxidized
phragmalin-type limonoid, and limonids 4 [6] and 5 [7] were known mexicanolide type compounds.
Limonoid 4 (14-Deoxyxyloccensin K) was first reported as a synthetic compound from natural xyloccensin
K, and we reported for the first time on its occurrence as a natural product [8]. We now wish to report the
isolation and structure elucidation of three new limonoids, chisomicine A 1, chisomicine B 2, and
chisomicine C 3, as well as the NO production inhibitory of the chisomicine A 1.
23
22
23
23
22
O
22
18
O
O
19
7
H
5
O
1
8
30
29
4
17
2
3
O
14
15
6
1 8
5
16
30
29
O
28
13
OH
10
20
21
28
4
18
O
15
12
16
11
O
O
2
3
O
1'
7
O
3'
2'
2'
5'
3'
4'
2
O
1
4
28
5'
O
21
12
20
11
13
H
O
16
15
MeO
O
O
22
18
17 O
14
8
30
2
3
4'
1
10
5
O
5'
9
19
6
20
13
H
23
O
22
21
HO
30
O
O
1'
2'
4'
20
4
2
3
O
1'
O
3'
1 8
O
5
14
23
O
13
OH
29
O
18
17
9
10
7
6
16
15
O
12
11
O
14
O
O
O
17
9
7
9
11
19
MeO
13
10
6
28
20
12
11
18
12
O
21
19
7
9
19
10
6
5
4
28
O
1
14
8
21
17 O
16
15
O
30
2
3
29
OH
29
3
4
5
2. Materials and Methods
2.1 General experimental procedures
2.1.1 Plant material
The barks of C. ceramicus were collected in 2000 from Hutan Simpan Bukit Enggang, Malaysia. The plant
species was identified by Mr. Teo Leong Eng with a Voucher specimen (No. KL 4973) and herbarium
specimen was deposited in the herbarium of the Chemistry Department, University of Malaya.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2.1.2 Extraction and isolation
The dried and powdered bark of C. ceramicine (900g) was extracted successively with methanol and the
methanol extract (200g) was partitioned with 10% aq MeOH and EtOAc.The EtOAc.- soluble materials
(10g.) were subjected to a silica gel column (hexane/ EtOAc. 1μ0→0μ1), in which a fraction eluted with
hexane / EtOAc 30%: 70% was further purified on a silica gel column with CH 2Cl2 - hexane - EtOAc
(5:3:2) to give chisomicine A 1 (250 mg; 2.5% yield). The second fraction with hexane / EtOAc 20%: 80%
was further purified on a silica gel column with EtOAc 65%: Acetone10% : Hexane25%, the first subfraction has been subjected to semi-preparative HPLC, developed with H2O-0.1%FA/MeOH-0.1%FA iso(25-75) Flow rate 2.5 ml/min. at RT 20.60 to give pure chisomicine B 2 (25mg; 0.25% yield). While Second
sub-fraction from the same column was subjected to the plate TLC with the solvent system EtOAc 65%:
Acetone10%: Hexane25%, to get pure chisomicine C 3 (16mg; 0.16% yield).
27
Chisomicine A (1): white, amorphous powderν [α] D -125 (c 0.7, εeOH)ν IR (KBr) max 2938, 1734, and
-1
1266 cm ν UV (εeOH) max (log ) 202 (4.15), and 214 (sh, 4.02)ν CD(εeOH) max 201 (Δ _36.2), 213
1
13
(0), 227 (7.73), 290 (1.24) nm; HNMR data (Table 1) and C NMR data (Table 2); ESIMS m/z 573 (M +
+
+
Na) ; HRESIMS m/z 573.2464 (M + Na) ; calcd for C32H38O8Na, 573.2464.
o
27
Chisomicine B (2): colorless needles; mp 176-178 Cν [α] D -66 (c 1.0,εeOH)ν IR (KBr) max 3391, 2972,
-1
1735, 1703, and 1268 cm νUV (εeOH) max (log ) 216 (3.λ2)ν CD (εeOH) max 205 (Δ 0), 20λ (0.71),
1
13
213 (0), 223 (-3.42), 236 (0), 245 (0.92) nm; H NMR data (Table 1) and C NMR data (Table 2); ESIMS
+
+
m/z 591 (M + Na) ; HRESIMS m/z 569.2706 (M +H) ; calcd for C32H41O9, 569.2751.
27
Chisomicine C (3): white, amorphous powder; [α] D -86 (c 1.0, εeOH)ν IR (KBr) max 3441, 2980, 1732,
-1
1718, 1706, and 1269 cm ν UV (εeOH) max (log ) 206 (4.16)νCD(εeOH) max 201 (Δ _4.86), 208 (0),
1
13
211 (0.44), 217 (0), 221 (-0.3), 227 (0), 235 (0.48), 263 (0.93) nm; H NMR data (Table 1) and C NMR
+
+
data (Table 2); ESIMS m/z 607 (M+ Na) ; HRESIMSm/z 607.2512 (M+ Na) ; calcd for C32H40O10Na,
607.2519
3. Results and Discussion
Chisomicine A 1 was afforded as white amorphous solid. The HRESIMS of chisomicine A 1 displayed a
+
pseudomolecular ion peak at 573.2464 (M+Na) , compatible to the molecular formula of C32H38O8Na. IR
-1
13
absorptions indicated the presence of carbonyl group at1734 cm . The C/DEPT NMR spectra revealed
2
3
thirty two carbon resonances due to four carbonyls, four sp quaternary carbons, three sp quaternary
2
3
3
3
carbons, six sp methines, four sp methines, five sp methylenes, and six methyls. Among them, two sp
3
2
methines ( C 76.8 and 80.2), one sp methyl ( C 52.0), and two sp methines ( C 141.7 and 142.8) were
ascribed to those bearing an oxygen atom.
Five partial structures a (C-2, C-3, and C-30), b (from C-5 to C-6), c (from C-9 to C-12), d (from C-22 to C1
1
23), and e (from C-3’ to C-4’) were deduced from H- H COSY analysis of 1 in CDCl3 (Figure1). The
presence of a bicyclo[5.2.1]dec-3-en-8-one unit containing the partial structure a was supported by HMBC
correlations as shown in Fig. 1. HMBC correlations for H-3, H-5, H3-28, H2-29 of C-4 ( C 43.3) gave rise
to the connectivity of the partial structures a and b through C-4 atom. The presence of a cyclopentanone
ring connected with the partial structure b was assigned by the HMBC correlations for H2-29 of C-1 ( C
220.6), C-5 ( C 40.5), and C-10 ( C 54.2), and for H-5 of C-1 and C-10. Connection among partial
structures a, b, and c could be assigned HMBC correlations for H3-19 of C-5, C-λ ( C 44.4), and C-10, and
for H-9 of C-8 ( C 131.0) and C-10. The presence of a methoxy carbonyl group connected to the partial
structure b was supported by the HMBC correlations for H 2-6 and H3-OMe of C-7 ( C 174.1). Partial
structure e constructing (E)-2-methylbut-2-enoic acid was attached at C-3 by the HMBC correlations for H3 and H-3’ of CC 167.1). The presence of a β-furyl ring at C-17 was also assigned by the HMBC
correlations as shown in Figure 1. In addition, the HMBC correlations for H3-18 of C-12 ( C 28.5), C-13
( C 37.8), C-14 ( C 131.6), and C-17 ( C 80.2), and for H-15 of C-8, C-13, C-14, and C-16 ( C 169.2)
indicated the presence of an isochromenone containing the partial structure c and a tetrahydropyran-2one ring. Thus, chisomicine A 1 was concluded to be an unique limonoid possessing a bicyclo[5.2.1]dec3-en-8-one ring system, an isochromenone, and a β-furyl ring at C-17.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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d
O
23
21
22
MeO
7
c
O19
11
b
5
13
9
10
6
O8
1
4 29
O
16
15
O
a
2
3
O
17
14
30
28
18 20
12
O
1'
2'
e
3'
1H-1HCOSY
5'
HMBC
4'
Figure 1 Selected 2D NMR correlations of Chisomicine A 1
NOESY correlations among H-2, H-3, and H-29b indicated that the ester at C-3 and Me-28 at C-4
assumed a -configuration. Furthermore, the relative configurations at C-5, C-13, and C-17 were
deduced from NOESY correlations among H-5, H-12a, and H-17 as shown in the computer-generated 3D
drawing as depicted in Figure 2. The relative configurations at C-9 and C-10 could be assigned by
NOESY correlations of H-9/H-30 and H3-19, and of H-30/H2-15.
Figure 2 Selected NOESY correlations for Chisomicine A 1
27
Chisomicine B 2, ([α] D -66 (c 1.0,MeOH) was isolated as a colourless crystal. The HRESIMS showed a
[M+H]+ peak at m/z 569.2076 corresponding to the molecular formula of C32H40O9. IR absorption implied
-1
1
the presence of esteric ketone group at 1731 cm . The H NMR spectrum showed the presence of βmono-substituted furan moiety and its position was confirmed by characteristic chemical shifts of H-17
singlet at δH 5.4. Furthermore, H-21 singlet at δH 7.7, H-22 at H 6.40 (d, J = 1.2 Hz), and H-23 at δH 7.4 (tlike). Additionally, two more proton signals have been appeared, one of them at H 4.8 (d, J=10.9) which
therefore indicated the presence of oxygen belong to H-3, while the second proton signal appeared at δH
3.25 (d, J=3.2) which is more up fielded in comparison with H-3 belongs to H-30, therefore indicates to the
9
presence of oxygen in the form of epoxy, it was in the agreement of 1D NMR data . Four methyl singlets
detected at δH 1.0 (Me-18), 1.0 (Me-19), 0.86 (Me-28), and 1.80 (Me-5′), meanwhile, one methyl doublet
(Me-4’) detected at ( H 1.3, J=9) and a methoxy singlet appeared at δH 3.69. One double bond proton
1
1
signals, H-3′, was detected at δH 6.97 (qd J=7, 1.5 Hz). H– H COSY cross signals observed (H-3′/H3-4′,
13
H-3/H-2, H-5/H2-6b, H2-12a/H2-12b, H2-15a/H2-15b, H2-6a/H2-6b, and H2-29a/H2-29b. The C/DEPT NMR
spectra revealed thirty two carbon resonances due to three carbonyls, two for ester at δC168.4 (C-1′) and
2
3
C 173.8 (C-7), and one for lactone (C-16) at δC (169.4), two sp quaternary carbons, five sp quaternary
2
3
3
carbons, four sp methines, seven sp methines, five sp methylenes, and six methyls. Among them, two
3
3
3
sp quaternary carbons ( C 80, 61.3), three sp methines ( C 78.6, 77.6, 59.6), one sp methyl ( C 52.0),
2
and two sp methines ( C 141.4 and 143.1) were ascribed to those bearing an oxygen atom. Figure 3
4
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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shows selected 2D NMR correlations for 2. HMBC correlations of H-17 to C- 20, C-21, and C-22 indicated
the presence of a β -furyl ring at C-17. The presence of a α-methyl crotonate at C-3 was confirmed based
on the HMBC correlation of H-3 ( H 4.8) to C-1′ ( C 168.4). Additionally, methyl propionate substituent at
C-5 found in typical A, B, D-seco limonoids, was observed from the chemical shifts ( H 3.69 and 2.30 for
MeO and H2-6 respectively and C 52-Oεe, C 34.2 C-6, and 173.75 C-7), the HMBC correlations of the
methoxy peak , H-5, and H2-6 to C-7 suggested that the methoxy group was attached to C-7 and methyl
propionate substituent attached to C-5. The position of Δ8–30 should be oxygenated in the form of epoxy,
it was confirmed by HMBC correlations of H-2 to C-2λ ( C 43.3), C-8 ( C 61.3), and C-30 ( C 59.6); H-30
to C-2 ( C 43.1) and C-1( C 80); and H-3 to C-30 ( C 5λ.6). In the -lactone ring (ring-D), the geminal
proton of H2-15 showed the HMBC correlations to carbons of C-8 ( C 61.3), C-14 ( C 44.8 ), and C-16 ( C
169.4. three methyls of C-18, C-19 and C-28 were attached to C-13, C-10, and C-4 respectively, by
HMBC correlations of H3-18 to C-12, C-13, C-14 and C-17, and of H3-19 to C-1, C-5, C-9, and C-10, while
H3-28 correlated C-3, C-4, and C-5. Thus the gross structure of 2 was suggested to possess phragmalintype skeleton with β-furan ring, -lactone ring, and α-methyl crotonate as shown in Figure 3.
Figure 3 Selected 2D NMR correlations of chisomicine B 2
27
Chisomicine B 3, ([α] D -86 (c 1.0, MeOH) was isolated as white amorphous powder. The HRESIMS
+
showed a [M+Na] peak at m/z 607.2512, corresponding to the molecular formula of C 32H40O10Na. IR
-1
1
absorption implied the presence of esteric ketone (1732cm ) groups. The H NMR spectrum showed the
presence of β-mono-substituted furan moiety and its position by characteristic chemical shifts of H-17
singlet at δH 5.38. Furthermore, H-21 singlet at δH 7.78, H-22 a broad singlet at H 6.46, and H-23 at δH
3
7.39 (t-like). Additionally, three down field sp proton signals have been appeared, one of them at H 4.8
(d, J=10.0) which therefore indicated the presence of oxygen belong to H-3, the other two were geminal
protons; one appeared at 3.93 (d, J= 9.6), while the second geminal proton appeared at δH 3.48 (d, J=
9
9.6), it was in the agreement of 1D NMR data . Additionaly, four methyl singlets signals were detected at
H 1.01 (Me-18), 0.63 (Me-19), 1.01 (Me-28), and 1.77 (Me-5’), meanwhile, one methyl doublet signals
2
(Me-4’) detected at ( H 1.6, J=7) and a methoxy singlet at ( H 3.69). Two sp proton signals, were
1
1
detected at H 6.92 as multiplet and at 5.48 (d, J=6.2) belong to H-3’ and H-30 respectively. H– H COSY
cross signals observed (H-3/H-2 and H-2/H-30, but no correlation found between H-3 and H-30 which is
1
1
approve of the position of carbon - carbon double bond between C-8 and C-30, meanwhile H– H COSY
cross signals observed (H-5/H2-6, H2-12a/H2-12b, H2-15a/H2-15b, H2-6a/H2-6b, and H2-29a/H2-29b, H13
3′/H3-4′). The C/DEPT NMR spectra revealed thirty two carbon resonances due to three carbonyls, three
2
3
2
3
3
sp quaternary carbons, five sp quaternary carbons, five sp methines, six sp methines, five sp
3
3
methylenes, and six methyls. Among them, two sp quaternary carbons ( C 97.3 and 72.9), two sp
3
2
methines ( C 75.3, 76.9), one sp methyl ( C 52.2), one methylene ( C 67.9), and two sp methines ( C
13
142.1 and 143.0) were ascribed to those bearing an oxygen atom. In addition, the C spectrum indicated
the presence of three carbonyls, two for ester at δC167.7 (C-1′) and C 173.96 (C-7), and one for lactone
5
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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13
(C-16) at δC (169.3). According to the C NMR spectral data, C-1 ( C 97.3) could be an acetal or
hemiacetal carbon and C-3, C-14, C-17, C-2λ, ( C 75.3, 67.9, 72.94, and 67.89, respectively) should be
oxygenated. Figure 4 shows selected 2D NMR correlations for 3.
HMBC correlations of H-17 to C- 20, C-21, and C-22 indicated the presence of a β-furyl ring at C-17. ).
The presence of a α-methyl crotonate at C-3 was confirmed based on the HMBC correlation of H-3 ( H
4.8) to C-1′ C 167.7). Additionally, methyl propionate substituent at C-5 found in typical A, B, D-seco
limonoids, was observed from the chemical shifts ( H 3.69 and 2.30 for MeO and H2-6 respectively and
C 52.2-Oεe, C 31.8 C-6, and 173.9 C-7), the HMBC correlations of the methoxy peak , H-5, and H2-6 to
C-7 suggested that the methoxy group was attached to C-7 and methyl propionate substituent attached to
C-5. There was a double bond between C- 8 and C–30 , that was confirmed by COSY and HMBC
correlations of H-2 to C-30 ( C 121.8), C-8 ( C 140,9), C-1 ( C 97.3 ), and C-3 ( C 75.3), in addition, H-30
7.
to C-1 ( C 97.3), C-λ( C 43.78), and C-14 ( C 72.94). It was in the agreement of 1D NMR data In the lactone ring (ring-D), the geminal proton of H2-15 showed the HMBC correlations to carbons of C-8 ( C
140.9), C-14 ( C 72.9), and C-16 ( C 169.3). Three methyls of C-18, C-19 and C-28 were attached to C13, C-10, and C-4 respectively, by HMBC correlations of (H3-18 to C-12, C-13, C-14 and C-17), (H3-19 to
C-1, C-5, C-9, and C-10), and ( H3-28 to C-3, C-4, C-5 and C-29).
Thus the gross structure of 3 was suggested to possess oxydized phragmalin-type skeleton with β-furan
ring, -lactone ring, and α-methyl crotonate as shown in Figure 4.
Figure 4 Selected 2D NMR correlations of chisomicine C 3
The absolute configuration of all three compounds could be assigned by comparing their experimental CD
spectra with the calculated CD spectra (CD calculations were performed by Turbomole 6.110 using RI-TDDFT-BP86/aug-cc-pVDZ11 level of theory on RI-DFT-BP86/SVP11 optimized geometries.). The
calculated CD spectra showed similar CD patterns to those of 1, 2, and 3 as shown in Figure 5 (should be
changed). Therefore, their absolute stereochemistries were proposed as shown in the structures.
1
Table 1 H NMR spectral data of compounds (1-3)
H
1
2
3
2
3
5
6a
6b
9
11a
11b
5.85 (1H, dd, 11.6, 6.4)
4.79 (1H, dd, 6.4, 1.6)
3.82 (1H, brd, 12.0)
2.52 (1H, dd, 16.0, 12.8)
2.35 (1H, m)
2.66 (1H, brd, 6.0)
1.87 (1H, brd, 14.4)
1.63 (1H, m)
2.94 (1H, dd, 10.9, 3.4 )
4.8 (1H, d, 10.9)
3.03 (1H, dd, 11, 2. 3)
2.33 (1H, dd, 17.2, 2.3)
227 (1H, m)
1.8 (1H, m)
1.9 (1H, brd, 11.1)
1.81 (1H, m)
2.96 (1H, m)
4.8 (1H, d, 10.0)
2.9 (1H, dd, 12.0, 10.0)
2.37 (1H, d, 11.3)
2.30 (1H, d, 11.3)
2.44 (1H, dd, 5.5, 4.6)
1.66 (1H, bd, 14.0)
1.51 (1H, m)
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
H
1
2
3
12a
12b
14
15a
15b
17
18
19
21
22
23
28
29a
29b
30
OMe
3'
4'
1.30 (1H, m)
1.04 (1H, m)
1.62 (1H, m)
1.34 (1H, d, 11.1)
2.04( 1H, dd, 6, 1.2)
2.5 (1H, dd, 18.4, 7.3)
2.3 (1H, dd, 18.4, 1.8)
5.4 (1H, s)
1.0 (3H, s)
1.0 (3H, s)
7.7 (1H, s)
6.4 (1H, d, 1.2)
7.4 (1H, t like,)
0.86 (3H, s)
2.0 (1H, d, 11)
1.33 (1H, dd, 11, 1.5)
3.25 (1H, d, 3.2)
3.69 (3H, s)
6.97 (1H, qd, 9.0, 1.6)
1.3 (3H, d, 9.0)
2.0 (1H, m)
1.17 (1H, m)
3.07 (2H, brs)
5.44 (1H, s)
1.09 (3H, s)
0.97 (3H, s)
7.54 (1H, s)
6.46 (1H, d, 1.2)
7.39 (1H, t like,)
1.13 (3H, s)
2.40 (1H, d, 17.6)
2.05 (1H, d, 17.6)
5.83 (1H, brd, 11.6)
3.72 (3H, s)
7.29 (1H, qd, 7.0, 1.6)
1.70 (3H, d, 7.0)
13
Table 1 C NMR Data of compounds (1- 3) in CDCl3.
C
1
2
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
28
29
30
OMe
220.6
135.5
76.8
43.3
40.5
33.8
174.1
131.0
44.4
54.2
19.1
28.5
37.8
131.6
33.0
169.2
80.2
16.4
22.9
120.7
141.7
109.9
142.8
22.6
46.4
129.1
52.0
80
43.1
78.6
44.8
39.4
34.2
173.8
61.3
41.6
45.1
21.5
33.7
36.0
44.8
27.3
169.4
77.6)
22.0
18.8
120.8
141.4
109.7
143.1
15.5
43.3
59.6
52.0
97
45.2
75.3
43.3
34.8
31.8
173.9
140.9
43.8
41.4
19.1
28.6
41.2
72.9
39.3
169.3
76.9
14.9
14.7
120.0
142.1
109.9
143.1
15.5
67.9
121.8
52.2
7
2.94 (1H, d, 18.8)
2.79(1H, d, 18.8)
5.38 (1H, s)
1.01 (3H, s)
0.63 (3H, s)
7.78 (1H, s)
6.46 (1H, s)
7.39 (1H, s)
1.01 (3H, s)
3.93(1H, d, 9.6)
3.48 (1H, d,9.6)
5.48 (1H, d, 6.2)
3.69(3H, s)
6.92 (1H, d, 7.0)
1.6 (3H, d, 7.0)
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
C
1'
2'
3'
4'
5'
1
167.1
127.9
139.5
12.0
14.3
2
168.4
128.0
139.3
14.2
12.1
3
167.7
127.4
140.0
14.7
11.8
Figure 5 Actual and simulated CD and UV spectra of chisomicine A
1
Chisomicine A 1 inhibited NO production in J774.1 cells dose-dependently stimulated by LPS and also
12
showed little effect on cell viability (Figure 6: IC50 20.2 ε). However chisomicne B 2, chisomicine C 3,
14-deoxyxyloccensin K 4 and proceranolide 5 did not show NO production inhibitory activity.
Figure 6 NO production ratio in J774.1 stimulated by LPS of chisomicine A 1.
References
1. (a) Taylor, D. A. H. In Progress in the Chemistry of Organic Natural Products; Herz, W., Grisebach, H.,
Kirby, G. W., Eds.; Springer, New York, 1984; Vol. 45. (b) Mulholland, D. A.; Parel, B.; Coombes, P. H.
Curr. Org. Chem. 2000, 4, 1011-1054.
2. (a) Zhang, H.; Wang, X.; Chen, F.; Androulakis, X. M.; Wargovich, M. J. Phytotherapy Res. 2007, 21,
731-734. (b) Roy, A.; Saraf, S. Biol. Pharm. Bull. 2006, 29, 191-201. (c) Carpinella, M. C.; Defago, M.
T.; Valladares, G.; Palacios, S. M. J. Agri. Food Chem. 2003, 51, 369-374. (d) Bray, D. H.; Warhurst,
D. C.; Connolly, J. D.; O'Neill, M. J.; Phillipson, J. D. Phytotherapy Res. 1990, 4, 29-35.
3. (a) Yin, S.; Wang, X. N.; Fan, C. Q.; Liao, S. G.; Yue, J. M. Org. Lett. 2007, 9, 2353-2356. (b) Zhang,
C. R.; Yang, S. P.; Liao, S. G.; Fan, C. Q.; Wu, Y.; Yue, J. M. Org. Lett. 2007, 9, 3383-3386. (c) Di, Y.
T.; He, H. P.; Liu, H. Y.; Yi, P.; Zhang, Z.; Ren, Y. L.; Wang, J. S.; Sun, Q. Y.; Yang, F. M.; Fang, X.;
Li, S. L.; Zhu, H. J.; Hao, X. J. J. Nat. Prod. 2007, 70, 1352-1355.
4. (a) Awang, K.; Lim, C. S.; Mohamad, K.; Morita, H.; Hirasawa, Y.; Takeya, K.; Thoison, O.; Hadi, A. H.
A. Bioorg. Med. Chem. 2007, 15, 5997-6002. (b) Mohamad, K.; Hirasawa, Y.; Lim, C. S.; Awang, K.;
8
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Hadi, A. H. A.; Takeya, K.; Morita, H. Tetrahedron Lett. 2008, 49, 4276-4278. (c) Mohamad, K.;
Hirasawa, Y.; Litaudon, M.; Awang, K.; Hadi, A. H. A.; Takeya, K.; Ekasari, W.; Widyawaruyanti, A.;
Zaini, N. C.; Morita, H. Bioorg. Med. Chem. 2009, 17, 727-730.
5. Bark of C. ceramicus (KL 4973) was collected from Hutan Simpan Bukit Enggang, Kedah, Malaysia in
2000. Bark of C. erythrocarpus (KL 5651) was collected from Johor, Malaysia in 2009. Voucher
specimens were deposited in the Herbarium of Chemistry Department, University Malaya.
6. Kim, J-G.; Cho, D.H.; and Jang, D. O.; Tetrahedron Letters 45 (2004) 3031-3033.
7. Kipassa, N. T.; Okamura, H.; Doe, M.; Morimoto, Y.; lwagawa, T; Nakatani, M. Hetrocycles 2008,
75(1), 157-164.
8. Najmuldeen,I.A.; Hadi, A. H.A.; Mohamad , M.; Awang, K.; and Ng, S. W.; Acta Crysta 2010, E66,
o1927.
9. Wu, J.ν Zhang, S.’ Xiao, Q.ν δi, Q.ν Huang, Jν δong, δ. and Huang, δ.ν Tetrahedron δetters 2004, 45,
591-593.
10. TURBOMOLE V6.1 2009, a development of University of Karlsruhe and Forschungszentrum
Karlsruhe
GmbH,
1989-2007,
TURBOMOLE
GmbH,
since
2007;
available
from
http://www.turbomole.com
11. (a) Eickorn, K.; Treutler, O.; Ohm, H.; Haser, M.; Ahlrichs, R. Chem. Phys. Lett. 1995, 240, 283289.(b) Becke, A.D. Phys. Rev. A. 1988, 38, 3098-3100. (c) Perdew, J.P. Phys. Rev. B. 1986, 33,
8822-8824. (d) Schafer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1992, 97, 2571-2577. (e) Weigend,
F.; Kohn, A.; Hattig, C. J. Chem. Phys. 2002, 116, 3175-3183
12. Aktan, F. Life Sci. 2004, 75, 639-653.
9
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
HMBC SPECTRA OF ALKALOIDS FROM LAURACEAE SPECIES: NINE STARS HALO-N THEORIES
1,2
1
O. Hanita , A.H.A. Hadi , Ahmad Laksamana Omar
3
1
Chemistry Department, Faculty of Science, University of Malaya,50603, Kuala Lumpur,Malaysia
Centre for Foundation Studies in Sciences, University of Malaya,50603, Kuala Lumpur, Malaysia.
3
Yayasan Gual Periok, Gual Periok, Kelantan, Malaysia.
2
E-mails: hanita74@um.edu.my, ahamid@um.edu.my
Abstract
Four theories related to the field of natural products were discovered by Dr. Halo-N and was published in
a book entitled Al Fathun Nawa, Volume 1, 2011. These theories are:
1.
2.
3.
4.
Nine Stars Halo-N Theory,
Nawiah 9 x 45 (1) Theory,
Nawiah 9 x 45 (2) Theory,
Halo-N 9.2 Homolength Theory
These theories have similarities with depiction of correlation spots in HMBC (Heteronuclear Multiple Bond
Coherence) spectrum, which is obtained through the NMR (Nuclear Magnetic Resonance) machine. The
HMBC spectra of various alkaloids isolated from Lauraceae species were studied and these spectra were
used to prove these theories. The theories were experimentally proven based on the HMBC spectra.
Keywords: Lauraceae, Nine Stars Halo-N Theory, HMBC spectra, alkaloids.
1. INTRODUCTION
1.1 NINE STARS HALO-N THEORY
“Each specialty of Mass of Bio-Nature will occur under the arrangement conducted by nine stars
(Called Code Nine Stars L System: 2.4.1.2.) in righteous equilibrium coordinate’ [1].
Figure 1 shows the findings of The Route of Mass [(7+2) = (9)] which is described in the Code Nine Stars
L System: 2.4.1.2. Thus, from the compass directions formed in nature, it is able to determine the
existence of a special mass, in which it could be a mass that had already been discovered or a new mass
which is yet to be discovered by bio-chemistry researchers.
Referring to Figure 1, the red horizontal line is the connecting line between the correlation points, consists
◦
of 7 correlation points (in yellow), which is in the clusters of 2, 4 and 1. Meanwhile, the vertical 90 angle
th
straight line is the connecting line from the horizontal line to the middle point of 2 correlation points, the 8
th
th
th
and the 9 spots, to form an L-shape. Any correlation point which is the nearest to the 8 and the 9
◦
th
correlation points, and touches the vertical 90 angle line, is the 10 correlation spot and the sought
‘special mass’. Thus, whichever HMBC spectrum for a natural product that matches the Nine Stars Halo-N
Theory will be a sign, that the natural product can be a vaccine for the humans.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Figure 1 Nine Stars L System: 2.4.1.2.
1.1 Nawiah 9 x 45 (1) Theory
“Positive or negative property of a carbon compound which is found through the Nine Stars HaloN Theory, can be determined by the position of any correlation points, in which it touches or
otherwise, the intersection point between a straight line drawn 45° from the 90° angle and a
straight line formed from the first correlation point connected to the middle point of the eighth and
ninth correlation points” [2].
Figure 2 describes the optical rotation property of a compound, in which it can be positive or negative,
from the Nawiah 9 x 45 (1) Theory’s point of view. The blue straight line (Nawiah δine 1) is the connecting
line between the first (1) correlation point and the middle point of the eighth (8) and ninth (9) correlation
points. Meanwhile, P is the intersection point between Nawiah Line 1 and a straight line with the angle
45°. If there is any correlation points located on the intersection point P, thus it describes that the
compound has a positive (+ve) property. On the contrary, if there is no correlation point on the intersection
point P, it implies that the compound has a negative (-ve) property. The positive or negative characteristic
of a compound is important to determine the effects of the compound as a vaccine. Vaccine with the
positive property will treat the external illness, while vaccine with the negative property will treat the
internal illness.
Figure 2 Nawiah 9x45 (1) Theory
1.3 Nawiah 9 x 45 (2) Theory
“Positive or negative property of a carbon compound found through the Nine Stars Halo-N Theory
can be determined by the existence of the correlation point located on the straight line formed
between the first correlation point and special mass / new mass.” [3].
Figure 3 describes the optical rotation direction of a compound, whether it is positive or negative, from
Nawiah λ x 45 (2) Theory’s point. The green straight line (Nawiah δine 2) is the straight line formed
between the first correlation point and special mass / new mass. If there is a correlation point located on
11
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
the Nawiah Line 2, then it means the compound has a negative (-ve) property. On the contrary, if there is
no correlation point located on the Nawiah Line 2, then it means the compound has a positive (+ve)
property. Once again the positive or negative property of a compound is important to determine the
usefulness of the compound as a vaccine. If a vaccine compound has a negative property, thus it can be
used to treat internal illness, where as a vaccine compound with a positive property can be used to treat
external illness.
Figure 3 Nawiah 9x45 (2) Theory
1.4 Halo-N 9.2 Homolength Theory
“The Single Compound or Mixed Compounds status of a carbon compound discovered through
the Nine Stars Halo-N Theory can be determined based on two homolength reference points
forming a triangular shape at the part of the New Mass (Special Mass) and another two
homolength reference points forming a triangular shape at the base of the first and second
correlation points for the Nine Stars Halo-N Theory.” [4].
Figure 4 shows the status of a compound as a mixed compound. Triangle A is formed between the eighth
and ninth correlation points with the new mass (special mass). Triangle B is formed between the first and
second correlation points with the second mass for the Nine Stars Halo-N Theory. If the triangular shape
of A is same as the triangular shape of B, thus the status of the compound discovered is mixed
compounds. On the contrary, if the triangular shape of B is not the same as the triangular shape of A, thus
the status of the compound discovered is single compound. The status of the compound in Figure 4 is
mixed compounds because the triangular shape of A is same as the triangular shape of B, in terms of
their distance and correlation points.
Figure 4 Halo-N 9.2 Homolength Theory
shows the status of a compound as mixed
compounds
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Figure 5 shows the status of the compound as a single compound. The triangular shape of B is not the
same as the triangular shape of A, as there is no correlation point at the base of triangle B.
Figure 5 Halo -N 9.2 Homolength Theory
shows the status of a compound as single
compound.
2. EXPERIMENTAL PROCEDURES
Studies on the validity and authenticity of Dr. Halo-N Theories were done based on the HMBC spectra of
the compounds of natural products isolated from Lauraceae species such as Phoebe tavoyana, Phoebe
grandis, Litsea petiolata and Dehaasia longipedicellata.
2.1 General experimental procedures
Spectroscopic measurements were performed as follows; Optical rotations were determined on Autopol
111 Automatic Polarimeter Machine with methanol and chloroform as solvent. UV spectra were obtained
using Shimadzu UV-160 Ultraviolet-Visible Spectrometer. IR spectra were obtained with CHCl3 on a
Perkin Elmer Spectrum 2000-FTIR Spectrometer. HR-ESI-MS were performed on a Shimadzu LC-MS1
13
IT—TOF spectrometer. H NMR (400MHz) , C NMR (400MHz), DEPT, COSY, HMQC and HMBC
spectra were acquired in a Bruker Avance 400 spectrometer using TMS as the internal standard and
CDCl3 as solvent.
3. RESULTS AND DISCUSSION
3.1 Nine Stars Halo-N Theory
3.1.1 Alkaloid (-) Norboldine
Figure 6 is the HMBC spectrum of alkaloid (-) Norboldine which had been matched with the Nine Star
Halo-N Theory. Based on the HMBC spectrum, the Nine Stars Halo-N is apparent with the arrangement of
2:4:1:2 - L System, which consists of correlation points connected with the red line. A corelation point
◦
which touched the vertical 90 angle straight line is the sought of new / special mass. Whichever HMBC
spectrum for a natural product that matches the Nine Stars Halo-N Theory will be a sign, that the natural
product can be a vaccine. The antimalaria activity of isolated compound (-) Norboldine was determined by
the procedure described by [5].
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Figure 6 HMBC spectrum of
alkaloid (-) Norboldine with
Nine Stars Halo-N Theory.
Table 1 Inhibition Growth Percentage of Plasmodium falciparum and Probit Analysis with SPSS 11.5
Sample
Norboldine
% Inhibition at Concentration ( g/mδ)
10
1
0.1
0.01
0.001
100
23.44
3.94
0.61
0
IC50 ( g/mδ)
1.49
(-) Norboldine was tested for in-vitro inhibitory activity against Plasmodium falciparum (Table 1). (-)
Norboldine showed potent inhibitory activity with the IC 50 value of 1.49µg/mL. It shown a good antimalaria
activity. The well known antimalaria drug, chloroquine has IC50 value of 0.006 µg/mL. Therefore, (-)
Norboldine proves that the Nine Stars Halo-N Theory is valid and is a true theory, since (-) Norboldine
exhibited strong activity.
3.1.2 Alkaloid (-) Lysicamine
Figure 7 is the HMBC spectrum of alkaloid (-) Lysicamine which had been matched with the Nine Star
◦
Halo-N Theory. A correlation point which touched the vertical 90 angle straight line is the sought of new /
special mass. Again, whichever HMBC spectrum for a natural product that matches the Nine Stars Halo-N
Theory will be a sign, that the natural product can be a vaccine. Based on the bio activity of the
compound, alkaloid (-) Lysicamine displayed potential activity of antimicrobial activity.
Figure 7 HMBC spectrum of
alkaloid (-) Lysicamine with Nine
Stars Halo-N Theory.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Table 2 shows the microorganisms strongly inhibited by the alkaloid (-) Lysicamine. Hence, this proves
that the Nine Stars Halo-N Theory is valid and true theory. The assessment of antimicrobial activity was
determine by measuring the diameter of a clear inhibition zone around the disc using a vernier caliper.
The mean diameter of inhibitory zone was measured to the nearest milimeter (mm). The result were
expressed as mean ± S.D. From our findings, alkaloid (-) Lysicamine exhibits a high degree of
antibacterial activity against these Gram-positive microorganisms. Tannins were found to be an excellent
source for antibacterial compound [6,7].
Table 2 The mean diameter of inhibitory zone (mm ± SD) of (-) Lysicamine against bacterial strains.
Inhibition diameter (mm ± SD)
Gram positive
Sample
Gram negative
Staphylococcus
epidermidis
Staphylococcus
aureus
Bacillus
Pasteurella
multocida
Enterobacter
cloacae
(-)Lysicamine
12±0
13.33±0.57
15.5±0.57
NI
NI
STREPTOMYCIN
20±0
13.66±0.57
21±0
21.33±1.15
NI
*NI – no inhibition observed. Doses of the samples were 10 µl (1mg/ml) per disc while antibiotic were 10 µg per
disc. ANTIBIOTIC: STREPTOMYCIN 10 µg/disc.
3.2 Nawiah 9 x 45 (1) Theory
3.2.1 Alkaloid (-) Norboldine
Figure 8 is the HMBC spectrum of alkaloid (-) Norboldine which had been matched with Nawiah 9X45(1)
and Nawiah 9 x 45(2) Theory. The Nawiah 9X45(1) is represented by a blue line and the Nawiah 9X45(2)
is represented by a green line. According to those theories, the positive or negative characteristic of a
compound is important to determine the effects of the compound as a vaccine. Vaccine with the positive
property will treat the external illness, while vaccine with the negative property will treat the internal illness.
The compound under study has a negative property because there is no a correlation point touches the
intersection point of Nawiah 9X45(1), and touches the line of Nawiah 9X45(2). Hence, alkaloid (-)
Norboldine is suitable to be used as a vaccine to treat human’s internal illness.
Figure 8 HMBC spectrum of
alkaloid (-) Norboldine with
Nawiah 9X45(1) and Nawiah
9x45(2) Theory.
15
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
3.1.1
Alkaloid (-) Lysicamine
Figure 9 is the HMBC spectrum of alkaloid (-) Lysicamine which had been matched with Nawiah 9X45(1)
and Nawiah 9X45(2) Theory. Again, the Nawiah 9X45(1) is represented by a blue line and the Nawiah
9X45(2) is represented by a green line. The compound under study has a negative property because
there is no correlation point touches the intersection point of Nawiah 9X45(1), and touches the line of
Nawiah 9X45(2). Hence, alkaloid (-) Lysicamine is suitable to be used as a vaccine to treat human’s
internal illness.
Figure 9 HMBC spectrum of
alkaloid (-) Lysicamine with
Nawiah 9X45(1) and Nawiah
9x45(2) Theory.
3.3 Halo-N 9.2 Homolength Theory
3.3.1 Alkaloid (-) Norboldine
Figure 10 is the HMBC spectrum of alkaloid (-) Norboldine which had been matched with Halo-N 9.2
Homolength Theory. The theory is meant to identify whether the alkaloid compound has the status of a
single compound or mixed compounds. The compound under study has a mixed compound since the
spectrum has two similar shape of triangles i,e triangle formed by second mass is similar shape with the
triangle formed by the special mass.
Figure 10 HMBC spectrum of
alkaloid (-) Norboldine with
Halo-N
9.2
Homolength
Theory.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
3.3.2 Alkaloid (-) Lysicamine
Figure 11 is the HMBC spectrum of alkaloid (-) Lysicamine which had been matched with Halo-N 9.2
Homolength Theory. The theory is meant to identify whether the alkaloid compound has the status of a
single compound or mixed compounds. The compound under study has a single compound since the
actual triangle shape at second mass is not the same with the triangle shape at special mass because the
spectrum has no correlation at the point supposed to be second mass.
Figure 11 HMBC spectrum of
alkaloid (-) Lysicamine with
Halo-N
9.2
HomolengthTheory.
4. CONCLUSION
Studies that had been carried out by using scientific methods through the correlation points in the 2
Dimentional HMBC spectrum for the selected compounds prove that the Nine Stars Halo-N theory and
three other related theories namely Nawiah 9X45(1) Theory, Nawiah 9X45(2) Theory and Halo-N 9.2
Homolength Theory are true and valid in the field of Natural Products. Those theories are purposely
created by Dr.Halo-N through interpretation of verses from Al Quran to facilitate studies and analysis of
natural products by scientist in seeking suitable vaccines for treating human’s illnesses. Indeed the
discovery of these theories from interpretation of verses of Al Quran by Dr. Halo-N gives a big blessing to
the world.
Acknowledgements
The authors wish to acknowledge HIR grant (F00009-21001), University of Malaya for the financial
support. We would like to thank to Mr. Che Yusof Che Mat for the technical assistance.
REFERENCES
[1]
Al-Fathun Nawa, 2011, Jilid 1. 1104.
[2]
Al-Fathun Nawa, 2011, Jilid 1. 1112.
[3]
Al-Fathun Nawa, 2011, Jilid 1: 1109-1110.
[4]
Al-Fathun Nawa, 2011, Jilid 1: 131-132.
[5]
Budimulya A.S, Syafruddin, Tapechaisri P, Wiliariat P, Mazruki S. (1997) The sensitivity of
Plasmodium protein synthesis to prokaryotic ribosomal inhibitors. Mol Biochem Parasitol 184:137141.
[6]
Scalbert, A. (1991) Antimicrobial properties of tannins. Phytochemistry. 30: 3875-3883.
[7]
Cowan, M.M. (1999) Plant Products as Antimicrobial Agents. Clinical Microbiology Reviews. 564582.
17
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
PRODUCTION AND CHARACTERISATION OF CELLULOSE AND NANO-CRYSTALLINE
CELLULOSE FROM KENAF CORE WOOD
1,*
1
1
Chan Chi Hoong , Chin Hua Chia , Sarani Zakaria , Ishak Ahmad
1
2
School of Applied Physics, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600
Bangi, Selangor, Malaysia
2
School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti
Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
Email: chihoongchan@gmail.com
Abstract
Natural fibres like kenaf has been studied extensively as a reinforcing phase which received major
attention recently due to its renewability, biodegradability, and higher strength comparable to other
synthetic fibres. In this study, nano-crystalline cellulose (NCC) was produced from kenaf core wood using
acid hydrolysis method. Kenaf core was alkaline treated in 4 wt% of sodium hydroxide and subsequently
bleached using sodium chlorite in acidic buffer. The resulting white, bleached kenaf core was hydrolysed
in 64 wt% sulphuric acid to obtain NCC. The resulting NCC suspension was characterised using X-ray
Diffraction (XRD), Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA),
Scanning Fourier Transform Infra Red (FTIR) analysis and Transmission Electron Microscope (STEM).
Hydrolysis in high concentration of acid further increases the crystallinity of kenaf core and reduces the
dimension of cellulose to nano scale. FTIR results show that with each subsequent treatment,
hemicellulose and lignin were removed while cellulose remains unchanged after hydrolysis treatment
which was reaffirmed by DSC result. The diameter of NCC obtained from kenaf core were found to be in
the range of 8.5 - 25.5 nm.
Keywords: Kenaf core, cellulose, nano materials, nano-crystalline cellulose
1. INTRODUCTION
Kenaf (Hibiscus cannabinus L., Malvaceae) is a herbaceous dicotyldenous plant consist of outer bast
which resembles soft wood fibres and a woody core which resembles hardwood (Pande, Roy, & Kant,
2000). Kenaf plant can grow very fast, reaching height of more than 3 m in 3 - 4 months which can be
sown now (Villar, Revilla, Gómez, Carbajo, & Simón, 2009; Webber III & Bledsoe, 2002). The annual yield
-1
of kenaf (whole stem) ranges between 12 - 30 tons ha depending on cultivars, soil type, climate, etc.
which is 3 times the maximum yield of Pinus radiata . (Villar, et al., 2009). Kenaf core composed of 60 65 wt% of whole stem kenaf (Pande, et al., 2000; Villar, et al., 2009). Kenaf plant is largely used for its
bast fibres which are superior in mechanical strength compare to kenaf core which made it largely
ignored.
In 2009, International Year of Natural Fibres highlights the importance of natural fibres and its impact
towards people. Natural fibres have a good mechanical strength comparable to synthetic fibres and most
importantly, it is renewable and sustainable. In recent years, one particular natural fibre derivatives
received major attention for its superior mechanical properties i.e. cellulosic nano fibres (Dufresne, 2010;
Eichhorn et al., 2010). It is most notably used as a reinforcing phase in composites and recently proven
can be used in a wide range of applications such as drug delivery excipient, transparent paper, iridescent
film, aerogel, etc. (Beck, Bouchard, & Berry, 2010; Jackson J.K. et al., 2011; Nogi, Iwamoto, Nakagaito, &
Yano, 2009; Sehaqui, Salajkova, Zhou, & Berglund, 2010)
In this study, kenaf core wood is used to produce nano-crystalline cellulose (NCC) using acid hydrolysis
method. Kenaf core wood is chosen due to its limited usage compared to bast fibres. On the other hand,
18
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
NCC from kenaf core wood source has not been prepared and characterised yet. The produced NCC will
be characterized using X-ray Diffraction (XRD), Differential Scanning Calorimetry (DSC) and
Thermogravimetric Analaysis (TGA), Scanning Fourier Transform Infra Red (FTIR) analysis and
Transmission Electron Microscope (STEM).
2. MATERIALS AND METHODS
2.1 Materials
Kenaf core powder of V36 cultivar is obtained from Rice and Industrial Crops Research Center (MARDI).
Chemicals such as NaOH, NaClO2 and H2SO4 are purchased from Merck, Acros and JT Baker
respectively
2.2 Sample Preparation
Five months old kenaf core wood was grinded and sieved to obtain kenaf core powder. It is then rinsed
once with distilled water and strained using cloth filter to remove impurities such as sand.
2.2.1 Alkaline Treatment
o
Alkaline treatment was conducted in NaOH solution of 4 wt% at 80 C for 3 times. The resulting fibres
were washed and strained using cloth filter until it reaches neutrality then oven dried. It is now deemed as
alkaline treated fibres.
2.2.2 Bleaching
o
Alkaline treated fibres were subjected to NaClO 2 bleaching of 1.7 wt% in acidic buffer at 80 C for 4 times.
The resulting fibres were strained using cloth filter until it reaches neutrality then oven dried. It is now
deemed as bleached kenaf core.
2.2.3 Acid Hydrolysis
Bleached kenaf core wood was hand grinded before acid hydrolysis. The grinded products were
hydrolysed in 64 wt% H2SO4. The high concentration of acid was removed through centrifugation at
10 000 rpm for 10 minutes until the solution was turbid. The resulting NCC suspension was dialysed
against cellulose membrane in deionised water until the suspension reaches a pH of around 5. NCC
suspension was freeze-dried to obtain NCC powder.
2.3 Analysis
NCC powder was sent for X-ray Diffraction (XRD), Differential Scanning Calorimetry (DSC) and Fourier
Transform Infra Red (FTIR) analysis whereas a known amount of NCC is suspended in water for
Scanning Transmission Electron Microscope (STEM) analysis.
2.3.1 X-ray Diffraction (XRD) Analysis
Kenaf core powder, alkaline treated kenaf core, bleached kenaf core and NCC were analysed using XRD
o
o
o
(Bruker AXS D8 Advance), CuKα1 from 2θ of 5 to 60 with a step size of 0.0250 .
The crystallinity index was calculated using Segal's method. It must be pointed out that Segal's method is
an empirical method to quickly determine the relative crystallinity. Crystallinity index is calculated using the
following equation.
CrI (%) = [(I002 – IAM) / I002] × 100 %
(Eq. 1)
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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2.3.2 Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA)
DSC and TGA (Mettler Toledo) analysis was conducted for bleached kenaf core and NCC. Samples were
o
o
heated at the rate of 10 C per minute to 600 C in N2 for DSC. For TGA, samples were heated at the rate
o
o
of 10 C per minute to 900 C in N2 gas to analyse its thermal stability.
2.3.3 Fourier Transform Infra Red (FTIR) Analysis
FTIR (Perkin-Elmer Spectrum 400) analysis was conducted for kenaf core powder, alkaline treated kenaf
-1
core, cellulose from kenaf core and NCC in the range of 4000 to 650 cm .
2.3.4 Scanning Transmission Electron Microscope (STEM) Analysis
The dimensions of nano-crystalline cellulose were evaluated using STEM, Hitachi SU8000 at 30 kV. A
drop of the diluted suspension of NCC was dropped onto copper lacey grids. From the micrographs, the
diameters of NCC were calculated with the aid of computer software. At least 50 measurements were
taken.
3. RESULTS
3.1 X-ray Diffraction
Figure 1 shows the XRD spectra of kenaf core wood powder, alkaline treated kenaf core, bleached kenaf
core and NCC. (002) plane of samples shifted slightly to the right with increasing treatment. The crystal
planes of cellulose are more profound with increasing treatment with the emergence of a doublet of (1 0 1)
and (1 0 -1). Both bleached kenaf core and NCC conform to peaks normally shown by cellulose such as
Avicel PH-101, a type of microcrystalline cellulose (Park, Baker, Himmel, Parilla, & Johnson, 2010).
Figure 1 XRD Spectra of (a) kenaf core wood powder, (b) alkaline treated kenaf core, (c) kenaf core
cellulose, (d) NCC
Crystallinity index (CrI) increases after alkaline treatment due to the removal of amorphous hemicellulose.
A combination of low NaOH concentration and relatively low temperature does not alter the cellulose
structure as shown by XRD results. Bleaching the alkaline treated fibres further increases the CrI by
removal of lignin. Further treatment by acid increases the CrI from 48 % for kenaf core powder to 75 % for
NCC samples.
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3.2 Differential Scanning Calorimetry and Thermogravimetric Analysis
The DSC thermogram of cellulose from kenaf core wood powder and NCC is shown in Figure 2. The first
o
o
endothermic peaks can be observed at the range of 32 C to around 130 C for kenaf core wood cellulose
o
samples, whereas for the NCC sample, the first endothermic peak can be observed at the range of 32 C
o
to around 140 C. The first endothermic peak is attributed to the loss of water. The area of first
endothermic peak for NCC is larger than cellulose from kenaf core wood powder is due to its the smaller
size which increases its surface area therefore increasing its water absorption. A second endothermic
o
peak can be observed at around 350 C for cellulose from kenaf core wood powder. The same result is
obtained for pure cellulose from a previous study (Yang, Yan, Chen, Lee, & Zheng, 2007). A small
o
endothermic peak can be observed at 200 C, no significant endothermic peaks can be observed at 350
o
C for NCC. This may be due to size of NCC is in nano scale now and some remaining sulphated groups
that acts as flame retardant (Roman & Winter, 2004).
Figure 2 DSC thermograms for (a) bleached kenaf core and (b) NCC
TG and DTG thermograms are shown in Fig. 3. The initial weight loss for both bleached kenaf core and
NCC are attributed to weight loss due to vaporisation of water. For bleached kenaf core, at higher
o
temperature, the weight loss is slow and grows shaper. The maximum weight loss happens at 347 C
where almost half or 23.8 % of sample weight still remains which shows a similar tendency with the earlier
DSC results. This value also corresponds well with commercial cellulose by Sigma-Aldrich (Yang, et al.,
o
2007) and Avicel (Rhim et al., 2010). At a temperature of higher than 400 C, a residue of 17.6 % still
remains.
On the other hand, for TG and DTG thermograms of NCC, the maximum weight loss happens at a low
o
temperature of 180 C compare to bleached kenaf core. This may be attributed to the remaining
unwashed sulphated groups present on NCC which lowered its degradation temperature (Roman &
Winter, 2004). The remaining NCC sample decomposes slowly with no significant DTG peak until the end.
Figure 3 (a) TG curves and (b) DTG curves for NCC and bleached kenaf core
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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3.3 Fourier Transform Infra Red
Fig. 4 shows the spectra for (a) kenaf core wood powder, (b) alkaline treated kenaf core, (c) bleached
kenaf core, (d) NCC. Different chemical treatments are expected to induce chemical changes.
Figure 4 FTIR spectra for spectra for (a) kenaf core wood powder, (b) alkaline treated kenaf core, (c)
kenaf core cellulose, (d) NCC
-1
The absorption peak at 1734 cm is attributed to the absorption of acetyl and ester groups in
hemicellulose in kenaf (Mehdi Jonoobi, 2010) and rice husk (Johar, Ahmad, & Dufresne, 2012). This peak
disappears after alkaline treatment. This shows that alkaline treatment is capable of removing
-1
hemicellulose. Absorption peak at 1593 cm is attributed to the aromatic symmetric stretching while
-1
absorption peak at 1503 cm is attributed to aromatic asymmetric stretching from lignin (Agarwal & Atalla,
2010; Yan, Xu, & Yu, 2009) .These peaks disappears after bleaching is conducted thus confirming lignin
has been removed successfully. Both peaks are not observed for bleached kenaf core and NCC.
-1
-1
-1
Meanwhile, absorption peak at 2900 cm , 1640 cm and 1160 cm are attributed to aliphatic alkyl,
adsorbed water on fibers and ether linkages from pyranose ring (Morán, Alvarez, Cyras, & Vázquez,
2008; Yang, et al., 2007). These peaks can be observed throughout to the final product, NCC which are
the characteristic peaks for cellulose.
No significant differences can be observed for kenaf core cellulose and NCC which indicates that the
bleached kenaf core structure remains unchanged after acid hydrolysis treatment.
3.4 Scanning Transmission Electron Microscopy
STEM micrographs in Figure 5 shows a single NCC fibre while Figure 6 shows an agglomerated NCC.
Acid hydrolysis is known to increase the crystallinity of cellulose (Bondeson, Mathew, & Oksman, 2006)
while a high concentration of acid can be used to produce NCC (Bondeson, et al., 2006; Dong, Revol, &
Gray, 1998). 50 measurements of NCC diameter are taken. The mean diameter of NCC sample was
found to be 15.2 nm in the range of 8.5 - 25.5 nm with a standard deviation of 3.68. The distribution of
diameter of NCC is plotted as shown in Figure 7.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Figure 5 Negatively stained single NCC on
lacey copper film
Figure 6 An agglomerated NCC
Figure 7 Diameter distribution of NCC
4. CONCLUSIONS
Kenaf core wood is alkaline treated and bleached. A suspension of NCC was prepared from bleached
kenaf core using acid hydrolysis method. The XRD shows that the structure of kenaf core wood and its
products did not change much after treatment. The peaks observed for bleached kenaf core and NCC
conform to commercial available cellulose sources. XRD also shows an increasing CrI with each
treatment from 48% to 75%, which suggest the removal of amorphous hemicellulose, lignin and
amorphous cellulose; these were confirmed using FTIR analysis. FTIR results show a successful
extraction of cellulose from kenaf core wood by removing its hemicellulose and lignin which can be seen
by the absence of hemicellulose and lignin designated peaks for bleached kenaf core and NCC. FTIR also
shows that a high concentration of acid hydrolysis did not alter the structure of kenaf core cellulose. DSC
and TGA results show that, bleached kenaf core obtained from kenaf core wood show the characteristics
thermal degradation of cellulose. On the other hand, from micrographs of STEM, a high concentration of
acid hydrolysis is capable of turning cellulose to nano scale now i.e. NCC. NCC can be produced from
kenaf core wood source with diameter ranging from 8.5 to 22.5 nm. It can be concluded that alkaline
treatment and sodium chlorite is capable of extracting cellulose from kenaf core wood. NCC can be
prepared using acid hydrolysis method and found to have a CrI of 75% with a mean diameter of 15.2 nm.
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REFERENCES
Agarwal, U. P., & Atalla, R. H. (2010). Vibrational Spectroscopy. In C. Heitner, D. Dimmel & J. A. Schmidt
(Eds.), Lignin and Lignans: Advances in Chemistry: Taylor & Francis.
Beck, S., Bouchard, J., & Berry, R. (2010). Controlling the Reflection Wavelength of Iridescent Solid Films
of Nanocrystalline Cellulose. Biomacromolecules, 12(1), 167-172.
Bondeson, D., Mathew, A., & Oksman, K. (2006). Optimization of the isolation of nanocrystals from
microcrystalline cellulose by acid hydrolysis. Cellulose, 13(2), 171-180.
Dong, X. M., Revol, J.-F., & Gray, D. G. (1998). Effect of microcrystallite preparation conditions on the
formation of colloid crystals of cellulose. Cellulose, 5(1), 19-32.
Dufresne, A. (2010). Processing of Polymer Nanocomposites Reinforced with Polysaccharide
Nanocrystals. Molecules, 15(6), 4111-4128.
Eichhorn, S., Dufresne, A., Aranguren, M., Marcovich, N., Capadona, J., Rowan, S., et al. (2010). Review:
current international research into cellulose nanofibres and nanocomposites. Journal of Materials
Science, 45(1), 1-33.
Jackson J.K., Letchford K., Wasserman B.Z., Ye L., Hamad W.Y., & H.M., B. (2011). The Use Of
Nanocrystalline Cellulose For The Binding And Controlled Release Of Drugs. International Journal
of Nanomedicine, 6(1), 321 - 330.
Johar, N., Ahmad, I., & Dufresne, A. (2012). Extraction, preparation and characterization of cellulose
fibres and nanocrystals from rice husk. Industrial Crops and Products, 37(1), 93-99.
Mehdi Jonoobi, J. H., Paridah Md. Tahir, Lukmanul Hakim Zaini, Syeed Saiful Azry, Majid Davoodi
Makinejad. (2010). Characteristics of Nanofibers Extracted from Kenaf Core. Bioresources, 5(4),
2556-2566.
Morán, J., Alvarez, V., Cyras, V., & Vázquez, A. (2008). Extraction of cellulose and preparation of
nanocellulose from sisal fibers. Cellulose, 15(1), 149-159.
Nogi, M., Iwamoto, S., Nakagaito, A. N., & Yano, H. (2009). Optically Transparent Nanofiber Paper.
Advanced Materials, 21(16), 1595-1598.
Pande, H., Roy, D. N., & Kant, S. (2000). Tear And Tensile Properties Of Soda Pulps From Kenaf Bast
Fibers. Tappi Journal, 83(6), 47.
Park, S., Baker, J., Himmel, M., Parilla, P., & Johnson, D. (2010). Cellulose crystallinity index:
measurement techniques and their impact on interpreting cellulase performance. Biotechnology
for Biofuels, 3(1), 10.
Rhim, Y.-R., Zhang, D., Rooney, M., Nagle, D. C., Fairbrother, D. H., Herman, C., et al. (2010). Changes
in the thermophysical properties of microcrystalline cellulose as function of carbonization
temperature. Carbon, 48(1), 31-40.
Roman, M., & Winter, W. T. (2004). Effect of Sulfate Groups from Sulfuric Acid Hydrolysis on the Thermal
Degradation Behavior of Bacterial Cellulose. Biomacromolecules, 5(5), 1671-1677.
Sehaqui, H., Salajkova, M., Zhou, Q., & Berglund, L. A. (2010). Mechanical performance tailoring of tough
ultra-high porosity foams prepared from cellulose I nanofiber suspensions. Soft Matter, 6(8), 18241832.
Villar, J. C., Revilla, E., Gómez, N., Carbajo, J. M., & Simón, J. L. (2009). Improving the use of kenaf for
kraft pulping by using mixtures of bast and core fibers. Industrial Crops and Products, 29(2–3),
301-307.
Webber III, C. L., & Bledsoe, V. K. (2002). Plant maturity and kenaf yield components. Industrial Crops
and Products, 16(2), 81-88.
Yan, T., Xu, Y., & Yu, C. (2009). The isolation and characterization of lignin of kenaf fiber. Journal of
Applied Polymer Science, 114(3), 1896-1901.
Yang, H., Yan, R., Chen, H., Lee, D. H., & Zheng, C. (2007). Characteristics of hemicellulose, cellulose
and lignin pyrolysis. Fuel, 86(12–13), 1781-1788.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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FORMULATION OF CURCUMIN LOADED STARCH NANOPARTICLES
Siti Nur Akmar*, Suk Fun Chin, Suh Cem Pang
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300,
Kota Samarahan, Sarawak (MALAYSIA)
Email: sn_akmar@yahoo.com
Abstract
Recently, various attempts have been made to overcome the limitation of poorly water-soluble drugs
through loading in polymeric nanoparticles (10-100 nm) in order to permits drug to be more effectively
delivered through the bloodstream which consists mostly of water. In this research, starch nanoparticles
were synthesized from native sago starch powder and these starch nanoparticles were used as the
loading agent, while curcumin was used as a poorly water-soluble model drug. Curcumin loading in sago
starch nanoparticles was investigated by using a simple nanoprecipitation method in water-in-oil (w/o)
microemulsion system as reaction medium. The types of reaction medium, types of surfactant, effect of
surfactant concentration, oil/ethanol ratio and initial curcumin concentration affected the characteristics of
the curcumin loaded starch nanoparticles in terms of particles size, morphology and loading efficiency
(LF). Dissolution of 1% (w/v) curcumin in 1:3 ratio of oleic acid to ethanol microemulsion system and in the
presence of 3% (w/v) Tween 80 surfactant in the formulation were found to be optimal in the preparation
process. Curcumin released from prepared starch nanoparticles formulation was slow and sustained over
four days.
Keywords: Starch nanoparticles, Curcumin, Loading efficiency, Accumulative release
1. INTRODUCTION
Starch is well-known, versatile, and inexpensive biodegradable polysaccharides that were be used in
medicine field as drug carrier material (Szepes et al., 2008; Ali & Al-Arifi, 2009; Wang et al., 2010). The
basic formula for starch is (C6H10O5)n and it consists of mainly two glycosidic macromolecules which are
amylose and amylopectin (Hui, 2006). Starch-based drug delivery systems have received great attention
as the results of their good hydrophilicity and biodegradability which important for controlled- and
sustained-release properties and also biocompatibility with tissue and cells (Lu et al., 2009; Moraes et al.,
2009).
Curcumin, a natural polyphenol found in the rhizomes of Curcuma longa has exhibited antioxidant, antiinflammatory, anti-survival, antiproliferative, anti-invasive and antiangiogenic activity (Kunnumakkara et
al., 2008). More recently, the clinical effects on antitumors properties of curcumin are currently being
studied in human clinical trials on various conditions and multiple myeloma (Aggarwal et al., 2003;
M.D.Anderson Cancer Center, 2004; M.D.Anderson Cancer Center, 2005). Unfortunately, these studies
have revealed that curcumin suffers from serious poor delivery characteristic since it is generally found
inactive in clinical trials primarily due to its water-insolubility and instability, and thus exceedingly poor
bioavailability (Anand et al., 2007).
To date, loading of curcumin in polymeric nanoparticles have the potential for imparting dispersibility of
curcumin in aqueous media, thus circumventing the pitfalls of poor solubility. Following this, Sahu et al.,
(2008) synthesized a novel polymeric amphiphile with methoxy poly(ethyleneglycol) as the hydrophilic and
palmitic acid as the hydrophobic segment to encapsulate hydrophobic compounds like curcumin in the
nanocarriers, making the drug soluble in an aqueous system. A different approaches was taken by
Mukerjee & Vishwanatha (2009) where they loaded the curcumin in poly(D,L-lactide-co-glycolide (PLGA)
nanospheres using solid/oil/water emulsion evaporation method resulted an encapsulation efficiency of
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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curcumin for about 90.88 ± 0.14%. In another important study, Mathew et al. (2012) synthesized watersoluble PLGA coated curcumin nanoparticles and coupled the nanoparticles with tet-1-peptide. They
found that curcumin encapsulated PLGA nanoparticles are able to destroy amyloid aggregates, exhibit
anti-oxidative property and are non-cytotoxic.
Therefore, research on encapsulation of curcumin by using starch nanoparticles is of current interest. In
this work the potential application of starch nanoparticles as loading agent and controlled release agent
for curcumin was investigated. The synthesis parameters that affected the encapsulation efficiency of
starch nanoparticles and release profile of curcumin from starch nanoparticles were investigated. These
results indicate that starch nanoparticles are a good loading and controlled release agent for curcumin.
2. MATERIALS AND METHOD
2.1 Materials
All chemicals were of reagent grade and were used without further purification. Ultrapure water (~18.2 M
Ω, 25 ºC) was obtained from Water Purifying System (EδGA, εodel Ultra Genetic), Cyclohexane and
absolute ethanol were obtained from HmbG Chemicals (Germany), oleic acid was obtained from Ajax
chemicals (Australia) while curcumin powder, Polysobate 80 (Tween 80) surfactant and sorbitan
monostearate (Span 60) surfactant were obtained from Merck (Germany). Native sago starch powder and
sunflower oil were obtained from a local grocery store.
2.2 Loading of Curcumin by using Starch Nanoparticles
Precipitation in water-in-oil (w/o) microemulsion technique was used to load curcumin in starch
nanoparticles. The solvent phase essentially consist of absolute ethanol as an organic solvent, oil (oleic
acid, sunflower oil and cyclohexane) as the continuous phase and surfactant (Tween 80 or Span 60) as a
emulsifying agent, while the non-solvent is mainly starch solution. About 0.2 mg of curcumin was
dissolved into 1:3 ratio of oil to absolute ethanol solution containing 3% (w/v) surfactant, and the mixture
was stirred for 1 hour. After 1 hour, 1 mL of starch solution (1% w/v) was added drop wise and stirred for
an addition of 1 hour. Curcumin was loaded into starch nanoparticles as the starch nanoparticles formed.
The starch nanoparticles loaded with curcumin were collected by centrifugation so that starch
nanoparticles loaded with curcumin were precipitated. The precipitate was washed four times with
ethanol, whereas, free curcumin was collected as supernatant. For loading of curcumin using starch
nanoparticles in ethanol solution without microemulsion system, the same synthesis conditions was used,
except the precipitation was carried out in the absence of microemulsion system.
2.3 Characterization of Curcumin Loaded Starch Nanoparticles
The UV-vis spectroscopic studies are carried out using a UV spectrophotometer (Jasco V-630) from 350500 nm. The starch nanoparticles loaded with curcumin were separated from the reaction medium by
centrifugation and the free curcumin was used to measure the absorption spectra.The UV spectra of the
curcumin showed a results of characteristic peak at 422 nm. SEM images were recorded using Scanning
Electron Microscope (SEM) (JEOL-SM 6390 LA) operating between 10-15 kV with a filament current of
about 60-70 µA. Powder samples were deposited on aluminium stubs and they were then coated with a
platinum layer using JEOL/JFC-1600 Auto Fine Coater.
2.4 Evaluation of Loading Efficiency
The percentage of curcumin loaded in starch nanoparticles preparation was determined by centrifuging
the nanoparticles and separating the supernatant. The supernatant was assayed by UV
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spectrophotometer (UV Jasco V-630 spectrophotometer) at 422 nm. Loading efficiency was calculated as
follows (Equation 1) (Dev et al., 2010; Mathew et al., 2010).
Loading efficiency (%) = [curcumin]tot – [curcumin]free
[curcumin]tot
X 100
Equation 1
Where [curcumin]tot is concentration of initially curcumin added and [curcumin] free is concentration of
curcumin present in supernatant after centrifugation.
2.5 Release Evaluation
In order to study the release of curcumin from starch nanoparticles, about 50 milligram of samples was
placed in a measured volume 15 mL of pH 7.4 phosphate buffer solution (PBS) (mimicking intestinal
medium) and pH 2.1 hydrochloric acid solution (mimicking gastric juice) at 37 ºC (mimicking human body
temperature). Free curcumin is completely insoluble in water, therefore at predetermined time intervals,
the solution was centrifuged to separate the released curcumin from the starch nanoparticles. The release
of curcumin was assayed spectrophotometrically at 422 nm. The percentage of curcumin released was
determined based on Equation 2 (Dev et al., 2010; Mathew et al., 2010).
Release of curcumin (%) = [curcumin]rel
[curcumin]tot
X 100
Equation 2
Where [curcumin]rel is concentration of release curcumin collected at time and [curcumin] to is total amount
of curcumin loaded in the starch nanoparticles.
2.6 Swelling Studies
The swelling profiles of the developed curcumin loaded starch nanoparticles were determined by
immersing a pre-weight dried curcumin loaded starch nanoparticles in 10 ml of buffer, pH 2.1 or 7.4 at 37
ºC. The swollen weights of the starch nanoparticles were determined at intervals. The swelling ratio was
then calculated based on Equation 3 (Singh et al., 2007; Akhgari et al., 2011).
Percent swelling (Ps) (%) = Ws – W d
Wd
X 100
Equation 3
Where W s is weight of swollen starch nanoparticles and W d is weight of dry starch nanoparticles
3. RESULTS AND DISCUSSION
3.1 Loading of Curcumin into Starch Nanoparticles
One of the major purposes of loading of curcumin into starch nanoparticles is to improve curcumin
dispersibility in aqueous media. As shown in Figure 1, curcumin is insoluble in aqueous media as
macroscopic flakes were observed to float in aqueous media. In contrast, curcumin loaded in starch
nanoparticles were fully dispersible in aqueous media. Thus, this clearly showed that loading of curcumin
in starch nanoparticles renders curcumin completely dispersibility in aqueous media. This is due to the
fact that when the size of starch nanoparticles is in nanosized ratio, it attributed to the large surface area
of starch nanoparticles which allow a greater interaction with the aqueous solvent which resulting an
increase in solubility in aqueous media (Sagar et al., 2007).
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Figure 1 Photographic image of (a) free
curcumin is poorly soluble in aqueous media,
and (b) the curcumin loaded in starch
nanoparticles is fully dispersible in aqueous
media
3.2 Optimization of Efficiency of Curcumin Loading in Starch Nanoparticles
3.2.1 Effect of Reaction Medium
The effect of different types of reaction medium on curcumin loading efficiency of starch nanoparticles
were studied by carried out the loading process in four different types of reaction medium namely
ethanolic solution (E), cyclohexane/ethanol microemulsion solution (C/O), sunflower oil/ethanol
microemulsion solution (SO/E) and oleic acid/ethanol microemulsion solution (OA/E). As shown in Figure
2, the efficiency of curcumin loading in starch nanoparticles presented the highest value of around 75.17 ±
0.03% (mean ± standard deviation (SD)) when it was precipitated into oleic acid/ethanol reaction medium
and a value of around 47.62 ± 0.02% (mean ± SD) when it was precipitated into sunflower oil/ethanol
microemulsion medium. Meanwhile, the curcumin loading efficiency of starch nanoparticles of 12.68 ±
0.06% (mean ± SD) was achieved with nanoprecipitation in ethanolic reaction medium, whereas
nanoprecipitation in cyclohexane/ethanol microemulsion reaction medium was a same trend with
nanoprecipitation in ethanolic reaction medium, since it had low loading efficiency which amounted to
16.85 ± 0.30% (mean ± SD).
From the results, the preparation of curcumin loaded starch nanoparticles in oleic acid/ethanol
microemulsion reaction medium presented the highest curcumin loading efficiency among microemulsion
tested. This is because oleic acid (C18) has a long alkyl chain lipid length and greater total chain number
than sunflower oil (C12) and cyclohexane (C6). The longer the alkyl chain length and the greater the total
chain number, the more hydrophobic of the reaction medium (Zhang et al., 2010).
Consequently, curcumin are more soluble and dispersed in the oleic acid/ethanol reaction medium which
resulted high efficiency of curcumin loading in starch nanoparticles. Besides, the preparation of curcumin
loaded starch nanoparticles in microemulsion reaction medium can enhance the curcumin loading
efficiency of starch nanoparticles than preparation in without microemulsion system due to good
properties of microemulsion system by reason of the presence of lipophilic domains of the system which
can dissolves a wide range of hydrophobic curcumin molecules (Malcolmson & Lawrence, 1990),
Figure 2 Effect of reaction medium on
loading efficiency of curcumin in starch
nanoparticles
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As can be seen in Figure 3, the smallest curcumin loaded starch nanoparticles were achieved by using
oleic acid/ethanol microemulsion system. This could be due to the longer the alkyl chain lengths of oil, the
particle size become smaller (Begum et al., 2012). Therefore, the oleic acid/ethanol microemulsion
reaction medium was employed for further optimization of curcumin loading efficiency of starch
nanoparticles.
Figure 3 SEM images curcumin
loaded
starch
nanoparticles
formulated
through
nano
precipitation
in
(a)
absolute
ethanolic solution, (b) cyclohexane/
ethanol, (c) sunflower oil/ ethanol,
and
(d)
oleic
acid/ethanol
microemulsion reaction médium
3.2.2
Effect of Types of Surfactant
The effect of different types of surfactant on loading efficiency of curcumin in starch nanoparticles was
studied by using two types of non ionic surfactant namely Sorbitan monostearate (Span 60) and
Polysorbate 80 (Tween 80). Both of these surfactants are non ionic, thus their ability as surfactants does
not depend on its ionic strength and they are non toxic in nature (Perez et al., 2009).
As shown in Figure 4, the efficiency of curcumin loading in starch nanoparticles was higher when Tween
80 (75.17 ± 0.03% (mean ± SD)) was used as a surfactant as compared to Span 60 (19.59 ± 0.23%
(mean ± SD)). These results demonstrated that Tween 80 is an effective surfactant which could be due to
a stable primary microemulsion was formed in the presence of Tween 80 as it is more soluble in water as
compared to Span 60 (Schmidts et al., 2009).
Figure 4 Effect of types of surfactant on
loading efficiency of curcumin in starch
nanoparticles
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3.2.3 Effect of Surfactant Concentration
The concentration of surfactant was observed to play an important role in the loading efficiency of
curcumin in starch nanoparticles. As can be seen in Figure 5, the loading efficiency of curcumin in starch
nanoparticles in the presence of surfactant in the microemulsion system was higher as compared to those
without surfactant which was at around 68.02 ± 0.03 (mean ± SD), 75.17 ± 0.03% (mean ± SD) and 77.88
± 0.01 (mean ± SD), while the loading efficiency of curcumin of 47.70 ± 0.09% (mean ± SD) was achieved
by starch nanoparticles without surfactant presented in the microemulsion system.
Figure 5 Effect of surfactant concentration
on loading efficiency of curcumin in starch
nanoparticles
The loading efficiency of curcumin in starch nanoparticles increased when the added amount of Tween 80
surfactant was increased form 1% to 3%. This could be due to an increase in surfactant concentration
may let to increase in amount of curcumin deposited at the surface and incorporate in the starch
nanoparticles since an enough surfactant presented to stabilize the interfacial tension of oleic acid/ethanol
microemulsion system (Jin et al., 1997).
3.2.4 Effect of Oil/Ethanol
The effect of oil/ethanol ratio on loading efficiency of curcumin in starch nanoparticles was shown in
Figure 6.
Figure 6 Effect of oil to ethanol volume on
loading efficiency of curcumin in starch
nanoparticles
It was found that on increasing the oleic acid (oil phase) to ethanol volume from 1:3 to 1:1 and 3:1
(oil/ethanol), the loading efficiency of curcumin in starch nanoparticles decreased approximately from
75.17 ± 0.03% (mean ± SD) to 14.03 ± 0.24% (mean ± SD) and 27.90 ± 0.07% (mean ± SD), respectively.
This is because a stable and homogeneous microemulsion cannot be form when oil content is too high.
Furthermore, the 3% (w/v) concentration of Tween 80 surfactant is not enough to stabilize the oleic
acid/ethanol microemulsion system, thus resulted in reduction of loading efficiency of curcumin in starch
nanoparticles. Figure 6 also shows that higher ratio ethanol to oil resulted in enhanced loading efficiency
of curcumin in starch nanoparticles. This could be due to the ethanol as cosurfactant can reduce the
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surface concentration of microemulsion system as their short hydrophobic chain and terminal hydroxyl
group can improve the interactions of surfactant monolayer at the interface of microemulsion system
(Alany et al., 2000).
3.2.5 Effect of Initial Curcumin Concentration
As can be seen in Figure 7, the loading efficiency of curcumin in starch nanoparticles descended
significantly from 68.77 ± 0.03% (mean ± SD) to 23.47 ± 0.03% (mean ± SD) and 13.07 ± 0.22% (mean ±
SD) as the initial curcumin concentration rose to 3 % w/v, respectively. This could be due to an increase
in initial curcumin concentration may contribute to a less polymer-drug interaction. Thus, lead to decrease
the efficiency of curcumin loading in starch nanoparticles (Kumar et al., 2011). In other words, as starch
nanoparticles has certain curcumin loading efficiency, addition of excess curcumin led to increase of
unencapsulated drug (free curcumin) which caused decrease of loading efficiency. After reaching
maximum curcumin loading efficiency, loading curcumin by starch nanoparticles cannot increase, which in
this study signified that starch nanoparticles reached its maximum point at 1% w/v initial curcumin
concentration.
Figure 7 Effect of initial curcumin
concentration on loading efficiency of
curcumin in starch nanoparticles.
3.3 Release of Curcumin from Starch Nanoparticles
The curcumin loaded starch nanoparticles which have been prepared in oleic acid/ethanol microemulsion
system in the presence of 3 % w/v Tween 80 surfactant was used for release studies since it showed the
highest efficiency of curcumin loading in starch nanoparticles among optimization parameters tested. The
release studies was performed in simulated gastric fluid (pH 2.1) and simulated intestinal medium (pH 7.4)
at 37 ± 0.5 ºC as a function of time with continuous stirring within 4 days. The pH 2.1 and pH 7.4 were
chosen to reflect the physiologic conditions of the gastrointestinal in body. The release profile of curcumin
from starch nanoparticles was presented in Figure 8.
Figure 8 Release profile of curcumin
from starch nanoparticles as a function
of time
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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It could be seen that curcumin released from starch nanoparticles was 46.51% at pH 2.1 and 56.34% at
pH 7.4 within 4 days (108 hours). This suggests that the release of curcumin from starch nanoparticles is
pH sensitive (Li et al., 2008). According to the results, the release of curcumin from starch nanoparticles
incubated in simulated gastric fluid (pH 2.1) is much slower than in simulated intestinal fluid (pH 7.4).
Similar results have been reported previously by Dev et al., (2010) who found that the in vitro drug release
of poly (lactic acid)/ chitosan nanoparticles showed a drug release rate of lower in the acidic pH when
compared to alkaline pH.
In this research, the release characteristics of curcumin from starch nanoparticles are dependent on the
swelling behaviour of the starch nanoparticles. The swelling behaviours of the starch nanoparticles in
buffer solution of pH 2.1 and 7.4 at 37 ºC are shown in Figure 9. The result shows that the release of
curcumin from starch nanoparticles at lower pH swelling medium was slower than that of the swelling
media of higher pH. This is attributed to the reason that a different pH of solution contributed to different
dipole interactions in the polymer chains, leading to the significant changes in the water uptake of these
starch nanoparticles (Singh et al., 2007). As a -COO group on starch nanoparticles will changes to COOH at low pH, this will reduce the swelling ability of starch nanoparticles, thus the release of curcumin
in pH 2.1 is slow as compared to in pH 7.4 (Wang et al., 2010).
Figure 9 Swelling profile of curcumin
loaded starch nanoparticles as a
function of time
4. CONCLUSIONS
In conclusion, we have successfully loaded curcumin into starch nanoparticles using a simple
nanoprecipitation technique. The loading efficiency was significantly affected by types of reaction medium,
concentration of surfactant, types of surfactant, oil/etanol ratio and initial curcumin concentration.
Dissolution of 1 % (w/v) curcumin in 1:3 ratio of oleic acid to ethanol microemulsion system by the
presence of 3 % (w/v) Tween 80 surfactant with 2 hours loading time was found to be optimal in the
preparation process. The loading of curcumin in starch nanoparticles affords a controlled release
mechanism under physiological pH and the curcumin can be released from starch nanoparticles for about
46.51 % at pH 2.1 and 56.34 % at pH 7.4 within 4 days (108 hours). Overall, our study suggested that
starch nanoparticles are a promising carrier and controlled release agent for curcumin and other
neutraceuticals.
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and drug release from polysaccharides matrix tablets based on pectin and inulin. Jundishapur
Journal of Natural Pharmaceutical Products, 6, 51 – 58.
Alany, R. G., Raders, T., Aqatonovic-Kustrin, S., Davies, N. M., & Tucker, I. G. (2000). Effects of
alcohols and diols on the phase behavior of quarternary systems. International Journal of
Pharmaceutics, 196, 141 – 145.
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Ali, A. E. H., & AlArifi, A. (2009). Characterization and in vitro evaluation of starch based hydrogels as
carriers for colon specific drug delivery systems. Carbohydrate Polymers, 78, 725 – 730.
Anand, P., Kunnumakkara, A. B., Newman, R. A., & Aggarwal, B. B. (2007). Bioavailability of
curcumin:
problems and promises. Molecular Pharmaceutical, 4, 807 – 818.
Begum, M. Y., Abbulu, K., Sudhakar, M., & Aneesa. (2012). Celexoib-encapsulated liposomes of long
alkyl chain lipids: formulation, characterization and in vitro performance. Der Pharmacia
Sinica, 3, 117 – 125.
Dev, A., Binulal, N. S., Anitha, A., Nair, S. V., Furuike, T., Tamura, H., & Jayakumar, R. (2010).
Preparation of poly (lactic acid)/chitosan nanoparticles for anti-HIV drug delivery applications.
Carbohydrate Polymers, 80, 833 – 838.
Hui, Y.H. (2006). Handbook of food science, technology and engineering. United States of
America:
CRC Press.
Jin, J. M., Parbhakar, K., & Dao, L. H. (1997). Model for water-in-oil microemulsions: surfactant effects.
Physical Review, 55, 721 – 726.
Kumar, D. A., Dharmendra, S., Jhansee, M., Shrikant, N., & Shiv, P. P. (2011). Development and
characterization of chitosan nanoparticles loaded with amoxicillin. International Research
Journal of Pharmacy, 2, 145 – 151.
Kunnumakkara, A. B., Anand, P., & Aggarwal, B. B. (2008). Curcumin inhibits proliferation, invasion,
angiogenesis and metastasis of different cancers through interaction with multiple cell signaling
proteins. Cancer Letters, 269, 199 – 225.
Li, P., Dai, Y. N., Zhang, J. P., Wang, A. Q., & Wei, Q. (2008). Chitosan-alginate nanoparticles as a
novel drug delivery system for nifedipine. International Journal of Biomedical Science, 4, 221 –
229.
Lu, D. R., Xiao, C. M., & Xu, S. J. (2009). Starch-based completely biodegradable polymer materials.
Polymer Letters, 3, 366 – 375.
Malcomlmson, C., & Lawrence, M. J. (1990). A comparison between nonionic micelles and
microemulsions as a means of imcorporating the poorly water soluble drug diazepam. Journal of
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Mathew, M. E., Mohan, J. C., Manzoor, K., Nair, S. V., Tamura, H., & Jayakumar, R. (2010). Folate
conjugated carboxylmethyl chitosan-manganese doped zinc sulphide nanoparticles for targeted
drug delivery and imaging of cancer cells. Carbohydrate Polymers, 80, 442 – 448.
Mathew, A., Fukuda, T., Nagaoka, Y., Hasumura, T., Morimoto, H., Yoshida, Y., Maekawa, I.,
Venugopal, K., & Kumar, D. S. (2012). Curcumin loaded PLGA nanoparticles conjugated with
tet-1 peptide for potential use in Alzheimer’s Disease. Plos ONE, 7, e32616.
M. D. Anderson Cancer Center. (2004). Trial of curcumin in advanced pancreatic cancer. Retrieved
May 18, 2012, from www.clinicaltrials.gov/ct2/show/NCT00094445
M. D. Anderson Cancer Center. (2005). Curcumin (diferuloylmethane derivative) with or without bioperine
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Moraes, C. M., Matos, A. P., Paula, E., Rosa, A. H., & Fraceto, L. M. (2009). Benzocaine loaded
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Perez, M. W., Gomez, A. T., Ruiz, M. J. G., & Rodriguez, M. A. (2009). Stability of emulsions for
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Singh, B., Sharma, N., & Chauhan, N. (2007). Synthesis, characterization and swelling studies of pH
responsive psyllium and methacrylamide based hydrogels for the use in the colon specific
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and drug delivery behaviour of starch-based hydrogels prepared via isostatic ultrahigh pressure.
Carbohydrate Polymers, 72, 571 – 578.
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Nanoscale Research Letters, 5, 917 – 925.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
PRODUCTION OF TROPANE ALKALOIDS FROM CELL SUSPENSION CULTURE OF
Hyoscymus niger L.
Chan Lai Keng and Rafidah Ishak
School of Biological Sciences, Universiti Sains Malaysia 11800 Penang, Malaysia
Email: lkchan@usm.my
Abstract
Friable callus could be induced form the leaf explants of the in vitro plantlets of Hyoscyamus niger on MS
medium supplemented with 2.0 mg/L picloram. The leaf-derived callus subsequently sub-cultured on MS
medium supplemented with 0.5 mg/L picloram became more friable with less granules but with higher
growth rate. The cell suspension culture of H. niger was initiated by transferring 0.5 g of the most friable
portion of the leaf-derived callus into 25 mL of the cell proliferation medium, MS liquid medium
supplemented with 0.5 mg/L picloram, 30 g/L sucrose and 0.1 mg/L myo-inositol. The growth pattern of H.
niger based on fresh and dried cell mass followed a typical sigmoid curve. Elicitations with calcium
chloride, yeast extract and casein hydrolysate gave different effects on the cell growth and production of
tropane alkaloids from the cell cultures of H. niger. Lower concentration of calcium chloride induced higher
cell growth. Only the production of scopolamine was increased when the concentration of calcium chloride
was doubled from the normal MS concentration (0.44 g/L). Elicitation with 3.0 g/L of yeast caused lower
cell growth while elicitation with 2.0 g/L yeast extracts caused significant increased in the production of
hyoscyamine and scopolamine. Elicitation with casein hydrolysate did not have any significant effect on
the growth of H. niger cells nor the production of hyoscyamine and scopolamine.
Keywords: callus, cell suspension culture, tropane alkaloids, picloram, Hyoscyamus niger
1. INTRODUCTION
Hyoscyamus niger is a European native plant of Solanaceae family. It is commonly known as black
henbane and it is also called the chicken murderer because when the fowls eat the seeds of this plant
they become paralysed and die. The seeds are also believed to be poisonous to children, rodents, and
fish (Haas, 1995). Other documented effects of henbane poisoning in humans are intoxication, stupor,
dilatations of the pupils, apoplexy, convulsions and even death. The poisonous property of this plant is
due to the presence of narcotic tropane alkaloids, hyoscyamine, scopolamine and atropine. Tropane
alkaloids are compounds known as muscarinic receptor antagonists. These substances interfere with the
parasympathetic nervous system because they chemically resemble the neurotransmitter acetylcholine
(Börsch-Haubold, 2007). In modern medicine, the applications of tropane alkaloids in pharmaceutical
include powerful bronchodilators to treat chronic bronchitis, as midriatics for dilation of pupil of the eye to
facilitate optical surgery and as antimuscarinic drugs to control Parkinson’s disease (De Luca, 2000).
However, since H. Niger cannot be cultivated in tropical countries like Malaysia, some of the
pharmaceutical companies in Malaysia have to import the dried plant parts to be used in the industry. The
application of in vitro plant culture techniques in the pharmaceutical industry is becoming more important
to ensure a sustainable supply of the source materials. Plant cell culture technique which is independent
of geographical and seasonal variation is an attractive solution to produce the meterials for the market
demand. This technique has been used for other plant species. Plant cells are biosynthetically totipotent,
which means that each cell in the culture retains complete genetic information of the parent plant. Thus,
cell culture is capable of producing the same range of chemicals found in the parent plant (Rao, 2002). As
a result, as long as the in vitro culture system of the cells can be established, the relevant chemicals
needed can be produced anywhere regardless of the climate difference. In accordance, this paper
reported the establishment of an in vitro cell culture system of H. niger as an alternative method for the
production of the tropane alkaloids.
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2. MATERIALS and METHODS
2.1 Establishment of cell suspension culture
To establish the H. niger cell suspension culture, 0.5 g of two weeks old friable callus were inoculated into
25 mL MS (Murashige and Skoog, 1962) liquid medium supplemented with 0.5 mg/L picloram (cell
proliferation medium) in 100 mL Erlenmeyer flasks. The cell suspension cultures were placed on a rotary
shaker (Certomat) and continuously agitated at 120 rpm, under 800 - 900 lux light illumination in the
culture room with a temperature of 25 ± 2 °C. After fifteen days of culture, the cells were harvested from
the liquid medium by filtering through two layers of filter paper (Whatman No. 1), assisted by air suction
pump (Aspirator 3A-S-Eyela, Raikikai Co. Ltd. Tokyo). The filtered cells were rinse once with double
distilled water and the fresh cell mass was then used for ilicitaion studies.
2.2 Effect of Elicitation on Hyoscyamus niger Cell Growth and production of tropane alkaloids
Sieved and filtered fresh cells (0.5 g) of H. Niger were inoculated in MS liquid medium supplemented with
0.5 mg/L picloram, 30g/L sucrose and 0.1 mg/L myo-inositol, the cell proliferation medium, with addition of
varied amount of elicitors (calcium chloride, yeast extracts and casein hydrolysate) stage wise. The cell
cultures were first added with calcium chloride with varying concentration, 0.22, 0.44, 0.88 and 1.32 mg/L.
Normal MS medium contains 0.44 g/L calcium chloride.
To study the effect of yeast extract, the fresh filtered cell (0.5 g) were inoculated in 25 mL of the cell
proliferation medium and 0.22 g/L calcium chloride, then added with 0, 0.5, 1.0, 2.0 and 3.0 g/L yeast
extract. For casien hydrolysate elicitation study, fresh filtered cells (0.5 g) were inoculated into 25 mL of
the cell proliferation medium, 0.22 g/L calcium chloride and without yeast extract but supplemented with 0,
0.5, 1.0, 2.0 and 3.0 g/L casein hydrolysate. For each elicitation treatment, 8 experimental units were used
and the experiment was carried out using complete randomized design. After 14 days of culture, the cells
were filtered and weighed. The data were analysed with One-Way ANOVA followed by comparison of
means using Tukey Test at p ≤ 0.05. The harvested cells for each batch were air-dried untill constant
weight was attained.
2.3 Chemical Analysis of Alkaloid Contents Hyoscyamus niger Cells
Extraction of tropane alkaloids from the elicitated H. niger cells was carried out using a modified method
adopted from Kursinszki (2005). The dried cells were then macerated to powder with mortar and pestle
and 200 mg powdered cells were soaked overnight in 25 ml extraction solution which made up of
chloroform, methanol and concentrated ammonium hydroxide (15:5:1, v/v/v). After 12 hour of soaking, the
samples were extracted twice in an ultrasonic bath at 27 ± 2°C for 20 minutes. The fractions were then
filtered under vacuum over 4-cm Buchner funnel with two pieces of filter paper (Whatman No. 5). The
filtrates were then mixed with 1.0 g anhydrous sodium sulphate (Na2SO4), re-filtered and evaporated to
dryness at 50 °C, under reduced pressure by a rotary evaporator (Eyela Rotary Vacuum Evaporator N-N
Series with Eyela Digital Waterbath SB-651, Tokyo Rikikai Co. Ltd.) couple with a water pump (Eyela
Aspirator A-3S, Tokyo Rikikai Co. Ltd.). Quantification of the alkaloids was carried out using gas
chromatography coupled with flame ionization detector (GC-FID), following the method reported by
Berkov and Pavlov (2004). Gas chromatography (GC) was carried out using a Hewlett Packard (HP: Palo
Alto, CA, USA) model 5890 chromatograph, equipped with HP-5 column (30m x 0.25mm i.d.ν 0.25 ε) and
a flame ionisation detector (FID). Each of the standard solutions and the cell extracts with a volume of 1
δ were injected and analysed in triplicates. The chromatograms of the extracts were then matched with
that of the standards to detect the presence of hyoscyamine and scopolamine based on the retention time.
Finally the hyoscyamine and scopolamine contents in each sample were determined by referring to the
calibration curve of the standards alkaloids.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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3. RESULTS AND DISCUSSION
3.1 Cell suspension culture of Hyoscyamus niger
The cell suspension culture initiated tends to form aggregates as the amount of cells increased. The
growth index of the first batch of culture was high (5.7 ± 1.0), and the cell cultures were made up of big
size of cell aggregates. Hence, the cells needed to be sieved at every sub-culture cycle to ensure that the
cell was uniform and optimum for cell growth. The growth pattern of H. niger cells based on fresh and
dried cell mass followed a typical sigmoid curve. The cell growth based on fresh cell mass consisted of a
lag phase of three days. This was followed by an exponential phase until the eighteenth day (1.3 ± 0.3 g)
after which the cell reached its stationary phase. The growth pattern based on dried cell mass showed
initial lag phase of three days and the cell growth started to increase exponentially up to day eighteenth
then followed with the stationary phase (Figure 1).
Figure 1 Growth pattern of
Hyoscyamus niger cell cultures
3.2 Effect of Elicitations on Hyoscyamus niger Cell Growth
Higher concentration of calcium chloride added into the cell proliferation medium (MS medium
supplemented with 0.5 mg/L picloram, 30g/L sucrose and 0.1 mg/L myo-inositol) resulted in lower cell
growth of H. niger (Figure 2). The cells grew in the culture medium containing 0.22 g/L calcium chloride
(halved the concentration in normal MS medium) had significantly increased the production of fresh cell
mass (7.576 ± 0.112 g) after fourteen days of culture. The culture medium containing the normal MS
concentration of calcium chloride (0.44 g/L) had lower cell mass (6.927 ± 0.153 g), followed by the culture
medium containing double concentration of calcium chloride (0.88 g/L) and triple (1.32 g/L); with cell mass
of 6.274 ± 0.085 g and 5.785 ± 0.138 g, respectively. The results followed similar trend for the dried cell
mass, except for the culture medium containing 0.88 g/L (double MS strength) and 1.32g/L calcium
chloride (triple MS strength) which had no significant increment in dried cell mass (Figure 2). Thus, 0.22
g/L calcium chloride halved the normal MS concentration of culture medium was used for subsequent H.
niger cell growth.
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Figure 2 Effect of calcium chloride
on the growth of Hyoscyamus
niger cells after 14 days of culture
Addition of 0.5 g/L to 2.0 g/L yeast extract to the cell proliferation medium (MS medium supplemented with
0.5 mg/L picloram, 30g/L sucrose, 0.1 mg/L myo-inositol and 0.22 g/L calcium chloride) did not have
significant effect on the growth of H. niger cells. When the concentration of yeast extract added was
increased up to 3.0 g/L, the cell growth was significantly lower compared to the cells cultured in the
medium added up to 1.0 g/L yeast extract. Data based on the dried cells biomass indicated that addition
of 0.5 g/L to 3.0 g/L yeast extract in the media did not affect the cell growth at all (Figure 3). Based on
these observations, it was concluded that addition of yeast extract to the medium did not improve the
growth of H. niger cells.
Figure 3 Effect of calcium chloride
on the growth of Hyoscyamus
niger cells after 14 days of culture
Based on the final biomass of the fresh cells, varied amount of casein hydrolysate added to the cell
proliferation medium (MS medium supplemented with 0.5 mg/L picloram, 30g/L sucrose, 0.1 mg/L myoinositol, 0.22 g/L calcium chloride but without yeast extract did not have any significant effect on the
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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growth of H. niger cells. However the dried cell mass showed a significant decrease when 0.5 g/L casein
hydrolysate was added into the cell proliferation medium (Figure 4). Thus, the addition of casein
hydrolysate did not improve the medium for Hyoscyamus niger cell growth.
Figure 4 Effect of Casein
Hydrolysate on the growth of
Hyoscyamus niger cells after 14
days of culture
3.3 Effect of Elicitations on production of tropane alkaloids
The total ions chromatograms scan of the standard of hyoscyamine and scopolamine showed that the
retention time (tR) for hyoscyamine was between 7.217 – 7.242 minutes and for scopolamine was about
7.948 – 7.951 minutes. The retention time of the standards were used as the reference for quantification
of the alkaloids content in the extracts of the elicitated H. niger cell suspension culture and the preparation
of a calibration curve for each alkaloid, hyoscyamine and scopolamine.
Different concentration of calcium chloride in the cell proliferation medium had no significant effect on the
production of hyoscyamine. In the medium supplemented with 0.44 g/L calcium chloride (the normal
concentration in MS medium) the content of hyoscyamine produced by the cells was 17.3 ± 5.2 g/g dried
cells. Halving the concentration of calcium chloride in the medium (0.22 g/L) did not cause any significant
effect on the contents of hyoscyamine produced. Similarly, increasing the concentration of calcium
chloride added to the medium up to 1.32 g/L did not show any significant effect on the contents of
hyoscyamine produced (Figure 5).
The content of scopolamine detected in the cells cultured in the medium supplemented with 0.44 g/L
calcium chloride (the normal concentration in MS medium) was 2.2 ± 0.2 g/g dried cells. The content of
scopolamine detected in the cells cultured in the medium supplemented with 1.32 g/L calcium chloride
(triple the normal concentration in MS medium) was 0.6 ± 0.2 g/g dried cells which was significantly
lower. However, the content of scopolamine produced by the cells cultured in the medium supplemented
with 0.22 g/L calcium chloride (halved the normal concentration in MS medium) and 0.88 (double the
concentration in MS medium) were not significantly different. Mean values for each parameter followed by
different alphabet were significantly different (Tukey, p ≤ 0.05)
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Figure 5 Effect of calcium chloride
concentration on the contents of
hyoscyamine and scopolamine in
Hyoscyamus niger cells
Elicitation with yeast extracts on the cell suspension cultures caused significant increase to the production
of hyoscyamine and scopolamine (Figure 6). In the control medium (without yeast extract) the content of
hyoscyamine and scopolamine produced by the cell were 4.3 ± 0.1 g/g dried cells and 5.2 ± 0.3 g/g
dried cells respectively. Addition of 0.5 to 1.0 g/L yeast extract into the culture medium did not affect the
production of the alkaloids in the cells. When the concentration of yeast extract added into the medium
was 2.0 g/δ, the production of hyoscyamine and scopolamine increased significantly to 20.λ ± 5.6 g/g
dried cells and 13.1 ± 2.6 g/g dried cells respectively.
Figure 6 Effect of yeast elicitation
on the contents of hyoscyamine
and scopolamine in Hyoscyamus
niger cells. Mean values for each
parameter followed by different
alphabet
were
significantly
different (Tukey, p ≤ 0.05)
Elicitation with casein hydrolysate on the cell suspension culture had no significant effect on the
production of hyoscyamine and scopolamine. In the control medium (without casein hydrolysate) the
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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content of hyoscyamine and scopolamine produced by the cells were 7.8 ± 1.7 g/g dried cells and 8.2 ±
0.5 g/g dried cells respectively. The increase in the concentration of casein hydrolysate added to the
medium up to 3.0 g/L did not show any significant effect on the contents of hyoscyamine and scopolamine
produced (Figure 7).
Figure 7 Effect of casein
hydrolysate elicitation on the
contents of hyoscyamine and
scopolamine in Hyoscyamus niger
cells. Mean values for each
parameter followed by different
alphabet
were
significantly
different (Tukey, p ≤ 0.05)
4. CONCLUSION
Tropane alkaloids, hyoscyamine and scopolamine, could be produced from the cell suspension cultures of
Hyoscyamus niger using cell proliferation medium supplemented with elicitors such as casein hydrolysate,
yeast extract or calcium chloride. The correct biotic elicitations of the cell cultures of H. niger was
necessary for the production of the tropane alkaloids. Hence it is a promising possibility of using cell
suspension culture as an alternative for the production of tropane alkaloids from H. niger, a temperate
plant not suitable to be grown in Malaysia.
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Murashige, T., & Skoog, F.A. (1962). A revised medium for rapid growth and bioassays with tobacco
tissue culture. Plant Physiology, 15, 473-479.
Rao, S.R. and Ravishankar, G.A., (2002) Plant cell cultures: Chemical factories of secondary metabolites.
Biotechnology Advances. 101-153.
41
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
EFFECT OF MEDIUM REPLENISHMENT IN CELL SUSPENSION OF Artemisia annua OF VIETNAM
ORIGIN
1
2
King Wey Heng , Derek Juinn Chieh Chan , Chan Lai Keng
1
1
School of Biological sciences, Universiti Sains Malaysia, 11800 Penang, MALAYSIA
School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Nibong Tebal, Pulau
Pinang
2
Email: lkchan@usm.my
Abstract
Initial studies conducted on plant cell suspensions of Artemisia annua of Vietnam origin in shake flasks
have proven to be a possible way of synthesizing artemisinin through plant cell culture technology. With
the high demand of artemisinin in tropical countries where malaria is widespread, there is a need for the
large scale production of this potent drug. However, A. annua suspension cultures cultivated in shake
flasks would not be economically feasible for commercialization of the end product. Based on the same
principles of cell culture techniques for secondary metabolite production on a larger scale in cell culture
tanks, process optimization is the key consideration in achieving desired culture conditions. With the
growth index of cell culture greatly reduced from shake flasks to cell culture tanks, we investigate the
possible factors to increase the productivity in large scale cultures through medium replenishment and
sucrose consumption, which may lead to future fed-batch cultures in cell culture tanks.
Key words: medium replenishment, cell culture technology, cell culture tank
1. INTRODUCTION
Plant cell cultures have proven to be able to generate secondary metabolites that offer the potential of
commercialization (Kieran et al., 1997). Secondary metabolites such as artemisinin produced from
Artemisia annua extracts are worth exploring as artemisinin possesses anti-malaria properties (Krishna et
al., 2008). Artemisinin is a sesquiterpene lactone that is high in efficacy against chloroquine-resistant
Plasmodium falciparum that causes cerebral malaria in tropical countries (Abdin et al., 2003). The activity
of the artemisinin drug is mainly due to the presence of the peroxide bridge which proves to be vital in its
activity against P. falciparum (Balint, 2001). Highest artemisinin content was found in the leaves of A.
annua of Chinese variety at 0.01-0.50 % (Nair et al.1986). Several setbacks of artemisinin production
include the lack of artemisinin consistency with low yields from A. annua grown in the field and the
unstable chemical synthesis of artemisinin. These setbacks have not only driven up the cost of artemisinin
but also prompted more efforts towards production of artemisinin through cell culture technology. However,
plant cells cultured in shake flasks would not be commercially viable for large scale production due to its
labour intensity nature and the high cost incurred subsequently. Nevertheless, large scale cultivation of
plant cells also comes with its own limitations such as the reduced productivity often attributed to shear
sensitivity, aggregate formation, oxygen requirement of the plant cells, and efficiency of mass transfer
which influences the growth rate, biomass yield and production of secondary metabolites (Taticek et al.,
1991). Previous reports shown that the incorporation of medium replenishment through substrate feeding
have proven to be able to enhance productivity in plant cell culture (Chan et al., 2010; Wang et al., 2000).
This is due to the removal of spent medium containing toxic secreted by-products that may hinder cell
growth or result in feedback-inhibition of other products in the culture medium (Rao & Ravishankar, 2002).
In the efforts to commercially produce these bioactive compounds from plant cell cultures at a larger scale,
we investigate the possibility of increasing productivity of A. annua cells through medium replenishment in
the culture at a shake flask level as a preliminary study towards substrate feeding in cell culture tanks.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2. MATERIALS AND METHODS
2.1 Preparation of cell suspension culture
Pre-selected callus line of Artemisia annua (2GTC2 clone) of friable nature were used for the preparation
of cell suspension culture. Callus of 2.50 ± 0.02 g was inoculated into 125 ml liquid Murashige and Skoog
-1
(1962) (MS) medium supplemented with 0.5 mg l-1 6- benzylamino purine (BA) and 0.5 mg l
-1
Naphtaleneacetic acid (NAA), 0.5 g l-1 casein hydrolysate and supplemented with 30 g l sucrose in 500
ml Erlenmeyer flasks. The medium pH was adjusted to 5.75 ± 0.02 prior to autoclaving at a temperature
-2
and pressure of 121oC and 1.05 kg cm respectively for 11 minutes using TOMY SS-325 Autoclave. The
o
cultures were kept in a culture room at 25 ± 2 C on a gyratory shaker at 100 rpm, under continuous
lighting using cool white fluorescent lights of approximately 1000 to 1500 lux. Cell fresh biomass was
determined after 16 days of culture. Measurement of dry weight was determined after the cells were airdried until constant weight. The data was analysed using one-way ANOVA followed by Tukey’s Test at p≤
0.05.
2.2 Growth kinetics of A. annua cells
2.2.1 Growth kinetic of A. annua cells for determination of growth pattern and residual sucrose
The A. annua cells of 2.50 ± 0.02 g were cultured in 500 ml Erlenmeyer flask containing 125 ml liquid MS
medium supplemented with 0.5 mg l-1 6-benzylamino purine (BA) and 0.5 mg l-1 Naphtaleneacetic acid
-1
(NAA), 0.5 g l casein hydrolysate and supplemented with 30 g l-1 sucrose. The pH was calibrated to 5.75
± 0.02 prior to autoclaving and was cultured throughout 28 days. Six Erlenmeyer flasks containing the
cells were harvested at 4 day intervals. The dissolved oxygen was measured at each harvesting interval
using a galvanic dissolved oxygen probe, YSI Pro20 with a measurement accuracy of ± 0.2 mg/L. The
dissolved oxygen probe was calibrated using a One-Touch calibration system according to the barometric
pressure. Fresh cell biomass was determined using vacuum filtration through a 0.2 m filter paper. The
supernatant of the liquid medium was collected for pH and residual sucrose determination.
2.2.2 Residual sucrose determination
The residual sucrose content was determined through colorimetric method of phenol-sulfuric acid (Dubois
et al., 1λ56). Supernatant in the liquid medium was collected from filtration through a 0.2 m filter paper
and diluted five times with distilled H2O. Approximately 0.1 ml of the diluted supernatant was transferred
into a test tube. Distilled H2O was then added to make up a total volume of 2 ml, followed by the addition
of 1 ml of 5 % phenol and 5 ml of 95 % sulphuric acid (H 2SO4). The test tubes were left to stand at room
temperature for 10 minutes. Then, they were transferred into a water bath of 30oC for 15 minutes. The
absorbance of the solution was read at 490 nm using a quartz cuvette in a UVMINI-1240
Spectrophotometer, Shimadzu Scientific Instrument. The readings of the absorbance were compared to
the standard curve determined through a series of dilution of the similar solution being tested.
2.3 Effect of medium replenishment volume on biomass and sugar utilization of A. annua cells
2.3.1 Effect of medium replenishment volume on fresh and dry cell biomass of A. annua
Medium replenishment was conducted on the A. annua cell cultures at four different replenishment
volumes, 25 % (31.25 ml), 50 % (62.50 ml), 75 % (93.75 ml) and 100 % (125 ml) of the total volume of the
liquid medium. The replenishment of the liquid medium was carried out on the 8th day of the culture, and
the biomass of the cells was determined at harvesting period, on the 16th day. The supernatant of the
culture at the harvesting period was collected for pH measurement and residual sucrose determination.
The cells were then air-dried until constant weight for the determination of their dry weight.
43
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
3. RESULTS AND DISCUSSION
3.1 Growth kinetics of Artemisia annua cells
The growth curve of A. annua was determined by measuring the fresh and dry cell biomass of the cells at
intervals of 4 days throughout a culture period of 28 days. From the results shown in Figure 1, it was
observed that from day 0 to day 4, there was only a slight increase of fresh weight from 2.500 ± 0.020 g to
2.947 ± 0.146 g, indicating the lag phase of the growth curve. The exponential phase was showed with the
noticeable increase in the fresh weight from day 4 to day 16, 2.947 ± 0.146 g to 10.587 ± 0.248 g
respectively. The fresh cell biomass obtained on day 20 was 10.766 ± 0.105 g, with a highest growth
index of 3.3 ± 0.04 indicated that the growth curve of the cells had reached their stationary phase as there
was no further increase in cell biomass after that. The growth curve of the cells began enter the death
phase at day 24 where the cell biomass decreased to 8.160 ± 0.455 g, and at day 28 a further decrease to
7.958 ± 0.310 g. Figure 2 shows the dissolved oxygen measured at each harvesting interval showed a
decreasing pattern from day 0 up to day 12, where the dissolved oxygen measured stabilized from day 12
onwards to day 28. This could be possibly due to the uptake of the oxygen for the survival and growth of
the cells increases as the cell biomass increases. Further studies on the specific oxygen uptake rate
(SOUR) are recommended to further understand the correlation of the oxygen uptake towards cell growth.
Based on the residual sucrose profile in Figure 2, the residual sucrose detected continued to show a
decreasing pattern throughout the 28 culture period, despite an unexpected peak in the residual sucrose
detected on day 24. It is observed that from day 4 to day 8 of the culture period, there is a noticeable drop
in the residual sucrose detected in the culture medium from 95 ± 1 % to 71 ± 3 %, indicating sucrose
consumption for the beginning of the exponential phase in the culture medium. Thus, day 8 was selected
to conduct further intermittent medium replenishment studies.
Figure 1. Growth kinetics of A. annua cells (2GTC2 clone) based on fresh weight, dry weight and growth
index throughout culture period of 28 days. The bar columns indicate the standard errors.
a
Growth index = [Final fresh weight (g) – Initial fresh weight (g)] / Initial fresh weight (g)
44
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Figure 2. Dissolved oxygen and residual sucrose profile of A. annua (2GTC2 clone) measured at
harvesting period throughout culture period of 28 days. The bar columns indicate the standard errors.
3.2 Effect of medium replenishment volume on biomass and sugar utilization of Artemisia annua
cells
In this study, medium replenishment volumes of 0 v/v %, 25 v/v %, 50 v/v %, 75 v/v %, and 100 v/v% were
conducted on the 8th day of the culture of the A. annua cells. Spent liquid medium in the culture was
replenished with fresh liquid medium to observe the effects of the medium replenishment towards the
growth of the cells in terms of fresh and dry cell biomass. The results indicated that there was an increase
in fresh cell biomass from 10.619 ± 0.789 g in the control flasks (0 v/v % medium replenishment) to 12.130
± 0.841 g with the medium replenishment of 25 v/v %.
However, the fresh cell biomass obtained with 50 v/v % and 75 v/v % medium replenishment were not
significantly different at Tukey HSD, α = 0.05 compared to the fresh cell biomass obtained at 25 v/v %
medium replenishment. It is interesting to note that at 100 v/v% medium replenished, the fresh cell
biomass was found to decrease to 7.929 ± 0.683 g. These results indicated that with the larger ration of
the medium removed containing more than half of the initial sucrose concentration such as 71 ± 3 % on
day 8 with reference to Figure 3, the cell growth was compromised. Studies conducted by Chan et al.
(2010) on the medium replenishment in cell culture of Cyperus aromaticus showed that this occurrence
was due to the decrease in sucrose utilization.
Based on the sucrose utilization percentage results obtained at the harvesting period in Table 1, we
observed that 25 v/v %, 50 v/v%, 75 v/v%, and 100 v/v% medium replenished, with sucrose utilization of
35.6 %, 34.4 %, 37.2 %, and 33.6 % respectively, all showed a drop in the sucrose utilization when
compared to the control group, at 46.1 %. This indicated that the medium replenishment affected the
sucrose utilization in the cell culture, but the magnitude of the volume of the medium replenished did not
have a significant effect on the sucrose utilization. The observed biomass yield obtained on an average for
-1
cultures with replenished medium were on an average 0.12 g DW g sugar, which was higher that the
-1
control group with 0.09 g DW g sugar. The increase in observed biomass yields in medium replenished
cultures compared to the control group indicated that the additional carbon source supplied through
medium replenishment played a role in the noticeable increase in cell biomass of the cultures. Therefore,
medium replenishment a minimum volume of 25 v/v% would be sufficient in attempts to increase the cell
45
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
biomass in the culture as the magnitude of volume of medium replenished did not significantly affect the
sucrose utilization as well as the observed biomass yield in the culture.
Figure 3. Effect of different volume of medium replenished in cell culture of A. annua (2GTC2 clone) on
the fresh weight, dry weight and growth index of the cells. Means with the same letters are not significantly
different based on Tukey Test (HSD, α = 0.05). The bar columns indicate the standard errors. Fresh
weight obtained at harvesting period, day 16. Growth index = [Final fresh weight (g) – Initial fresh weight
(g)] / Initial fresh weight (g)
a
b
Table 1. Effect of different volume of medium replenished in cell culture of A. annua (2GTC2 clone) on the
sugar utilization and on the observed biomass yield.
Culture parameter
Control
(0% v/v)
25 % v/v
50 % v/v
75 % v/v
100 % v/v
Sugar utilization %c
46.1 ± 3.8 a
35.6 ± 1.7 b
34.4 ± 2.7 b
37.2 ± 2.5 b
33.6 ± 2.9 b
YX/S (g DW g-1 sugar)d
0.09 ± 0.01 a
0.12 ± 0.01 bc
0.13 ± 0.01 c
0.12 ± 0.01 bc
0.11 ± 0.01 c
Means with the same letters are not significantly different based on Tukey Test (HSD, α = 0.05). The bar
columns indicate the standard errors. Calculated based on harvesting period, Observed biomass yield,
YX/S
c
d
4. CONCLUSION
The incorporation of medium replenishment strategy in cell cultures of A. annua (2GTC2 clone) has
shown an enhancement in the cell biomass with the highest obtained at 12.130 g with 25 v/v% medium
replenishment. Based on the overall observed biomass yields, the cell biomass increase is correlated with
the increase in carbon source supplied in the culture media. This study demonstrates the possibility of
incorporating medium replenishment strategies in further work for the scale-up process from shake flasks
to cell culture tanks, with the potential for fed-batch production.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
References
Abdin, M. Z., Israr, M., Rehman, R. U., & Jain, S. K. (2003). Artemisinin, a novel antimalarial drug:
biochemical and molecular approaches for enhanced production. Planta Medica, 69, 289-299.
Balint, G. A. (2001). Artemisinin and its derivatives: An important new class of antimalarial agents.
Pharmacology and Therapeutics, 90, 261-265.
Chan, D. J. C., Ahmad, A. L., & Chan, L. K. (2010). Improvement of biomass and juvenile hormone III
(JH III) production from Cyperus aromaticus cell suspension culture via in situ membrane
filtratrion technology. Acta Physiologiae Plantarum, 32, 1153-1159.
Kieran, P. M., MacLoughlin, P. F., & Malone, D. M. (1997). Plant cell suspension cultures: some
engineering considerations. . Journal of Biotechnology, 59, 39-52.
Krishna, S., Bustamante, L., Haynes, R. K., & Staines, H. M. (2008). Artemisinins: their growing
importance in medicines. Trends in Pharmacological Scineces, 10, 520-527.
Nair, M. S. R., Acton, N., & Klayman, D. L. (1986). Production of artemisinin in tissue cultures of
Artemisia annua. Journal of Natural Products, 49(3), 504-507.
Rao, S. R., & Ravishankar, G. A. (2002). Plant cell cultures: Chemical factories of secondary
metabolites. Biotechnology Advances, 20, 101-153.
Taticek, R. A., Moo-Young, M., & Legge, R. L. (1991). The scale-up of plant cell culture: Engineering
considerations. Plant Cell, Tissue and Organ Culture, 24, 139-158.
Wang, H. Q., Yu, J. T., & Zhong, J. J. (2000). Significant improvement of taxane production in
suspension cultures of Taxus chinensis by sucrose feeding strategy. Process Biochemistry, 35,
479-483.
47
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
MORPHOLOGICAL EFFECT OF SODIUM AZIDE ON Artemisia annua OF VIETNAM ORIGIN
1
1
Leow E Shuen , Chan Lai Keng and Nadali Babeian Jelodar
2
1
School of Biological Sciences, University Sains Malaysia, 11800 Penang (MALAYSIA)
Department of Plant Breeding, College of Agriculture, University of Mazandaran, P.O.Box 578 Sari
(IRAN)
2
E-mail: leow_e_shuen@hotmail.com
Abstract
The mutagenecity of sodium azide (NaN3) was studied on seeds of Artemisia annua (A. annua) for
alteration in plant morphology. Seeds treated with 1-5 mM NaN3 and untreated seeds were germinated on
full strength Murashinge and Skoog medium. Low percentage (<3%) of putative mutant plant had shown
morphological abnormalities during the first two subculture cycles. However, these abnormalities subsided
through each cycle. The height, size of the leaves and internodes of putative mutant plants were recorded
and no significant difference were observed after four subculture cycles. Nonetheless, the glandular
trichome, which is the sole site for artemisinin production, had shown increased in trichome density when
observed under scanning electron microscope (SEM). The highest glandular trichome density was found
on the leaves of A. annua at 2mM NaN3. Therefore, NaN3 is a potential mutagen to create variability in
plant breeding of A. annua and hence producing new variety of A. annua with enhanced artemisinin.
Keywords: Artemisia annua; Morphology; Sodium Azide mutagenesis; SEM
1. INTRODUCTION
Artemisia annua L. (A. annua) is an aromatic annual herb belongs to the family Asteraceae, native to Asia,
mainly in China but it has been widely distributed throughout the temperate region (Simon et al., 1990). A.
annua is generally higher than two metres, single-stemmed with alternate open branching. The aromatic
leaves are ranged from 2.5 to 5cm in length and highly dissected. 10-celled glandular biseriate trichomes
and 5 cell filamentous (T) trichomes are found in both leaves and flowers (Ferreira & Janick, 1995).
A.annua has gained much attention in the past 30 years due to its capability in synthesizing a compound
named atemisinin. Artemisinin, a sesquiterpenoid lactone endoperoxide which has shown the most
prominent anti-malarial effect against multi-drug resistant Plasmodium spp (Klayman, 1985; Qinghaosu
Antimalaria Coordinating Research, 1979). In accordance to this, studies had shown that the secreting 10celled biseriate glandular trichomes on the leaves of A. annua are the major site for biosynthesis and
accumulation of artemisinin (Duke & Paul, 1993). Duke and co-workers had further proven these glandular
trichomes as the solitary site for artemisinin and artemisitene production (M. V. Duke, Paul, Elsohly, Sturtz,
& Duke, 1994). Therefore, some research efforts have been devoted to trichome formation and biosynthesis
of antimalarial drug artemisinin in the past decade. Plant morphological characters are another plausible
indication for selection of A.annua with high artemisinin content. A. annua clones with high artemisinin yield
are characterized by tall robust plants having long internodes, open and extended branching, dense leaves
and thick stems (Elhag et al., 1992). Owing to the importance of this medicinal plant, mutational approaches
have been incorporated to create variation and increase artemisinin yield. The use of sodium azide (NaN3)
mutagen (an alkylating agent) to enhance artemisinin content in the callus cells had shown promising
results and considered as a feasible approach (Al-Qurainy & Khan, 2010). Detection and analysis of nrDNA
using Internal Transcribed Spacer (ITS) sequencing on sodium azide-induced regenerated shoots of A.
annua had further proven NaN3 as a good mutagen for causing point mutation (Al-Qurainy et al., 2011).
However, no study has been reported on the plant morphology of sodium azide-induced Artemisia annua.
Thus, we evaluated the induced variability of the traits in the leaf trichomes.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2. MATERIALS AND METHODS
2.1 Seed treatment with NaN3
A. annua seeds of Vietnam origin were get from field in Vietnam. The seeds were treated with 1 – 5 mM of
NaN3 solution (diluted in 0.5M citrate phosphate buffer; pH3.2) for 2 hours incubation period. Seeds with
only citrate phosphate buffer without NaN3 solution were used as control. The treated and untreated
seeds were washed three times to remove excess NaN3 and buffer solution with distilled water.
Subsequently, they were sterilized by soaking seeds with 70% alcohol for 10mins. After that, alcohol was
removed and seeds were soaked in 20% Clorox for another 10 minutes. Clorox was removed and the
seeds were rinsed with autoclaved distilled water for three times. Seeds were blotted dry with filter paper.
Subsequently, treated and untreated seeds were germinated immediately on MS solid medium
(Murashige and Skoog, 1962) and placed under constant temperature of 25 ± 2°C and light intensity of
about 32.5 mol m-2 s-1. The MS medium was prepared without any plant regulators, supplemented with
30g/l sucrose, 8/l of agar (Algas, Chile) and the pH was adjusted to 5.75 before autoclaving (Tommy 325)
at temperature of 121°C for 11 min under 1.05 kg/cm2 pressure.
2.2 Effects of NaN3-induced seed on trichome morphology of A. annua plantlets
Leaves of putative mutant plantlets were fixed in McDowell-Trump fixative prepared in 0.1 M phosphate
buffer or cacoadylate buffer (pH 7.2) at 4°C for 1 day. Subsequently, the leaves were washed with 0.1 M
phosphate buffer for 3 times, 10 min for each wash before post-fixing in 1% Osmium tetroxide (OsO4)
solution at room temperature for 1.5 h. The specimens were then rinsed to remove excess OsO4 with
distilled water for 20 minutes followed by dehydration process using 50% ethanol, 75% ethanol, 95%
ethanol and 100% ethanol. The dehydrated tissues were soaked in hexathyldisilazane (HMDS) for 10 min.
HMDS from the specimen vial was removed and the specimen vial with the tissue was left in the
dessicator to air dry at room temperature. The air-dried specimens were then mounted by double-sided
adhesive tape on SEM specimen stubs, and coated with thin gold layer (40 to 60 mm) using Bio-Rad SEM
coating system(University Sains Malaysia). Leaf morphology was observed with the aid of Scanning
Electron Microscope (SEM) at 5 kV.
3. RESULT AND DISCUSSIONS
Low percentage of abnormalities (3%) arose in putative mutant A. annua plantlets grown from the treated
seeds. Common abnormalities observed during the initial growth and first subculture cycle. However, the
number of abnormal plants had gradually reduced and disappeared with each subculture cycle. Possible
explanation of the gradual reduced in chimerism is the diplontic drift (random loss) (Balkema, 1972) or
ontogenic loss (directed loss due to the position of the mutation and normal development) as in-vitro
micropropagation results in a quick dissolution of the chimera upon each subculture cycle and facilitates
the recovery of genetically solid and stable mutants (Donini and Micke, 1984; Sonnino et al., 1986). For
that reason, data was recorded and analyzed after the fourth to obtain the “real” stable results.
Although the abnormalities subsided and the putative mutant plants had not shown significant
morphological difference after four subcultures, results had shown significant increase in foliar glandular
trichome numbers (Table 1; Figure 1). Given that glandular trichome is the sole site of artemisinin
production as mentioned earlier, the increase of trichome numbers has indirectly reflected the increase of
artemisinin level. This agrees with the finding (Al-Qurainy & Khan, 2010) which had reported that callus
induced from foliage leaves of NaN3 treated seeds had shown higher artemisinin level compared to the
normal ones. They found out that the maximum artemisinin occurred at 3mM though the most effective
concentration of our study being 2mM. Different treatment conditions such as pH, temperature and
different cultivars used utterly affects the mutagenic effect. Al-Qurainy andKhan (2010) have shown the
insufficiency of one hour incubation period in NaN3 mutant production for A. annua seed treatment. Hence,
the increase of incubation period to 2 hours can be another reason that leads to different outcome.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Table 1 Effect of treated and untreated seeds on the number of glandular trichomes per mm2 of A. annua
leaf on both leaf adaxial and abaxial.
Concentration
Adaxial
Abaxial
Control
17 ± 1c
19 ± 2b
1mm
30 ± 1bc
49 ± 8a
2mM
61 ± 10a
47 ± 3a
3mM
41 ± 6b
27 ± 6b
4mM
22 ± 5c
32 ± 5b
5mM
32 ± 1c
32 ± 2b
Note. Mean values for each concentration followed by the same alphabet are not significantly different
(Duncan’s εultiple Range Test, p = 0.05).
Figure 1 Leaf (adaxial) microstructure of (a) untreated (b) 2mM NaN 3-treated A. annua after fourth
subculture cycle by SEM (100x). Capitate gland circled in red represents one of the glandular trichomes.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
4. CONCLUSION
NaN3 could be utilized to create variability in plant breeding of A. annua that ultimately increases the
possibility of producing new variety of A. annua with high artemisinin.
References
Al-Qurainy, F., Al-Hemaid, F. M., Khan, S., Ajmal Ali, M., Tarroum, M., & Ashraf, M. (2011). Detection of
sodium azide-induced mutagenicity in the regenerated shoots of Artemisia annua L., using
internal transcribed spacer (ITS) sequences of nrDNA. Pakistan Journal of Botany, 43(4),
2183-2186.
Al-Qurainy, F., & Khan, S. (2010). Mutational approach for enhancement of artemisinin in Artemisia
annua. Journal of Medicinal Plant Research, 4(17), 1714-1726.
Balkema, G. H. (1972). Diplontic drift in chimeric plants. Radiation Botany, 12(1), 51-55.
Duke, M. V., Paul, R. N., Elsohly, H. N., Sturtz, G., & Duke, S. O. (1994). Localization of artemisinin
and artemisitene in foliar tissues of glanded and glandless biotypes of Artemisia annua L.
International Journal of Plant Sciences, 155(3), 365-372. doi: 10.1086/297173
Duke, S. O., & Paul, R. N. (1993). Development and Fine Structure of the Glandular Trichomes of
Artemisia annua L. International Journal of Plant Sciences, 154(1), 107-118.
Donini, B., & Micke, A. (1984). Use of induced mutations in improvement of vegetatively propagated
crops. Induced mutations for Crop Improvement in Latin America. IAEA Tech. Doc. 305, 79-98.
Elhag, H. M., El-Domiaty, M. M., El-Feraly, F. S., Mossa, J. S., & El-Olemy, M. M. (1992). Selection
and micropropagation of high artemisinin producing clones of Artemisia annua L. Phytotherapy
Research, 6(1), 20-24.
Ferreira, J. F., & Janick, J. (1995). Floral morphology of Artemisia annua with special reference to
trichomes. International Journal of Plant Sciences, 156(6), 807-815.
Klayman, D. L. (1985). Qinghaosu (Artemisinin): An antimalarial drug from China. Science, 228(4703),
1049-1055.
Murashige, T., & Skoog, F.A. (1962). A revised medium for rapid growth and bioassays with tobacco
tissue culture. Plant Physiology, 15, 473-479.
Qinghaosu Antimalaria Coordinating Research, G. (1979). Antimalaria studies on Qinghaosu. Chinese
Medical Journal, 92(12), 811-816.
Simon, J.E., Charles, D., Cebert, E., Grant, L., Janick, J., & Whipkey, A. (1990). Artemisia annua L.: A
promising aromatic and medicinal. Advances in new crops. Timber Press, Portland, OR, USA.
522-526.
Sonnino, A., Ancora, G., & Locardi, C. (1986). In vitro mutation breeding of potato: the use of
propagation by microcuttings. International Symposium Nuclear techniques and in vitro culture
for plant improvement. IAES, Vienna, 385-394
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
A COMPARISON OF PRODUCTION OF SOMATIC EMBRYO FROM THREE SELECTED COCOA
(Theobroma cacao L.) CLONES
1*
Norhashimah, A.R. , Rebicca, E.
2
1,2
Department of Plant Science and Environmental Ecology,
Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak.
*Email: arnorhashimah@gmail.com
Abstract
Theobroma cacao L. is currently in the third ranking important commodity in Malaysia after oil palm and
rubber. As to increase the best quality of cocoa production with faster propagation, the study of tissue
culture for cocoa was carried out. The objectives of this study were to compare between two protocols for
three selected cocoa clones, to discover the best protocol for better multiplication of cocoa production and
to compare which cocoa clones give the best response in somatic embryogenesis. This study is important
in order to discover the best protocol to optimize the production of somatic embryos. Three cocoa clones,
KKM 1, MCB C5 and MCB C8 were studied in this experiment. The staminodes were cultured on
Pennsylvania State and Nestle protocol for the comparison of somatic embryogenesis. Based on the
®
result, for 10% Clorox treatment, the highest percentage of responsive explants producing somatic
®
embryo is clone MCB C8 cultured on Nestle protocol with 87.2%. For 20% Clorox treatment, the highest
percentage of responsive explants producing somatic embryo is clone MCB C8 cultured on Nestle
protocol with 25.5%. Overall, Pennsylvania State is the best protocol since it is suitable to produce
somatic embryos for all selected clones.
Keywords: Theobroma cacao, somatic embryogenesis, somatic embryo, Pennsylvania State protocol,
Nestle protocol
1. INTRODUCTION
Theobroma cacao L. or cocoa is in the third ranking importance commodity in Malaysia after oil palm and
rubber. Cocoa is required in high demand since it is the importance source of chocolate production.
Cocoa is member of Sterculiaceae family. Malaysian Cocoa Board (2009) reported that cocoa trees are
flowering throughout the year especially during rainfall season. According to Malaysian Cocoa Board
(2009), cocoa clone is produced via vegetative propagation of individual trees with high yielding progeny
and the clones produced has similar morphological characteristics of the tree, cocoa pod, cocoa beans
and flowers. The cocoa clones that have been study were KKM 1, MCB C5 and MCB C8.
Micropropagation has become a reliable approach for large-scale rapid plant multiplication, which based
on plant cell, tissue and organ culture on well-defined tissue culture media under aseptic conditions (Jain,
2007). The advantages of tissue culture methods include the possibility of rapidly generating asexually
propagated, uniform plants with highly valued genetic traits. In this experiment, two protocols for cocoa
somatic embryogenesis (Pennsylvania State Protocol and Nestle Protocol) were used to induce somatic
embryos. Novak and Brunner (1992) defined that somatic embryogenesis (SE) is a process of single cell
or a group of cells initiating the developmental based on plant cell totipotency that leads to regeneration of
non-zygotic embryos capable of germinating to form complete plants. Traore (2003) stated that, somatic
embryogenesis provides powerful tool for fast propagation of large number of cocoa plants from
agronomically-valuable genotypes. Secondary somatic embryogenesis is a developmental pathway
whereby new somatic embryos are initiated from primary somatic embryos. The production of cocoa
somatic embryos varies greatly depending on the genotype and individual floral explants (Li et al., 1998;
Tan and Furtek, 2003). The objective in this study is to compare between two protocols for three selected
cocoa clones. The specific objective in this study is to discover the best protocol for better multiplication of
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
cocoa production. In addition to the specific objective is to compare which clones of cocoa will give better
response in the tissue culture experiment.
2. MATERIALS AND METHODS
2.1 Samples Collection
Samples of unopenned immature flowers (about 2 to 3 weeks old) were collected from Cocoa Research &
Development Centre, Kota Samarahan. Staminodes, needle-like structures fused with a stamen at the
base extracted from flowers buds. Flower buds collected in the early morning, between 8 and 10 am. The
flower buds were obtained by using small forcep and avoid from touch with hand to reduce the
contamination. During transportation, the flowers soaked in the bottle containing distilled water to prevent
the flowers became dry and kept fresh before cultured.
2.2 Surface sterilization
®
Before surface sterilization treatment were conducted, the Clorox solution was added with two drops of
™
Tween 20 , a wetting agent to improve the disinfestation by acting as a surfactant thereby removing the
surface contaminants like soil and dust. There were two treatment conducted in this experiment. For the
®
first treatment (experiment 1), the flowers were immersed in the 10% (wt/vol) Clorox for 10 minutes with
®
gentle agitation. After immersion, the Clorox solution was discarded. For the second treatment
(experiment 2), the flowers were immersed in the 70% ethanol for one minute with gentle agitation. After
®
that, the ethanol were discarded, then the flowers were immersed in the 20% (wt/vol) Clorox for 10
®
minutes with agitation. After immersion, the Clorox solution was discarded. After that, the flowers for both
treatments were rinsed three times with autoclaved distilled water. After the last rinse, the containers were
covered in order to maintain the flowers moisture. After two weeks in culture, the staminode explants were
observed and result was recorded.
2.3 Induction of somatic embryo for Pennsylvania State protocol
Staminodes explants were prepared by drying the immature flowers on a sterile paper, then the immature
flowers were sliced by using sterile scalpel. Staminodes (five per floral) were extracted from the upper
part of the flower bud. Primary somatic embryogenesis was employed according to the protocol
established by Li et al. (1998). Ten out of twenty staminodes explants were cultured on Petri dishes of
Primary Callus Growth (PCG) medium that contained Driver and Kuniyuki Walnut (DKW) basal salts as
described by Driver and Kuniyuki (1984) and Tulecke and McGranahan (1985). While the rest of the ten
staminodes were cultured on Nestle protocol media. The Petri dishes were sealed with parafilm and
maintained in the dark at 25ºC. After 14 days, explants were subcultured onto Petri dishes containing
Secondary Callus Growth (SCG) media and maintained for another 14 days. Somatic embryos were
induced by transferring the callus onto Petri dishes containing Embryo Development (ED) medium.
Cultures were incubated in the dark for 14 days. Subsequent subcultures were carried out at intervals of
14 days by transferring the explants onto fresh ED medium. The percentage of staminodes producing
embryos over the total number of cultured explants were recorded. Secondary somatic embryos were
induced as described by Maximova et al. (2002). Mature primary embryos with developed cotyledons
were selected. Well-developed cotyledons that pinkish or light yellow were used because of the high
embryogenic potential. Care was taken to avoid using very young cotyledons which are transparent or
old, thickened hairy cotyledons with highly visible trichomes. The cotyledons were separated from the
2
embryo hypocotyls and sliced with a sterile scalpel into approximately 4 mm pieces. The explants were
cultured on the SCG medium. The Petri dishes were sealed with parafilm and maintained for 14 days.
Then, cotyledons explants were subcultured onto ED medium. The explants were maintained by
transferring explants on fresh ED medium under the same conditions at intervals of 14 days to induce
secondary somatic embryos. All images were taken by using light microscope.
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2.4 Induction of somatic embryo for Nestlé protocol
Flower explants were prepared by drying the immature flowers on the sterile paper, then slicing them by
using sterile scalpel. Staminodes (five per floral) were extracted from the upper part of the flower bud.
Protocol for production of somatic embryos was developed by Fontanel et al, (2002). Ten staminodes
explants from the same flower used for Pennsylvania State media were cultured for induction of primary
somatic embryos on the Petri dishes containing Induction 1 (IND1) media. The Petri dishes were sealed
with parafilm and maintained in the dark at 25ºC. After 14 days, the explants were transferred onto fresh
IND1 Petri dishes and maintained for another 14 days. Then, the cultures were transferred onto Petri
dishes containing Induction and Expression (INDexp) medium for 14 days. The explants were maintained
by transferring the cultures onto fresh INDexp medium and maintained under the same condition at
intervals of 14 days until primary somatic embryos were induced and matured. The percentages of
staminodes producing embryos over the total number of cultured explants were recorded. For induction of
secondary somatic embryos, cotyledon explants were selected from mature primary somatic embryos.
Well-developed and high embryogenic potential cotyledons which were pinkish or light yellow were used.
The cotyledons were separated from the embryo hypocotyls and sliced with a sterile scalpel into
2
approximately 4 mm pieces. The cotyledon explants were cultured in the Callus Multiplication (CM2)
medium. The Petri dishes were sealed with parafilm and maintained for 14 days. For expression of
secondary somatic embryos, the cultures were transferred onto Expression (EM2) medium. The Petri
dishes were sealed with parafilm. The cultures were maintained by transferring onto fresh EM2 medium at
intervals of 14 days until production of secondary somatic embryos. All images were taken by using light
microscope.
3. RESULTS AND DISCUSSION
3.1 Surface sterilization treatment
®
For experiment 1 (10% Clorox treatment), cocoa clone KKM 1 was not experimented, so there was no
result for KKM 1 cocoa clone. After two weeks of staminodes culture initiation, the percentage of axenic,
®
responsive and callusing staminodes explant for Experiment 1 (10% Clorox concentration treatment) and
®
Experiment 2 (20% Clorox concentration treatment) were observed and recorded. From the Table 1
below, the percentage of axenic, responsive and callusing staminodes explant of experiment 1 showed
mostly 100 % axenic and responsive explants while experiment 2, the figure showed the percentage of
axenic, responsive and callusing was lower than experiment 1 with percentage range from 74% to 96%.
Table 1: Percentage of axenic, responsive and callusing staminode explants for experiment 1 and
experiment 2.
Percentage of axenic and responsive staminode explants (%)
Experiment
MCB C5
MCB C8
KKM 1
Penn State
Nestle
Penn State
Nestle
Penn State
Nestle
Experiment 1
98.75
100
100
100
-
-
Experiment 2
74
82
96
94
88
96
®
Based on the result, the explants under 10% Clorox treatment showed that, there was 1.25% of
staminode explant from Penn State Protocol (PS) MCB C5 was contaminated where other staminodes
explants expanded and started producing callus at the edge cut of staminodes. While for staminodes
®
explants undergone 20% Clorox treatment showed that, there were no initial contamination occured but
some staminodes were not producing callus and blackish. Some staminode explants produced callus after
®
two to three weeks later. From the observation, after the 20% Clorox treatment was carried out the flower
buds surface became bruises and the colour of flowers surface were also faded. This indicated that the
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
®
®
appropriate concentration of Clorox was 10% Clorox (for 10 minutes) treatment because the
®
concentration of Clorox did not damaged the explants as well helped to produce the high percentage of
®
axenic cultures. High concentration of Clorox used in the treatment caused damage to the staminode
explants. According to Salwa et al. (2011), surface sterilization treatment showed high concentration of
®
Clorox gave the high percentage of mortality of the explants. It is important to be cautious since surface
sterilant is also toxic to the explant tissues. Therefore concentration of the sterilizing agent and duration of
the treatment should be optimum to minimize tissue mortality of the explants due to over sterilization.
3.2 Somatic embryogenesis of three selected cocoa clones
An overview of the production somatic embryos from the selection of flower buds until the production of
secondary somatic embryos were presented in the Figure 1. Staminodes derived from unopened
immature flowers (2 to 3 weeks old) were cultured on the Primary Callus Growth (PCG) and Induction 1
(IND1) media. The staminodes started to expand to about twice of their original size after two week and
callus initiated at the edge cut of the staminodes explants. After three to four weeks, the callus developed
over the entire staminodes while cultured on Secondary Callus Growth (SCG) and IND1 media. More
creamy white of globular callus clusters continued to develop after subcultured on the Embryo
Development (ED) and Induction and Expression (INDexp) media.
a
b
c
Ca
Ca
PSE
f
e
d
Ce
SSE
Ca
Figure 1 An overview of the production somatic embryos. Callus (Ca) initiated from the cut edge of
cultured cocoa staminodes explant after two weeks (a) (bar = 1 mm). Formation of embryogenic callus
after six weeks (b) (bar = 1.5 mm). Production of primary somatic embryo (PSE) at cotyledonary stage
developed among callus (c) (bar = 1.5 mm). Well-developed cotyledons selected from primary somatic
embryos were cultured on Callus Multiplication (CM2) and Secondary Callus Growth (SCG) media (d) Ce=
Cotyledonary explant (bar = 2 mm). Induction of callus of cotyledonary explants after two weeks cultured
(e) (bar = 2 mm). The secondary somatic embryos at cotyledonary stage (f) (bar = 2 mm).
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Figure 1b showed the embryogenic callus appeared prior to the production of somatic embryos. Some
callus produced small adventitious roots. Primary somatic embyos at cotyledonary stage development
were produced subsequently after the callus was induced from the staminode explants (Figure 1c). The
earliest somatic embryos at globular stage were apparent within seven weeks after culture initiation,
which was after three weeks cultured on ED and INDexp media. For induction of secondary somatic
embryos, well-developed cotyledon of matured primary somatic embryos which were pinkish or light
yellow in colour were sectioned and cultured on Secondary Callus Growth (SCG) and Callus Multiplication
(CM2) media for two weeks (Figure 1d). The cotyledonary explants started to expand and the callus was
induced at the cut edge of cotyledons explants and multiply over the cotyledonary explants (Figure 1e).
Then the explants were transferred on Embryo Development (ED) and Expression (EM2) media. From the
observation, the production of secondary somatic embryos were faster than production of primary somatic
embryos which was after about one week subcultured on ED and EM2 media. The secondary somatic
embryos started to appear among the cotyledonary explants. The image of normal secondary somatic
embryos refers to Figure 1(f).
Several factors affected the production of somatic embryos in this research. Firstly, the type of cocoa
flowers bud used. In this study, the unopenned cocoa flowers bud were used and the unopenned flowers
helped to reduce the percentage of contamination. The staminodes in the unopenned cocoa flower were
not exposed to bacteria and fungi. The physiological age of flowers bud used also affecting the production
of somatic embryos. Too young or too old flowers can reduce the production of somatic embryos. In this
study, the 2 to 3 weeks old flowers bud gave high percentage of production somatic embryos.
Study by Tan and Furtek (2003) proved that the staminodes derived from unopenned, 2 to 3 weeks old
flowers yielded the highest percentage of explant producing somatic embryos compared to unopenned, 1
to 2 weeks old cocoa flowers bud and openned, 3 to 4 weeks old cocoa flowers bud. In addition, flower
part used as explants also has effect on the production of somatic embryos. Staminodes explants gave
high percentage of somatic embryos production. Research by Tan and Furtek (2003) proved that
staminodes were the best explants for somatic embryogenesis compared with anthers and petals base.
The cocoa flower buds explants collected early in the morning at between 8.00 am to 10.00 am and then
kept in the bottle containing distilled water also helped to increase the production of somatic embryos.
The cocoa flower bud explants were maintained since the staminodes explants still in cold, fresh and
healthy condition during the cultures.
Based on the previous research by Tan and Furtek (2003), the observation made proved that cocoa
flower explants collected at the beginning of the rainy season, especially after prolonged dry period, tend
to have a higher efficiency of somatic embryogenesis. The general health of the mother plant and season
of the year also contribute to embryogenesis frequencies in cocoa (Tan and Furtek, 2003).
3.3 Types of callus produced
The production of callus varies among these three selected cocoa clones. From the observation, the
cocoa clone MCB C5 produced solely white compact callus while the other two cocoa clones MCB C8
and KKM 1 produced yellow friable callus at initial cultures and turned to black and seems like died but
th
the callus did not die until weeks 15 , because the callus were able to generate multiple number of
somatic embryos. Different from white compact callus of cocoa clone MCB C5, the large white compact
callus enlarged every week but produced only small number of somatic embryos.
Based on the study conducted by Tan and Furtek (2003), callus was mainly produced in two types which
were white compact callus or yellow friable callus. Maximova et al. (2002), reported that two
morphologically distinct types of callus are produced. The first type of callus consists of elongated cells
and appeared white and shows that somatic embryos almost never generated from this type of cell callus
clusters. The second type callus consists of round cells and appeared light to dark brown and friable.
These cell callus clusters are often found in association with somatic embryos.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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3.4 Stage development of somatic embryos
There were four stages in the development of cocoa somatic embryos, the stages of somatic embryos
until maturity were presented in Figure 2. Primary somatic embryos developed rapidly on the ED and
INDexp media. After cultured for three weeks in the ED and INDexp media, the primary somatic embryos
were produced and appeared in globular shape (Figure 2a). Figure 2b showed the somatic embryos
developed into heart shape within five weeks after subsequently transferred on the fresh ED and INDexp
media. After 6 to 7 weeks in the ED medium and INDexp medium, the somatic embryos developed to
torpedo shape (Figure 2c). The torpedo shape developed to cotyledonary stage (Figure 2d) after 8 to 9
weeks in ED medium and INDexp medium. The stage development of secondary somatic embryos was
similar to primary somatic embryos.
a
c
b
d
Figure 2 Stages development in cocoa somatic embryo. (a) Globular shape stage (bar = 0.5 mm). (b)
Heart shape stage (bar = 1.5 mm). (c) Torpedo shape stage (bar = 1.5 mm). (d) Cotyledonary stage
development (bar = 2 mm).
The primary somatic embryos produced in two morphological features which were normal somatic
embryos and abnormal somatic embryos. The normal primary somatic embryos developed with welldefined hypocotyls with two cotyledons while the abnormal primary somatic embryos developed with
either multiple cotyledons or fusion of hypocotyls. Alemanno et al. (1997), divided somatic embryos into
three categories. First category consisted of normal somatic embryos that exhibited clear bipolarity, welldefined shoot and root apices and two cotyledons. Second category was abnormal somatic embryos that
fused at the hypocotyls or had multiple cotyledons. The third category was abnormal somatic embryos
that had a protoderm but did not show bipolar organization.
Li et al. (1998), reported the abnormal somatic embryos with fusion of hypocotyls and with
underdeveloped or extra cotyledons were observed. However, morphological abnormalities are not
necessarily indicative of the subsequent development of an abnormal plant. Li et al. (1998) reported that
normal cocoa plants were recovered from abnormal somatic embryos, but they developed at a slower rate
in vitro than somatic embryos with normal morphological characteristics. These abnormal somatic
embryos were capable of producing secondary somatic embryos on subsequent cultures (Li et al., 1998).
This study showed among the normal primary somatic embryos, two types of somatic embryos produced
were translucent somatic embryos and whitish somatic embryos. The translucent somatic embryos
produced short hypocotyls and developed big cotyledons which were beneficial for induction of secondary
somatic embryos while the whitish somatic embryos produced long hypocotyl and small cotyledons.
Further observation showed that, this types of somatic embryos produced small roots.
Li et al. (1998), reported that there were two types of somatic embryos were identified among the somatic
embryos produced. The first type of somatic embryo had a yellowish and translucent appearance and
also had an expanded somatic embryo axis with large yellow to pink cotyledons. During extended periods
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of cultured on ED medium, the first type somatic embryos did not produce roots and remained dormant
with cotyledon expansion. While the second type of somatic embryo was whitish and had an opaque but
defined embryonic axis with small and white cotyledons. These somatic embryos produced roots with
elongated hypocotyls on ED medium at maturity.
3.5 Production of somatic embryos
The production primary somatic embryos and secondary somatic embryos for experiment 1 and
experiment 2 are showed in the Table 2. The data for total explants producing primary somatic embryo
th
until week 15 and data for total explants producing secondary somatic embryos from second until six
weeks were recorded.
Table 2 Production of primary and secondary somatic embryos for Experiment 1 and Experiment 2.
SE
Experiment 1
MCB C5
Nestle
PSE
Penn
State
12
SSE
3
Experiment 2
MCB C8
Nestle
0
Penn
State
13
0
4
MCB C5
Nestle
68
Penn
State
5
90
2
MCB C8
KKM 1
Nestle
0
Penn
State
2
Nestle
12
Penn
State
2
0
2
28
4
13
5
PSE=Primary Somatic Embryo SSE= Secondary Somatic Embryo
The total explants producing somatic embryos varies among these three selected cocoa clones. Cocoa
clone MCB C8 (Nestle Protocol) produced the highest explants producing primary somatic embryos
production. Cocoa clone MCB C8 (Nestle Protocol) was also gave rise to the highest explants producing
secondary somatic embryos compared to cocoa clones MCB C5 and KKM 1. After subcultured in ED and
EM2 media for one week, the first production of secondary somatic embryos was observed among the
cotyledonary explants and increased every week while early production of primary somatic embryo was
after three weeks cultured on ED and EM2 media. Total explants producing primary somatic embryos until
the 15th weeks was lower.
Li et al. (1998), found that secondary somatic embryos produced more uniform and developed in shorter
time when compared to primary somatic embryos. Secondary somatic embryogenesis was generated to
increase the number and quality of somatic embryos (Maximova et al., 2002). Maximova et al. (2002)
reported that only matured primary somatic embryos with well-developed cotyledons were selected for
secondary somatic embryogenesis. Cotyledons with high embryogenic potential are light yellow or pink in
color. Young cotyledons which were transparent or white, thickened hairy cotyledons with long and visible
trichomes were not used in the study.
3.6 Primary somatic embryogenesis of selected cocoa clones
Based on the result in Table 3, the highest percentage of responsive explants producing primary somatic
embryos for experiment 1 and 2 was cocoa clone MCB C8 (Nestle Protocol) with 87.2% (experiment 1)
and 25.5% (experiment 2). For both experiment, clone MCB C5 (Nestle Protocol) showed 0% of
responsive explants producing primary somatic embryo. Clone MCB C8 performed well in both
Pennsylvania State Protocol and Nestle Protocol. The result showed all selected cocoa clones cultured on
Pennsylvania State Protocol were able to generate somatic embryos. Overall, the best performance was
clone MCB C8 cultured on Nestle Protocol (experiment 1).
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Table 3 The percentage of responsive explants producing primary somatic embryos for experiment 1 and
experiment 2.
% RE
producing
PSE
Experiment 1
Experiment 2
MCB C5
Penn
Nestle
state
MCB C8
Penn
Nestle
state
15.6
16.3
0
MCB C5
Penn
Nestle
state
87.2
10
0
MCB C8
Penn
Nestle
state
4
KKM 1
Penn
Nestle
state
25.5
4
10.2
RE = Responsive Explants. PSE = Primary Somatic Embryos.
Differrent types of callus produced have effect on the production of somatic embryos. White compact
callus (MCB C5) produced low percentage of primary somatic embryos. While the yellow friable callus
(MCB C8 and KKM 1) produced high percentage of primary somatic embryos. The genotypes group of
these cocoa clones were not study in this research. Therefore further study on the genotypes group
should be carried out in order to prove that, the different genotypes of cocoa clones will affect the
percentage of production of somatic embryos for these three selected cocoa clones. According to
Chengalrayan (1998), the ability to form somatic embryos was believed to be under genetic control. Tan
and Furtek (2003) study on several cocoa clones showed the different percentage of somatic embryos
production although carried out on the same media. Thus, the genotypic effect was observed in primary
somatic embryogenesis in those selected cocoa clones. Maximova et al. (2002), reported that all
genotypes tested were able to produce secondary embryos. The results showed that the methods
developed by Maximova et al. (2002) support secondary somatic embryogenesis in a wide variety of
genotypes although the efficiencies may vary with genotypes.
3.7 Secondary somatic embryogenesis of selected cocoa clones.
Table 4 shows the production of secondary somatic embryos for experiment 1 and 2 started from the
second week until the sixth week. For cocoa clone MCB C5 (Nestle Protocol) in experiment 1 and 2, there
was no cotyledons of primary somatic embryo cultured for induction of secondary somatic embryos
because there was no production of primary somatic embryos.
Table 4 Production of secondary somatic embryos for experiment 1 and experiment 2.
Week
nd
2
th
4
th
6
Experiment 1
Penn State
Nestle
MCB
C5
2
2
3
MCB
C8
3
3
4
MCB
C5
-
MCB
C8
50
74
90
Experiment 2
Penn state
MCB
C5
1
1
2
MCB
C8
1
1
2
Nestle
KKM
1
1
2
4
MCB
C5
-
MCB
C8
13
21
28
KKM
1
9
9
13
The results shows that the highest production of secondary somatic embryos for cocoa clone MCB C8
cultured on Nestle Protocol with range 50 to 90 explants (experiment 1) and 13 to 28 explants
(experiment 2). All selected cocoa clones cultured on Pennsylvania State Protocol were able to generate
secondary somatic embryos although the percentages were low. Overall, the best performance for
production of secondary somatic embryos was cocoa clone MCB C8 cultured on Nestle Protocol
(experiment 1). Secondary embryogenesis is the process of multiple identical secondary embryo
produced from a single somatic embryo. Secondary embryogenesis procedure provides a continuous
source of embryos without the need to start from floral tissue. Maximova et al. (2002) reported that a
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
procedure was described for secondary somatic embryogenesis of cocoa in order to increase the
efficiency and quality of somatic embryos produced.
According to Maximova et al. (2002), primary and secondary somatic embryogenesis of cocoa resulted
from two distinct ontogenetic pathways. Primary somatic embryos are multicellular in origin and
unsynchronised, while secondary somatic embryos are unicellular in origins and synchronised. Unicellular
somatic embryos have the potential utility for clonal propagation of elite genotypes of cocoa for clonal
breeding trials programmes and useful for scale-up of new varieties for large-scale propagation programs.
The single cell origin of cocoa secondary somatic embryos can offer a system useful in the the
development of genetic transformation. The secondary somatic embryo inherited the characteristics of its
mother plant. As an experimental system, secondary somatic embryogenesis has certain advantages
such as high multiplication rate, independence of an explant source and repeatability (Traore, 2003).
4. CONCLUSIONS
Two protocols (Nestle Protocol and Pennsylvania State Protocol) are used to study the somatic
embryogenesis for three selected cocoa clones (MCB C5, MCB C8 & KKM 1). The best performance for
®
somatic embryogenesis is MCB C8 (Nestle Protocol) undergone 10% Clorox treatment (experiment 1). In
this study, cocoa clone MCB C8 is discovered to be best cultured on Nestle Protocol which able to induce
high number of somatic embryos. Cocoa clone MCB C8 can also produce somatic embryos on
Pennsylvania State Protocol. Therefore, MCB C8 is the cocoa clone that has better quality for propagation
of cocoa somatic embryogenesis. From the study, cocoa clones cultured on Pennsylvania State Protocol
for both experiment 1 and 2 are able to produce somatic embryos although in low percentage. Thus,
Pennsylvania State Protocol is the best protocol to produce somatic embryos for all selected cocoa
clones.
ACKNOWLEDGEMENT
The authors are grateful to the staff at Cocoa Research & Development Centre, Kota Samarahan,
especially Mr Osli Bin Pozan, for the helps with the cocoa flowers sampling and also to the staff of
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and Technology,
UNIMAS, for their technical assistance.
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Maximova, S.N., Alemanno, L., Young, A., Ferrière, N., Traoré, A. and Guiltinan, M.J. (2002). Efficiency,
genotypic variability and cellular origin of primary and secondary somatic embryogenesis of
Theobroma cacao L. In Vitro Cellular and Develomental Biology Plant. 38: 252-259.
Novak, F.J. and Brunner, H. (1992). Plant breeding: Induced Mutation Technology for Crop Improvement.
IAEA BULLETIN. 4 : 25-33.
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Salwa, S.S., Saad, S. M., El-Shamy, M.A. and Asmaa, E.A.E. (2011). Propagation of Cerbera odollam
Plant by Using Tissue Culture Technique. Journal of Horticultural Science and Ornamental Plants 3
(3): 276-282.
Tan, C.L. and Furtek, D.B. (2003). Development of an In Vitro Regeneration System For Theobroma
cacao From Mature Tissues. Plant Science. 164 : 407–412.
Traore, A., Maximova, S.N. and Guiltinan, M.J. (2003). Micropropagation of Theobroma cacao
L.using
somatic embryo-derived plants. In Vitro Cellular and Developmental Biology Plant. 39 : 332-337.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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EFFECTS OF MS MEDIUM AND MACROELEMENTS ON MULTIPLE SHOOTS FORMATION OF
Lobelia chinensis Lour.
1
Thong Weng Hing and Pang Wei Wei
2
1
2
INTI International University (MALAYSIA)
METTA Space Continuous Education Center (MALAYSIA)
Email: wenghing.thong@newinti.edu.my
Abstract
Lobelia chinensis is an important herb in traditional Chinese medicine. Due to over development and
deforestation as well as infection by pathogen, it is now under the threat of extinction. Conventional
vegetative propagation is too slow to achieve the commercial quantities required. Tissue culture could
scale up the propagation rapidly and economically. This study was carried out to investigate the effects of
Murashige and Skoog (MS) medium and major elements at different levels on the in vitro regeneration of
L. chinensis. The nodal segments of L. chinensis were grown on MS medium and MS medium
supplemented with different levels of phosphorus (0, 1.0, 2.0, 4.0 and 6.0 mM), calcium (0, 0.5, 1.0, 2.0
and 3.0 mM), magnesium (0, 0.5, 1.0, 1.5 and 2.0 mM), potassium (0, 12.5, 25.0, 50.0 and 75.0 mM) and
different ratio of nitrate and ammonium ranging from 0.25:1 to 4:1. The MS medium induced 3.5 shoots
per explants. The most number of multiple shoots, 11.7 shoots per explants, was achieved in MS medium
+
supplemented with NO3 /NH4 at ratio 0.25:1. After acclimatization, the plants grew well in the field.
Keywords: Tissue culture, MS medium, multiple shoots formation, macroelements.
1. INTRODUCTION
Lobelia chinensis (family Lobeliaceae), commonly known as Chinese lobelia herb, is an important herb in
traditional Chinese medicine. L. chinensis can serve as a respiratory stimulant (Liu and Peng, 1994) and
is used to treat schistosomiasis, fever and remove tumour (Wang, 2001; Tada et al., 1995).
L. chinensis is not commonly found in wild and easily infected by some pathogens (Thong et al., 2012).
Due to over development and deforestation, it is now under the threat of extinction. Conventional
vegetative propagation is too slow to achieve the commercial quantities required. Tissue culture could
scale up the propagation rapidly and economically. In vitro culture is an important method for efficient
clonal propagation of plant species (Aranda-Peres and Martinelli, 2009).
There are many factors influence the in vitro growth of microplantlets, such as plant growth regulators,
temperature, light quality which varied with organs, plant genotype and the phase of plant growth. (Niedz
and Evens, 2007; Chand and Singh, 2004; Morard and Henry, 1998). In addition, the micronutrients and
macronutrients also play an important role in micropropagation of woody plants (Andreu & Marìn, 2005).
However, the optimum concentration of minerals of the basal culture medium may vary between different
species or genotype (Goncalves et al., 2005; Williams, 1991).
This study was carried out to investigate the effects of Murashige and Skoog (MS) medium and major
elements at different levels on the in vitro regeneration of L. chinensis.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2. MATERIALS AND METHODS
2.1 Plant Material
In this study, the in vitro cultured Lobelia plantlets were used as plant material.
2.2 Culture conditions
The pH of the MS médium was adjusted to 5.7-5.8 using 0.01 M NaOH or 0.01 M HCl before autoclaving
-2
at 121C under 1.2 kg cm for 15 min. Gelrite 0.25% (w/v) was included as a gelling agent. All cultures
were incubated at 252C under a 16 hours photoperiod provided by cool-white fluorescent lamps and 8
hours of darkness.
2.3 Effect of MS médium on micropropagation of L. chinensis
Experiments were carried out to determine the effect of MS medium on multiple shoot formation of L.
chinensis. A 4-6 mm nodal segments of L. chinensis were aseptically inoculated on the MS medium. Ten
nodal segments were used for each treatment and the experiment was repeated three times. The
responses of the explants were observed and the number of shoots formed per explant was evaluated
over a period of eight weeks.
2.4 Effect of Macroelements on micropropagation of L. chinensis
Experiments were carried out to determine the effect of macroelements on multiple shoot formation of L.
chinensis. All test tubes used in this study were rinsed with 0.1 M HCl and double distilled water prior use.
The media were prepared separately by deleting each of the macroelements. The macroelements studied
were phosphate, calcium, magnesium, potassium and nitrogen. NaH 2PO4.H2O salt was used to supply
phosphate ion. Initial phosphate concentrations in the medium were at 0, 1.0, 2.0, 4.0 and 6.0 mM,
separately. Calcium ion was supplied as CaCl2.2H2O salt and initial concentrations applied were at 0, 0.5,
1.0, 2.0 and 3.0 mM, respectively. Magnesium ion was supplied as MgSO 4.7H2O salt. Initial magnesium
concentrations in treatment media were prepared at 0, 0.5, 1.0, 1.5 and 2.0 mM, respectively. KNO 3 was
replaced by KCl to supply the potassium ion. Potassium was supplied into the media with initial
concentrations at 0, 12.5, 25.0, 50.0 and 75.0 mM, separately. The ammonium concentration was
maintained at 20.0 mM, while nitrate concentrations were varied at 12.5, 25.0, 50.0 and 80.0 mM besides
medium without supplemental nitrate and ammonium. The ammonium was supplied as (NH 4)2SO4 salt. A
30 samples were used for each treatment and the experiment was repeated three times. The responses
of the explants were observed and the number of shoots formed per explant was evaluated over a period
of eight weeks.
2.5 Acclimatization and Planting Plantlets in the Field
Acclimatization was carried out to harden in vitro plantlets before transferring to the field. Uniformed
rooted micropropagated lobelia plantlets were selected from the glass vessels and transferred to a growth
chamber with air temperature of 252C and a photoperiod of 16 hours. Inside the growth chamber, each
plantlet was immediately transplanted into individual commercial plastic pots containing a steam-sterilized
mixture of sand: soil (1:1, v/v). After transplanting, the plantlets were subjected to an acclimatization
treatment of seven days of high humidity. At day eight, the vigorously grew and uniformed size lobelia
plantlets were transferred to the glasshouse. All pots were fertilized twice a week with 100 mL of MS
nutrient solution. Total survival rate of plantlets were determined at day 30 after transferred to glasshouse.
2.6 Statistical Analysis
Analysis was performed using Statistical Analysis System (SAS) (SAS Institute Inc., Cary, NC, 1985).
Statistical differences were tested by Duncan’s multiple range test for the means. The significance level
was set up at p<0.05.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
3. RESULTS AND DISCUSSIONS
3.1 Effect of MS medium on Multiple Shoot Formation
When the nodal segments of L. chinensis were cultured on MS medium, it was observed that an average
of 3.5 shoots per explants was achieved. Basal medium was known to influence the growth response of
various plants in different ways. This was considered to be a consequence of the differences mainly in
their salt components and proportions (Bhojwani and Razdan, 1983). It was observed that the MS medium
induced in general the highest proliferation rates and the best shoot growth of Malus sieboldii and hybrids,
with an average of 3.6 shoots/explants (Ciccotti et al., 2008). Likewise, it was reported that shoot
proliferation of Dionaea muscipula was best on MS medium which induced 10.7 leaves/explant (Jang et
al., 2003). Similarly, it was observed that for regeneration of Eucommia colmoides and Amelanchier
arborea, MS medium increased culture growth and more shoots were produced on MS medium than on
woody plant medium (Chen et al., 1995; Brand, 1993). It was reported that the best response in terms of
shoot multiplication of Tylophora indica, English Shrub Rose “Heritage” and Chlorophytum borivilianum
were achieved on MS medium containing plant growth regulator (Faisal et al., 2007; Ozel and Arslan,
2006; Dave et al., 2003). Moreover, Gerbera jamesonii (Shabanpour et al., 2011) Stevia rebaudiana
(Ibrahim et al., 2008), Exacum Styer Group plantlets (Unda et al., 2007), Capsicum sp. (Peddaboina et al.,
2006), Rauvola tetraphylla (Faisal et al., 2005), Zingiber petiolatum (Prathanturarug et al., 2004) and
Citrus aurantium (Al-Ababneh et al., 2002) were cultured on MS basal medium. It was noticed that the
lobelia plantlets rooted well on MS médium. This was in agreement with the findings of Skirvin and Chu
(1979).
3.2 Effect of Macroelements on micropropagation of L. chinensis
When the nodal segments of L. chinensis were cultured in MS medium supplemented with varied
concentrations of macroelements, different regenerative responses were exhibited. The means of shoots
formed were significantly different in all the treatments tested.
In the omission of phosphate, the growth of the shoots was retarded and the shoots gradually turned
brown. The roots formed were thin and white brownish in color. Phosphate deficiency was characterized
by reduced growth (Krishna, 2002; Gupta, 1999; Anon, 1998). However, high phosphate interfered with N
absorption and micronutrient absorption such as Zn, Fe, or Co (Anon, 1998). It was observed that more
shoots were produced with the elevated phosphate concentration in the medium. The roots produced
were long and greenish in color. When supplied with 1.0 mM phosphate, the average number of shoots
formed was 4.1 shoots per node. The shoot number increased to 6.2 and 7.1 shoots in media fortified with
2.0 and 4.0 mM phosphate, respectively. The shoot multiplication was most effective in medium
containing 6.0 mM phosphate which induced 9.0 shoots. Similarly, white lupin (Lupinus albus L.)
developed proteoid roots when grown in phosphate deficient condition. These short, lateral, densely
clustered roots were adapted to increase phosphate availability (Gilbert et al., 1999). Likewise, He and
colleagues (1989) observed that phosphate played an important role in the formation of leaf structures on
the callus of wheat. Sharma and Thorpe (1999) had found a correlationship between the numbers of
shoots produced by explant with the amount of phosphorus absorbed by explants of many tree species. In
addition, Ružić et al. (2000) reported that the growth and multiplication of sweet cherry rootstock Gisela 5
depended on the uptake of N and P from the culture medium. This plant variety showed the best growth
and development on MS 2X and MS media with the highest N and P uptake. Smith et al. (2000) also
found that phosphate was rapidly consumed by shoot forming explants in the aspect of initiation and the
growth of shoots. It was reported that more than half of the phosphorus pool was consumed by day 20
during shoot initiation of tobacco culture (Bar-Yousef et al., 1995).
The nodal segments of L. chinensis produced two to three leaves and died after two weeks in MS medium
2+
2+
devoid of calcium ion (Ca ). Increasing the concentration of Ca resulted in higher number of shoots
2+
formed per explant. At 0.5 mM Ca , 4.1 shoots per explant were produced. MS media supplemented with
2+
1.0 and 2.0 mM Ca induced 5.1 and 6.3 shoots, respectively. The highest shoots number, 6.6 shoots,
2+
was achieved at 3.0 mM Ca . The roots produced were green in color. Similarly, the use of higher levels
of Ca and Mg in the Bacto-Agar medium improved the quality of rose shoots, and in Gelrite medium
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
increased the rose multiplication rate (Podwyszynska and Olszewski, 1995). Aranda-Peres and Martinelli
(2009) also found that Ca is a critical nutrient for plant growth and absorption of other minerals. Higher Ca
concentration (12mM) induced significantly higher fresh and dry weight in bromeliad plantlets. Calcium
play an important role in cell growth, cell elongation, cell división, cell signaling, translocation of sugar,
stabilizes and protect the plasma membrane and could act as a secondary messenger (Hirschi, 2004;
Reddy, 2001; Schroeder et al., 2001). In addition, Ca2+ can strengthen cell walls and protect the plant
species from biotic and abiotic stress (Aranda-Peres and Martinelli, 2009). Hepler and Wayne (1985)
2+
observed that low concentration of Ca led to reduced cell division in roots and suggested that the mitosis
2+
2+
and cytokinesis were regulated by Ca ion. Ca deficiency caused inhibition of bud growth, browning and
death of root tips, cupping of maturing leaves, weak growth, blossom end rot of many fruits, pits on root
vegetables, apples and pears (Zekri and Obreza, 2003; Gupta, 1999; Anon, 1998; Wignarajah, 1995). In
2+
2+
excess, Ca interferes with boron (B), Mg or K absorption. High Ca usually causes high pH which
precipitate many of the micronutrients so that they become unavailable to the plant (Gupta, 1999; Anon,
1998).
2+
No nodal explant survived in the MS medium without magnesium ion (Mg ). The lowest number of shoots
2+
formed per explant, 3.9 shoots, was induced at 0.5 mM Mg . Meanwhile, medium supplemented with 1.0
2+
and 1.5 mM Mg induced 5.4 and 6.8 shoots, respectively. The highest shoots number, 8.1 shoots, was
2+
achieved at 2.0 mM Mg . The roots formed at all the concentrations were green in color. Magnesium was
needed in physiological processes including photosynthesis, respiration and biosynthesis of protein, DNA
and RNA (Fontes et al., 1999). Bauhinia forficata donor plants treated with low magnesium nutrient
solution showed less protein production (Defavari, 2000, cited in Borgatto, 2002). Deficiency symptoms of
magnesium were reduced growth, interveinal chlorosis (yellowing between the ribs of the leaf; appeared
striped). Magnesium in excess would interfere with calcium and potassium uptake as well as reduced
growth (Anon, 2003; Gupta, 1999).
+
After growing for two weeks, the shoots became brown and dead in the potassium (K ) deficient MS
medium. Benlloch-González et al. (2008) also found that potassium starvation favoured stomatal
conductance and transpiration, as well as inhibiting shoot growth of olive trees. More shoots were induced
+
with the increasing concentration of K . An average of 3.1 and 6.7 shoots per explant was produced in the
+
+
medium supplemented with 12.5 and 25.0 mM K , respectively. At 50.0 mM K , 8.6 shoots were achieved.
+
+
The maximum shoots number, 10.1 shoots, was induced at 75.0 mM K . The roots formed at 12.5 mM K
+
was light green with a bit brownish. The color of roots was light green at 25.0 and 50.0 mM K and green
+
at 75.0 mM K . Similarly, Naik and Sarker (1998) had shown the promoting effect of potassium on
microtuber number and size of potato, and it was cultivar specific. Kothari and colleagues (2004) observed
+
that higher level of K was essential for plant regeneration and increased the number of regenerated
+
shoots of Eleusine coracana. Plant tissue must maintain an adequate K concentration to function
+
optimally. K was needed as a vacuolar osmoticum and enzymes co-factor in the cytoplasm (White, 1997).
+
K had been found to act more as a regulator in various systems of plants (Anon, 2003; Zekri and Obreza,
+
2003; Krishna, 2002; Liu and Zhong, 1996). K deficiency caused reduced growth, inhibition of protein
synthesis, shortened internodes, necrotic spots in the leaf, reduction of lateral bud breaks and tendency to
wilt readily (Anon, 2003; Zekri and Obreza, 2003; Gupta, 1999; Walker et al., 1998; Wignarajah, 1995).
Low concentration of cytosol potassium affected the potassium-dependent metabolic processes (Leihh
+
and Jones, 1984). In excess, K caused N deficiency in plants and may affect the uptake of other positive
ions such as B, Ca and Mg. N/K balance was important. High N/low K favored vegetative growth; low
N/high K promoted reproductive growth such as flower and fruit (Gupta, 1999; Anon, 1998).
-
+
In the absence of both nitrate (NO3 ) and ammonium (NH4 ) ions, none of the nodal explants survived.
+
When the ratio of NO3 /NH4 was 1:1.6 and 1:08, 3.8 and 7.4 shoots per node were produced,
respectively. The number of shoots formed increased to 9.1 shoots at ratio 1:0.4. The shoot proliferation
was most effective at the ratio of 1:0.25 which induced 11.7 shoots. The color of the roots was light green
+
−
at lower concentration and green at higher concentration. Similarly, it was reported that a NO 3 :NH4 ratio
of 1:0.2 produced 1.5, 4 and 2.2 fold increases in the mean multiplication rates than MS medium for three
different garlic clones, respectively. The differences in the multiplication rates could probably be due to an
increase in the N level supplied as nitrate (Luciani et al., 2001). It was reported that highest root
production of Eucalyptus marginata was obtained when the medium contained 7.5 mM nitrogen in a ratio
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
−
−
+
+
of 2NO3 :1NH4 (Woodward et al., 2006). Hyndman et al. (1982) observed that when the NO3 :NH4 ratio
increased from 0.1 to 3.0, there was an increase in the number of roots/explant in R. hybrida cv. Improved
Blaze. In addition, they also found that reduced concentration of KNO3 and NH4NO3 concentration was
+
crucial for root growth. The ratio of NO3 /NH4 , was an important factor on callus response towards auxin,
N uptake and pH regulation during plant tissue culture (Fracago and Echeverrigaray, 2001). Brand (1993)
reported that the number of shoots increased linearly with increases in medium NO 3 content. Higher level
of NO3 was found to promote plant regeneration, might be due to the effect of NO 3 on organic acid
biosynthesis, which was required for shoot induction (Kothari et al., 2004). Potassium nitrate (KNO3) with
concentrations of 1.5 folds of the MS medium produced the maximum number of cell rows and sizes of
potato (Iranbakhsh et al., 2011).In contrary, MS medium with half nitrate concentration was the most
efficient for shoot induction and MS medium was the best for shoot proliferation for Gerbera jamesonii
(Shabanpour et al., 2011). Anandarajah and McKersis (1990) reported that incorporation of increased
ammonium tended to reduce survival after desiccation, plant conversion and seedling vigor of Medicago
sativa. It was reported that a higher multiplication rates in different rose cultivars when the ammonium
content of the medium was reduced (Valles and Boxus, 1987; Curir et al., 1986). In contary, Naik et al.
(2011) found that high concentration of NH4NO3 induced the number of adventitious shoots of Bacopa
monnieri.
An abundant supply of essential nitrogen (N) compounds was required in each plant cell for normal cell
division, growth and respiration (Zekri and Obreza, 2003) and for the biosynthesis of macromolecule,
RNA, DNA and protein for the new cell (Colmer and Bloom, 1998). Avilla et al. (1998) and Mashayehki
(2000) reported the role of nitrogen as a signal molecule of plant growth which enhanced the gene
expression for the nitrate uptake and utilizing enzyme such as nitrate reductase and nitrite reductase
enzymes. Nitrogen affected the absorption and distribution of practically all other elements and enhanced
micronutrients uptake and utilization (Zekri and Obreza, 2003; Gupta, 1999). Most of the genotypes of
Malus sieboldii showed the highest proliferation rates and the best growth when cultured on the MS
modification with the highest content of nitrogen (Ciccotti et al., 2008). A shortage of this essential
element inhibited plant growth while an excess of the element was wasteful (Anon, 2003; Krishna, 2002;
Lata et al., 2002; Gupta, 1999; Wignarajah, 1995). Plant could take up and metabolize nitrogen either as
+
NO3 or NH4 (Wiren, 2000; Dubey and Pessarakali, 1995; Wignarajah, 1995). Although plant cells in
culture might grow on NO3 alone, the pH of the medium (5.0–6.0) was usually more stable and better
+
results were obtained when the medium contained both NO 3 and NH4 ions as sources of N (Rout et al.,
2000).
3.3 Acclimatization And Planting Plantlets In The Field
After acclimatization, plantlets transferred to glasshouse exhibited 100% survival. They grew well and
exhibited morphological characters similar to wild plants. They developed new leaves and flowered
normally. Similarly, most of the Malus sieboldii and hybrids achieved survival rates between 90–100%
(Ciccotti et al., 2008).
4. CONCLUSION
The macronutrients namely nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg)
and S (sulfur) were required in all types of plant cultures, but the optimal concentration of each might vary
with plant species (Rout et al., 2000).The current study has shown that macronutrients at different
concentrations played an important role in proliferation of L. chinensis. Among these macronutrient,
nitrogen showed the most impact on the micropropagation of this medicinal herb.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
VIRUS DETECTION AND ELIMINATION IN COCOA (Theobroma cacao L.) THROUGH
SOMATIC EMBRYOGENESIS
1
Rebicca Edward and Andrew Wetten
2
1
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak.
2
School of Biological Sciences, Harborne Building, University of Reading, Reading RG6 6AS, UK
Email: erebicca@frst.unimas.my
Abstract
Cacao swollen shoot virus (CSSV) is a major pathogen that has seriously constrained cocoa production in
West Africa, particularly Ghana and Nigeria. The aim of this study was to assess the efficacy of cocoa
somatic embryogenesis to produce virus-free clonal propagation material both for replanting and to
facilitate the safe international exchange of germplasm. Polymerase Chain Reaction (PCR)-based
screening, is employed in this study because of its capacity for CSSV detection prior to the appearance of
visual symptoms. Degenerate PCR primers were developed in order to improve the CSSV-strain
dependence of earlier tests. The degenerate primers were capable of detecting 37 out of a putative 56
CSSV strains, four more than the sequence specific primers. For tissue culture studies, cocoa staminodes
cultures were established from flowers of CSSV-infected cocoa genotypes CL 19/10 strain 1A and
Amelonado Plant 2 to produce callus, primary and secondary somatic embryos, with genotype AMAZ 15
used as a virus-free control. PCR-based CSSV detection proved that virus could be detected at callus,
primary somatic embryos and secondary somatic embryo stages, indicating that the progress of the virus
was progressively impeded. These findings support the use of somatic embryogenesis as a mean of
improving CSSV-free clonal propagation of cocoa.
Keywords: Cacao swollen shoot virus, cocoa, somatic embryogenesis, Polymerase Chain Reaction,
staminodes.
1. INTRODUCTION
Cacao swollen shoot virus (CSSV) has a long history in Ghana. The first report of the condition caused by
CSSV known as Cocoa Swollen Shoot Disease (CSSVD) in Ghana was made in 1936 (Steven, 1936). All
affected trees and neighbouring contact trees were cut down and burned, even before identifying the
pathogen as a virus (Steven, 1936). Early identification of cacao swollen shoot disease is important since
its viral infection at present cannot be cured. Failure to detect the infection of trees is known to be a
problem and it has been established that latently infected trees – those not yet showing symptoms - can
act as a source for further transmission (Legg, 1982). CSSVD was later discovered in other West African
cocoa producing countries: Nigeria in 1944 (Thresh, 1959), Ivory Coast in 1946 (Mangenot et al., 1946),
Togo in 1949 (Partriot et al., 1978) and Sierra Leone in 1963 (Attafuah et al., 1963). The disease was also
reported to be found in Sri Lanka and in Indonesia (Java and North Sumatra) (Thresh et al., 1988).
However, neither the disease nor its pathogen has been reported in the Amazon Basin (South America),
from where cocoa was originally exported into West Africa. Quarantine measures to prevent the spread of
CSSV by the movement of infected cocoa pods and cutting materials, is now possible with the improved
PCR-based detection for the virus (Muller et al., 2001). Genomic variability of CSSV isolates collected
from different West African locations indicate however that without appropriate precautions PCR-based
screening could result in false positives. Therefore, the aim of development of strain-independent CSSV
detection by PCR in this study, would not only contribute to the quarantine procedures for the diagnosis of
CSSV in cocoa trees, but also to inform the wider field of safe plant germplasm exchange. This study also
aims to examine the efficacy of somatic embryogenesis for the interruption of virus movement during
clonal multiplication of cocoa.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2. MATERIALS AND METHODS
2.1 CSSV detection by Polymerase Chain Reaction (PCR)
2.1.1 CSSV isolates
A total of 56 putatively distinct isolates of CSSV were obtained from the Cocoa Museum, Cocoa Research
Institute of Ghana (CRIG). The trees were maintained in a segregated area separated from the nearest
neighbouring cocoa trees by insect proof netting. The isolates originate from widely distributed cocoa
growing areas across Ghana and were classified by symptom expression according to the presence or
absence of swellings and the absence or presence of (and intensity) leaf symptoms (Ollennu, 2001).
Among the most visually CSSV-infected cocoa leaf samples were those that come from Ghana’s Eastern
region which is reported to be the region where the most virulent strain of the causal virus occurs (Dale,
1962). This is followed by Western and Ashanti regions, while only one infected leaf sample derives from
the Central region.
All leaf samples from Cocoa Museum, Cocoa Research Institute of Ghana were frozen at -80ºC as soon
as they were delivered to the University of Reading via courier. Positive controls used in this experiment
were two cocoa genotypes from CL 19/10 strain 1A and ICS 68. They were symptomatic leaves from
CSSV-infected cocoa trees maintained in an insect-proof netting cage within a glasshouse at the
University of Reading. Another two positive controls used were CSSV-infected cocoa seedlings
maintained separately at the Plant Science Glass house (strains New Juaben and Kpeve). For the
negative control, leaf material was collected from cocoa genotype AMAZ 15 from the International
Intermediate Cocoa Quarantine Centre, University of Reading. DNA extraction was conducted using the
®
basic protocol described by manufacturers of the DNeasy Plant Mini Kit (Qiagen Ltd., UK) with some
minor modifications.
2.1.2 PCR amplification
For the first generation primer, PCR primer pairs were designed based on regions of 100% homology
across the six published CSSV DNA sequences of the CSSV genome available from the National Center
for Biotechnology Information database (NCBI) (Accession nos.: AJ534983.1, AJ608931.1, AJ609019.1,
AJ609020.1, AJ781003.1 and L14546.1) (Hagen et al., 1993; Muller and Sackey, 2005). The primers were
generated using software, Geneious version 5.4 (Biomatters Ltd.) and manufactured by Sigma, UK as
follows: CSSV forward primer (AACCTTGAGTACCTTGACCT) and the CSSV reverse primer
(TCATTGACCAACCCACTGGTCAAG). The primer product is approximately 375 base pair (bp)
depending on viral strain and runs from position 350 to 725 bp on accession AJ608931.1. Whereas for the
second and third experiments, CSSV second generation primers with a mixture of nucleotide bases
designed using software, Geneious version 5.4 (Biomatters Ltd.) consisted of a mixture of 48 variables
with
redundant
nucleotide
bases
as
followsμ
CSSVε13uniFμ
5’
(ACAGCTATGACCATGAGYATHCARGARTGGTAYGA)
and
CSSVε13uniRμ
5’
(AAAACGACGGCCAGTCAYTGNCCNACCCAYTGRTC). [R = G or A, Y = T or C, H = A or C or T, N = G
or A or T or C]. A final volume of 10 µl for each PCR reaction mixture consisted of a ratio 5:3:1:1
comprising 5 µl of master mix containing Taq polymerase and dNTPs (Multiplex PCR kit, Qiagen, UK), 3
µl of Nanopure water, 1 µl of primer solution containing 2 µM of CSSV forward and reverse primers and 1
µl of DNA sample (concentration range of 4-16 ng/µl).
Once all of the PCR tubes contain all 4 of the reactants in their correct ratios the PCR tubes were and
then placed into a PCR thermocycler which facilitates the amplification of the DNA. The amplification cycle
o
o
o
conditions used were as follows: 94 C for 30 s to denature the DNA, 57 C for 90 s to anneal, 72 C for 60 s
o
for elongation. The cycle was repeated 35 times with a final extension at 10 C indefinitely until removed
from the machine.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2.2 CSSV elimination through somatic embryogenesis
2.2.1
Plant materials
Unopened immature flowers from three cocoa genotypes infected with CSSV, namely CL 19/10, ICS 68
and Amelonado Plant 2 maintained in the insect-proof netting glasshouse were collected as research
samples. Healthy cocoa flowers, as negative control were collected in the University of Reading Cocoa
Intermediate Quarantine Unit. Floral buds from these trees were collected for the induction of somatic
embryogenesis.
2.2.2 Induction of somatic embryos
Induction of cocoa somatic embryos was generated according to the protocol of Li et al. (1998). After the
surface sterilization procedure was conducted, the flower buds were sliced perpendicular to their
longitudinal axis about 1/3 of the flower length from the base with a sterile scalpel blade. Staminode (five
per floral bud) explants were extracted from the upper part of the flower bud. Twenty staminode explants
were placed into a Petri dish containing primary callus growth (PCG) medium and sealed with sealing film
o
(Nescofilm). Cultures were maintained in the dark at 25 C for 14 days. Explants were then transferred into
a Petri dish containing secondary callus growth (SCG) medium, sealed and cultured for another 14 days
under the same conditions. Callus usually formed at the cut edge of each staminode by the end of this
culture period. Explants were then transferred to Petri dishes containing embryo development (ED)
o
medium and cultured in the dark at 25 C for 14 days. Subsequent subcultures were carried out at intervals
of 14 days, by transferring explants onto fresh ED medium to induce somatic embryos which arose via the
callus. Secondary somatic embryos were induced as described by Maximova et al. (2002). Mature primary
embryos with developed cotyledons were selected and the cotyledons were separated from the embryo
hypocotyls and sliced into 4×4mm pieces. Cotyledon explants were placed in Petri dishes containing SCG
o
medium, sealed with Nescofilm (Azwell, Osaka, Japan) and cultured in the dark at 25 C for 14 days.
Explants were subcultured onto fresh ED medium every 14 days and maintained under the same
conditions to generate secondary somatic embryos. Secondary embryos were produced within two to
three months after culture initiation, with minimal additional callus development.
2.2.3 DNA extraction and PCR amplification
Total genomic DNA of the experimental materials was extracted using a slightly modified DNeasy Plant
mini kit (Qiagen Ltd., UK) protocol. The DNA was extracted from the leaves of CSSV infected cocoa trees
(CL 19/10) and a CSSV-free (AMAZ 15); callus tissues induced from the floral buds of the CSSV infected
cocoa trees; somatic embryos induced from the callus tissues and secondary somatic embryos induced
from the primary somatic embryos. PCR was run as described in section 2.1.2.
3. RESULTS AND DISCUSSIONS
3.1 CSSV detection by Polymerase Chain Reaction (PCR)
Results of the PCR detection of CSSV presented in Table 1 show leaf samples of CSSV-infected cocoa
plants from the Cocoa Museum, Cocoa Research Institute of Ghana (CRIG). To confirm the results, PCRs
were repeated for DNA samples that gave negative results and faint positive bands, both for the first
generation CSSV primers and also degenerate primers CSSVM13uni. This means that PCRs for both
primers were only repeated when two consecutive results were not the same.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Table 1 PCR screening of leaf samples of CSSV-infected cocoa plants from CRIG.
First generation CSSV
CSSVM13uni F&R
Genotype
primer F&R
No.
PCR trial
1
2
3
1
2
1
Abo Boya (CC) - WR
2
Achechere (CC) - W/R
+
3
Achiasi W/R
+ (faint)
+
4
AD 135 ER
+
+
5
AD 7 E/R
+
+
6
AD 75/ER
7
Adiembra/CC WR
+
+ (faint)
8
Agyepomaa
+
+
+
+
9
Aiyim (CC) – WR
+
+
+
10 Amanfie W/R
+
+
+
11 Anibil (CC) WR
12 Asamankese Isolate
+ (faint)
+
+
13 Ayiboso – W/R
+
+
+
14 Bakukrom /CC/W/R
+
+
+
15 Bechem B/A
+
+
+
+
16 Bisa
+
+
17 Bobiriso/Juaso 1 ASH
+
+
18 Bosomtwe/I J ASH
+
+
+
19 Bosomuoso 2 W/R
+
+
20 Datano W/R
21 Dawa/1H/ER
+
+ (faint)
22 Dochi/IG/ER
+ (faint)
23 Enchi E1/A/3 W/R
+ (faint)
+ (faint)
24 Gavepetodzi - U/R
+
+
+
25 Jamesi – W/R
+
+
+
26 Koben - ASH
+
+
+
27 Kofi Pare Isolate (1A)
+
+
+
+
28 Konongo (IK) ASH
+
+
+
29 Kpeve Isolate
30 Krofa / Juansa F2T2
+
+
+
31 Kwadzo Kumkrom J2/A
32 Kwakoko Juansa North A/R
+
+
+
33 Kwaku Anyan T1 B/A
+ (faint)
34 Madjeda Nkwanta Agogo/F1/T2/ASH
+
+
+
35 Mampong (1m) - ER
+ (faint)
+ (faint)
36 Miaso Isolate
+
+
+
37 N1 Isolate
+
+
38 New Juaben Isolate (1A)
+
+
+
+
39 Nkrankwanta Isolate
+
+
+
40 Nsaba Isolate
+
+
41 Oyimso Agogo 5 ASH
+
42 Pa Men (1e) - ER
+
+
43 Peki - U/R
+
+
+
+
44 Punekrom – W/R
45 Sankore T3/3
+
+
+
46 SS 365B Isolate
+
+
+
47 SS167 – E/R (mildstrain)
48 Suhuma W/R
+
+
+
+
49 Surowno /WR
+
+
+
50 Tafo Yellow
+
73
3
+
+
-
+
+
+
+
-
+
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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No.
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52
53
54
55
56
Genotype
PCR trial
Tease Adeakyi
Tease Atomsu-Abuom
Techimantia outbreak 3T-15
Virus AD 14/ER
Virus AD 196
Worawora
First generation CSSV
primer F&R
1
2
3
+
+
+
+
+
CSSVM13uni F&R
1
+
+
+
+
2
+ (faint)
+ (faint)
+
+
+
+
3
+ (faint)
For the first generation primer, leaf samples were assessed in two batches. In the first PCR trial, a batch
of 24 CSSV-infected cocoa leaf samples was received from CRIG. This was the initial experiment and 7
out of 24 cocoa leaf DNA samples were PCR positive with CSSV. The second PCR trial was the result for
the batch of 32 CSSV-infected cocoa leaf samples and repeated trial for the former batch of 24 cocoa leaf
samples. 33 samples were positive with CSSV and 3 samples gave faint bands. For the third trial, the 3
samples that gave faint bands were repeated and the 20 samples that gave negative amplifications were
also repeated. Given the inclusion of a positive CSSV control, the results showed that all samples in the
third trial were not positive with CSSV with this primer pair. Whereas for the second generation primer
(degenerate primer CSSVM13uni), 36 out of 56 cocoa DNA leaf samples were positive with CSSV in the
first PCR trial, while 1 sample gave a faint band using the degenerate primer pair. The second PCR trial
was to repeat the first trial. 37 samples gave positive amplification and 7 samples gave faint bands. The
third trial was repeated to confirm the results in the first and second trials and results were similar to those
from the second trial.
The first generation primers designed for CSSV detection based on the alignment of the six published
nucleotide sequences derived from the virus strain 1A isolates (Hagen et al., 1993; Muller and Sackey,
2005) suggested that more than half of the 56 CSSV-infected leaf samples from Cocoa Research Institute
of Ghana (CRIG) gave positive CSSV detection. Further screening experiments with CSSV second
generation primers, which consisted of a mixture of 48 variable redundant nucleotide bases, suggested its
capability to detect 37 strains of the 56 CSSV-infected leaf samples, four more virus strains than the first
generation primers (33 strains). Thus, the design of the second generation CSSV primers showed its
potential for improving the quarantine procedures for the international exchange and long term
conservation of cocoa germplasm. Further research might be devoted to improvement of the PCR
performance by developing a real-time PCR assay for CSSV detection. The quantitative real-time PCR
assay might be possible for the reproducible and specific detection of CSSV from the banana and plantain
explants, as reported for the detection of episomal Banana streak virus (BSV) in banana and also in
plantain (Delanoy et al., 2003). An initial study for developing a real-time PCR assay for cocoa was carried
out by Quainoo (2006). His findings showed that real-time PCR assays are appropriate for the detection
and estimation of the CSSV concentrations in the cells of cocoa, which conforms to the findings of realtime PCR developed for the rapid detection of episomal BSV in banana (Delanoy et al., 2003).
3.2 CSSV elimination through somatic embryogenesis
3.2.1 Viral screening of cocoa embryogenic tissues derived from CSSV infected cocoa trees
Statistical significance of differences of the effect of cocoa genotypes on virus elimination was assessed
by Chi-square (SPSS software version 19, IBM SPSS Statistics).
PCR-based CSSV detection on callus samples derived from staminodes (Table 2) showed that CL 19/10,
ICS 68 and Amelonado Tree 2 (Plant 2) were tested positive with CSSV. The most CSSV detection was
found with CL 19/10 calluses (60%), followed by calluses of ICS 68 (36.66%) and Plant 2 (30%). From the
chi-square test, the P-value was 0.047. Therefore, there was significant difference between the cocoa
genotypes on virus elimination.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Table 2 Virus screening for 4 weeks old callus tissues derived from staminodes of CSSV-infected cocoa
trees.
Genotype
CL 19/10
ICS 68
Amelonado Plant 2
Number of callus tissues
tested
30
30
30
PCR Positive test results
(18/30) 60%
(11/30) 36.66%
(9/30) 30%
Results of the PCR detection of CSSV presented in Table 3 indicated that half of the primary somatic
embryos of CL 19/10 tested positive for CSSV. Primary somatic embryos of ICS 68 and Plant 2 gave
positive results at 40.90% and 40.74% respectively. From the chi-square test, the P-value was 0.728.
Therefore, no significant difference was found in the effect of cocoa genotype on virus elimination.
Table 3 Virus screening of primary somatic embryos of CSSV-infected cocoa trees.
Number of primary somatic embryos
Genotype
Positive test results (%)
tissues tested
CL 19/10
30
(15/30) 50%
ICS 68
22
(9/22) 40.9%
Amelonado Plant 2
27
(11/27) 40.74%
Not all secondary somatic embryos induced from cotyledonary explants of primary somatic embryos
infected with CSSV gave positive results. From Table 4, 19.23% of secondary somatic embryos of CL
19/10 were PCR positive for CSSV, followed by secondary somatic embryos of Plant 2 (17.4%). All
secondary somatic embryos of ICS 68 tested PCR negative for CSSV. From the chi-square test, the Pvalue was 0.035. Therefore, significant difference was found in the effect of cocoa genotype on virus
elimination. Somatic embryogenesis from cocoa staminodes induced from two CSSV-infected trees, CL
19/10 and ICS 68, successfully resulted in the production of a number of clonal plantlets that could be
weaned to the glasshouse. These CSSV PCR-negative plantlets have not shown any symptoms of CSSV
infection in over one year since weaning.
Table 4 Virus screening of secondary somatic embryos induced from primary somatic embryos of CSSVinfected cocoa trees.
Number of
Number of embryos
secondary
Positive test
Weaned plantlets
Genotype
converted to plantlets
somatic
results (%)
(%)
(%)
embryos tested
CL 19/10
26
(5/26)19.23%
(12/26) 46.15%
(10/26) 38.46%
ICS 68
10
0
(2/10) 20%
(2/10) 20%
Amelonado Plant 2
23
(4/23) 17.4%
0
0
A recent report suggested that somatic embryogenesis was capable of the progressive interruption of the
movement of CSSV from primary somatic embryos to secondary somatic embryos (Quainoo, et al., 2008).
It is notable that the findings in Chapter 4 concur with and expand upon that report. From the callus
derived from the CSSV-infected staminodes, to the primary somatic embryos and later to the secondary
somatic embryos, the presence of CSSV was reduced, indicating that the progress of the virus was
progressively impeded. While the precise point at which the virus was eliminated is not yet known data
from this study showing the apparent paucity of plasmodesmata between the cells comprising the cocoa
embryogenic callus suggest this as a likely area for inhibition of viral movement. These findings can
contribute to the further improvement of the crop by generating disease free materials before they are
widely distributed.
During the course of this study, one of the major findings would be the investigation on effectiveness of
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
the application of somatic embryogenesis technique to interrupt movement of CSSV. Somatic
embryogenesis is indeed effective for virus elimination in cocoa and it has been demonstrated to function
for a range of cocoa genotypes. This also means that a likely mechanism for the interruption of CSSV
movement has been identified. Other major findings would be the initial study of the plasmodesmata in
cocoa embryogenic cells. The interesting result on the declining of the plasmodesmata frequency from
staminode to callus and to somatic embryos is worthy of further investigation.
4. CONCLUSIONS
In conclusion, using CSSV primers to the conserved sequence regions, one can determine the presence
of CSSV from the CSSV-infected leaf samples. Similarly, the use of degenerate primers to target the
conserved sequence regions of other strains of CSSV, has slightly improved the PCR-based CSSV
detection. The outcome of these trials is potentially useful with respect to future breeding work aimed at
improving the crop in West Africa as a means to generate clean test materials. It will also be of value for
the accelerated testing of cocoa materials for CSSV before they are distributed and conserved globally.
ACKNOWLEDGEMENT
This work is supported by a scholarship from the Ministry of Higher Education of Malaysia. The authors
are grateful to the staffs at Cocoa Research Institute of Ghana for the leaf samples, staffs at Cocoa
Intermediate Quarantine Unit, and Harborne Building and University of Reading, UK for their technical
assistance.
REFERENCES
Attafuah, A., Blencowe, J. W. and Brunt, A. A. (1963) Swollen shoot disease of cocoa in Sierra Leone.
Tropical Agriculture (Trinidad), 40:229-232.
Dale, W.T. (1962) Virus diseases. In: Agriculture and Land Use in Ghana (Ed, Wills, J. B.) Oxford
University Press, London, pp. 286-316.
Delanoy, M., Salmon, M., Kummert, J. (2003) Development of real-time PCR for the rapid detection of
episomal Banana streak virus (BSV). Plant Disease, 87:33-38.
Hagen, L.S., Jacquemond, M., Lepingle, A., Lot, H. and Tepfer, M. (1993) Nucleotide sequence and
genomic organization of cacao swollen shoot virus. Virology, 196:619-628.
Legg, J.T. (1982) The Cocoa Swollen Shoot Research Project at the Cocoa Research Intitute, Tafo,
Ghana, 1969-1978. Overseas Development Administration, London.
Li, Z., Traore, A., Maximova, S. and Guiltinan, M. (1998) Somatic embryogenesis and plant regeneration
from floral explants of cacao (Theobroma cacao L.) using thidiazuron. In vitro Cellular
and
Developmental Biology-Plants, 34:293-299.
Mangenot, G., Alibert, G. and Basset, A. (1946) Sur les caractères du swollen shoot en Cote-d’lvoir.
Review international Botany Application Agriculture Tropical, 283:13.
Maximova, S.N., Alemanno, L., Young, A., Feffiere, N., Traore, A. and Guiltinan, M. J. (2002) Efficiency,
genotopic variability, and cellular origin of primary and secondary somatic embryogenesis of
Theobroma cacao L. In vitro Cellular and Developmental Biology-Plant, 38:252-259.
Muller, E. and Sackey, S. (2005) Molecular variability analysis of five new complete cacao swollen shoot
virus genomic sequences. Archives of Virology, 150:53-66.
Muller, E., Jacquet, E., and Yot, P. (2001) Early detection of cacao swollen shoot virus using polymerase
chain reaction. Journal of Virology Methods, 93:15-22.
Ollenu,
L.A.A.
(2001)
Synthesis:
case
history
of
cocoa
viruses.
Retrieved
from
http//www.iita.org/info/viology/pdf_files/33-49.pdf.
Partriot, M., Amefia, Y.K., Djiekpor, E.K. and Bakar, K.A. (1978) Le “swollen shoot” du cacaoyer au Togo:
inventaire preliminaire et première estimation des partes causes par la maladie. Café cacao The,
XXII: 217-228
Quainoo, A.K. (2006) Germplasm conservation of cocoa (Theobroma cacao L.) and virus elimination
through tissue culture. PhD thesis, University of Reading, UK.
76
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Quainoo, A.K., Wetten, A.C. and Allainguillaume, J. (2008) The effectiveness of somatic embryogenesis in
eliminating the cocoa swollen shoot virus from infected cocoa trees. Journal of Virological Methods,
149(1):91-96.
Steven, W.F. (1936) A new disease of cocoa in the Gold Coast. Gold Coast Farmer, 5(122):144.
Thresh, J.M. (1959) The control of cacao swollen shoot disease in Nigeria. Tropical Agriculture (Trinidad)
36:35-44.
Thresh, J.M., Owusu, G.K., Boamah, A. and Lockwood, G. (1988) Ghanaian cocoa varieties and swollen
shoot virus. Crop protection, 7(4):219-231.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
APORPHINE ALKALOIDS AND ANTIOXIDANT ACTIVITY OF PHOEBE GRANDIS (NEES) MERR.
O. Hanita
1,2*
1
, M.M.Azrul , A.Hamid A. Hadi
1
1
Chemistry Department, Faculty of Science, University of Malaya,50603, Kuala Lumpur,Malaysia
Centre for Foundation Studies in Sciences, University of Malaya,50603, Kuala Lumpur, Malaysia.
2
*E-mail: hanita74@um.edu.my
Abstract
The genus Phoebe of the family Lauraceae is of wide tropical occurrence but has been little studied
chemically. Antioxidant activity was determined by two methods – reducing power (FRAP) and DPPH. The
present study of the hexane, dichloromethane, methanol and alkaloid extracts from the leaves of Phoebe
grandis (Nees) Merr showed that these extracts have shown a good scavenging activity. The antioxidant
activity was higher for alkaloids compared to other fractions. Activity- guided fraction led to the isolation of
four compounds and these compounds were lysicamine 1, N-methyllitsericinone 2, N-methyllitsericine 3
and dicentrinone 4. Compounds 1 and 4 belong to the oxoaporphine type alkaloid and compounds 2 and
3 belong to proaporphine type alkaloid.
Keywords: Phoebe grandis, Aporphine alkaloids, Antioxidant, FRAP, DPPH.
1. INTRODUCTION
Phoebe belongs to the family of δauraceae. The plant also known as “Medang” and there are an
estimated of 50 species in the Pantropic, in which 23 species are found in Malaysia [1]. Phoebe plants
are known as a rich source of aporphinoid and other isoquinoline alkaloids [2-9]. Biological screening on
the crude alkaloidal extract of the leaves of Phoebe grandis for antiplasmodial activity has shown positive
-1
result: IC 50 <8µgmL [10]. Therefore, we have launched a chemical investigation on the extract.
Antioxidant activity methods have been used to monitor and compare the antioxidant activity such as
DPPH radical scavenging activity and Ferric reducing antioxidant power (FRAP) assay. Further
investigation of the leaves has now led to the isolation of two oxoaporphine alkaloids, lysicamine 1 and
dicentrinone 4; and two proaporphine type alkaloids, N-methyllitsericinone 2 and N-methyllitsericine 3.
Structural elucidation was done mainly by 1D and 2D NMR, IR, UV, MS.
2. MATERIALS and METHODS
2.1 General Methods
All solvent, except those used for bulk extractions (distilled) are AR grade. Glass and aluminium
supported silica gel 60 F254 plates were used for TLC and preparative TLC respectively. TLC spots were
visualised under ultra-violet light (254 nm and 365 nm) followed by spraying with the Dragendorff’s
reagent for alkaloidal screening. Silica gel 60, 70-230 Mesh ASTM (Merck 7734) and silica gel 60, 230400 Mesh ASTM (Merck 9385) were used for column and flash chromatography, respectively. HR-ESI-MS
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1
13
were performed on a Shimadzu LC-MS-IT—TOF spectrometer. H NMR (400MHz) , C NMR (400MHz),
DEPT, COSY, HMQC and HMBC spectra were acquired in a Bruker Avance 400 spectrometer using
TMS as the internal standard and CDCl3 as solvent.
2.2 Plant Material
The plant materials were collected from Hutan Simpan Bukit Serting, Negeri Sembilan, Malaysia (19 Feb
2008) by the phytochemical group of the Chemistry Department, University of Malaya. Voucher specimen
(KL 5540) is deposited in the Herbarium of the Department of Chemistry, University of Malaya, Kuala
Lumpur, Malaysia and in the Herbarium of the Forest Research Institute, Kepong, Malaysia. The plant
samples (KL 5540) were identified by Mr L.E. Teo and Mr Din.
2.3 Extraction and Separation
Amount of 3.7kg of dried and milled leaves of the plant Phoebe tavoyana (Meissn.). Hk.F. were first
defatted with hexane for 3 days at room temperatura, then filtered. After that they were moistened with
15% of NH4OH, and exhaustively extracted with CH2CI2 by Soxhlet extractor for about 18 hours. The
CH2CI2 extract were reduced to 500 ml followed by acidic extraction using 5% HCI until εayer’s test is
negative. The combined extracts were then basified with concentrated ammonia solution to pH 10 -11
and re-extracted with CH2CI2. The CH2Cl2 fractions were washed with distilled H2O and dried over
anhydrous sodium sulphate. The solvents (dichloromethane extracts) were evaporated to dryness under
reduced pressure to give the yield. The CH2CI2 extract were evaporated to dryness to yield 13.50 g of
crude alkaloid.
This CH2CI2 crude alkaloid was subjected to column chromatography over silica gel using hexane :
CH2CI2 (50:50) and various ratio of CH2CI2 and MeOH (100:0, 98:2, 95:5, 93:7, 92:8, 90:10, 85:15, 80:20,
70:30 and 50:50) and finally with pure MeOH. TLC was used to monitor the fractions collected which to
be grouped into series of fractions. Extensive column chromatography was used to isolated the pure
alkaloid.
3. RESULTS and DISCUSSION
3.1 Compound 1
Compound 1, was obtained as a yellow amorphous solid. The crude alkaloid was subjected to column
chromatography over silica gel using the solvent system of 97:3 (CH 2Cl2:CH3OH). The mass spectrum
revealed a molecular ion peak at m/z 291, which corresponded to the molecular formula of C 18H13NO3.
Other significant fragmentations observed were at m/z 277. The UV spectrum showed maxima absorption
at 236, 267, 360 and 396 nm indicating the existence of a highly unsaturated oxoaporphine chromophore
-1
1
[11,12]. The IR spectrum showed a conjugated ketone peak at 1665 cm [12, 13, 14, 15]. The H
spectrum showed two distinct methoxyl peaks at 4.00 and 4.08 which was probably situated at C-1 and
C-2. No methylenedioxy peak was observed. H-3 appeared as a singlet situated at 7.21. Two doublets
(J=5.24 Hz) typical of the H-4 and H-5 signals of an oxoaporphine were observed at 7.78 and 8.88
respectively. The H-5 proton appeared at lower field compared to H-4 due to the neighbouring N- atom
adjacent to C-5. Four aromatic protons on ring D resonated at four aromatic proton signals. A very
downfield signal λ.16 (1H, dd, J1=8.36 Hz, J2=0.68; H-11). In addition a dd was observed at 8.57 (1H,
dd, J1=7.88 Hz, J2=1.40 Hz; H-8) which experienced a deshielding effect from the neighbouring C-7
carbonyl group. Another two multiplet or dt proton 7.75 (1H, dt, J1=8.52 Hz, J2=1.64Hz; H-λ) and 7.56
(1(H, dt, J1=8.52 Hz, J2=1.64Hz; H-10).
13
The
C-NMR spectrums gave a total of eighteen carbons which validated the molecular formula.
13
13
Analysis on the C NMR gave nine quaternary carbons. Others values of chemical shift for C NMR
given in the Table 1. Hence, compound 1 is oxoaporphine alkaloid can be deduced to be lysicamine 1 and
the spectral data were in full agreement with the literature values [16,17,18,19,20]. (refer Table 2)
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1
Table 1 H (δ ppm) Spectral Data of Lysicamine 1 and dicentrinone 4
Lysicamine, 1
Position
1
1a
1b
2
3
3a
4
5
6a
7
7a
8
9
10
11
11a
1-OCH3
2-OCH3
H
Dicentrinone 4 [21,22]
Observed (Nita,2012)
7.21 (s)
7.78 d (J = 5.24Hz)
8.88 d (J = 5.24Hz)
8.57 dd (J = 7.88 Hz, 1.40Hz)
7.75 dt (J = 8.25Hz)
7.56 dt (J = 7.96Hz, 1.08Hz )
9.16 dd (J = 8.36Hz, 0.68Hz)
4.00 (s)
4.08 (s)
Position
1
1a
1b
2
3
3a
4
5
6a
7
7a
8
9
10
11
11a
9-OCH3
10-OCH3
O-CH2-O
H
observed (Nita,2012)
6.57(1H,s)
7.81 d (J=5.4 Hz)
8.87 d (J=5.4 Hz)
8.06 (s)
8.82 (s)
4.04
4.08
5.97 d (J=0.72 Hz)
5.94 d (J=1.48 Hz)
13
Table 2 C (δ ppm) Spectral Data of Lysicamine 1
Position
Observed
1
1a
1b
2
3
3a
4
5
6a
7
7a
8
9
10
11
11a
OCH3
13
C (δ ppm)
151.0
118.8
121.2
155.8
105.4
134.5
122.6
144.0
144.3
181.7
133.3
127.8
133.4
127.9
127.4
131.04
55.2
59.7
literature values [17]
literature values [23]
145.3
119.6
122.0
152.0
106.4
135.3
123.4
145.0
156.7
182.5
132.0
128.7
128.7
134.2
128.3
134.7
56.0
60.5
152.2
120.0
122.3
157.0
106.6
145.4
123.8
145.2
135.7
182.9
132.2
129.0
129.1
134.6
128.6
134.5
56.4
60.8
3.2 Compound 2
N-methyllitsericinone, 2 was isolated as an amorphous solid. The structure proposed could be the
proaporphine type of alkaloids.
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-1
For the IR, there is very significant strong peak at 1470.92 cm shown the presence of carbonyl group
-1
-1
(ketone) and two medium absorption peaks at 1044.56 cm and 1254.45 cm for ether group (C - O
1
stretch). The H-NMR spectrum displayed a pair of doublet peaks at (5.897 ppm and 5.90 ppm; J =
1.24Hz) and (5.854 ppm and 5.858 ppm; J = 1.60Hz) that correspond to methylenedioxy group at position
C-1 and C-2 respectively. A singlet peak appeared at 6.50 ppm represented proton attached to a benzene
ring at C-3 position. There is a strong singlet peak at about 2.40 ppm indicated a methyl group attached to
N atom. The aliphatic protons appeared between 1.7 ppm – 3.3 ppm.
13
The C-NMR spectrum of this alkaloid showed the presence of eighteen carbons and results from the
DEPT experiment showed that there are seven methylene carbons (CH 2) and one methylenedioxy group.
The presence of the ketone group at C-10 position indicated at 211.65 ppm that is most deshielded peak
and N-methyl group at 43.47 ppm. Quaternary carbon at C-7a position appears at 46.03 ppm. The
chemical shifts for this known alkaloid are shown in Table 3.
13
1
Table 3 Chemical shifts of C-NMR, H-NMR and DEPT for N-methyllitsericinone, 2
1
13
H (J,Hz)
C ( , CDCl3)
Position
DEPT
1
148.14
C
2
140.71
C
3
106.47
CH
6.50 s
2.94 m
4
27.43
CH2
2.74 m
3.15 m
5
54.99
CH2
2.54 m
6a
65.70
CH
3.32 bd s
2.61 m
7
44.54
CH2
1.79 m
7a
46.03
C
2.50 m
8
36.45
CH2
1.94 m
2.72 m
9
38.60
CH2
2.49 m
10
211.65
C
2.14 m
11
39.00
CH2
2.06 m
2.17 m
12
34.59
CH2
2.04 m
N-CH3
43.47
CH3
2.41 s
Methlenedioxy
5.856 d (1.60)
100.63
CH2
(O-CH2-O)
5.898 d (1.24)
3.3 Compound 3
N-methyllitsericine, 3 was isolated as amorphous solid. The mass spectrum showed a molecular ion peak
+
at m/e 302.05 [M] which is correlated to molecular formula C18H23O3N. The structure proposed is an
aporphine type of alkaloid. The difference for this alkaloid with N-methyllitsericinone 2 is substituent at
position 10 (C-10) where hydroxyl group and ketone group occur for the alkaloids respectively.
For the IR spectrum, it showed a broad peak indicated the presence of hydrogen bonding, which is OH at
position C-10. The others peaks were same with alkaloid 2 because they have similar structure, only
1
differs in position C-10. The H-NMR for this alkaloid show a pair of doublet peaks at (5.863 ppm and
5.866 ppm; J = 1.20Hz) and (5.905 ppm and 5.901 ppm; J = 1.6Hz) that correspond to methylenedioxy
group at position C-1 and C-2. A singlet peak at 6.46 ppm clearly showed that proton attached at position
C-3 on the benzene ring. Strong peaks at about 2.40 ppm showed that methyl group is attached to N
atom. For this alkaloid, there is one singlet peak at 4.72 ppm correspond to proton at position 10 (H-10).
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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There is also about wide peak at 3.99 ppm for the hydroxyl group at position 10 (10-OH). This
phenomenon occurs because the proton attached to oxygen is labile, so their neighbouring proton can’t
‘see’ them clearly. The aliphatic protons appear at low range (1.2 ppm – 3.3 ppm).
13
The C-NMR spectrums for this alkaloid show the presence of eighteen carbons. The N-methyl and
methylenedioxy group occur at 43.22 ppm and 100.48 ppm respectively. There is a quaternary carbon
peak (position C-7a) appeared at 46.6 ppm. This quaternary carbon is important for the proaporphines
13
type of alkaloids. The chemical shifts of C-NMR for this known alkaloid are shown in Table 4.
13
Table 4 C-NMR chemical shifts of N-methyllitsericine, 3
13
Position
C ( , CDCl3)
1
148.25
2
140.79
3
105.93
4
29.70
5
54.93
6a
65.71
7
27.33
7a
46.60
8
128.95
9
124.01
10
77.21
11
31.70
12
30.21
N-CH3
43.22
Methylenedioxy (O-CH2-O)
100.48
3.4 Compound 4
Compound 4 was isolated as fine pale yellow needles; and its molecular formula was established as
+
C19H13O5N, by LCMS mass spectrum indicated the [M] peak is at 335.8420.
-1
-1
For IR spectrum, there are two strong absorption peaks at 2962cm and 2925cm (C-H aromatic) and a
-1
medium peak at 1453cm (C=C aromatic) which indicated the presence of aromatic ring. Strong peaks at
-1
-1
1724cm prove the appearance of carbonyl (ketone) group. A medium peak appear at 1666cm showed
-1
-1
-1
the presence of imine group while three strong peaks at 1261cm , 1096cm and 1020cm indicated the
-1
1
presence of ethers groups (C-O). Para substituent was proved by the peak at 799cm . The H-NMR
spectrum showed there are two strong singlet peaks at 4.038 ppm and 4.084 ppm indicated the presence
of two methoxy groups at position C-9 and C-10.
There are a pair of doublet peaks at 5.97 ppm, d, J = 0.72 Hz; and 5.94 ppm, d, J = 1.48 Hz indicated the
appearance of methylenedioxy group at position C-1 and C-2. A singlet peak at about 6.57 ppm shown
proton attached to benzene at position C-3. Two singlet peaks at 8.06 ppm and 8.82 ppm indicated the
presence of two protons at para position with respect to each other. This is true because only para
substituent could give singlet peak while meta and ortho substituents surely give dd peak. It is because
the proton at para substituent has no correlation with other proton, and two ortho-coupled aromatic
protons at position H-4 and H-5 ( H = 7.81 (1H, d, J=5.4 Hz)ν and H = 8.87 (1H, d, J=5.4 Hz). These data
are shown in Table 1, together with comparison with data from the literature, established the structure of
compound 4 as that of the known aporphine alkaloid dicentrinone 4 [24,25].
3.5. Antioxidant activity
3.5.1 DPPH assay
DPPH was used to determine the proton radical scavenging action of dichloromethane and alkaloid
extracts of the leaves of Phoebe grandis (Nees) Merr, because it possesses a proton free radical
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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and shows a characteristic absorbance at 517 nm. DPPH is a stable free radical at room
temperature and accepts an electron or hydrogen radical to become stable diamagnetic molecule.
The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm,
which is induced by antioxidants. Table 5 and Figure 1 illustrates a decrease in the concentration of
DPPH radical due to the scavenging ability of the soluble constituents in the dichloromethane and
alkaloid extracts of the leaves of Phoebe grandis (Nees) Merr, and the standard ascorbic acid, as a
reference compound, presented the highest activity at all concentrations. The IC 50 values were
found to be 70 µg/ml ascorbic acid and >1000µg/ml for both dichloromethane and alkaloid extracts of
the leaves of Phoebe grandis (Nees) Merr. The 50% inhibition concentration (IC 50) value is obtained
using serial dilutions. A lower IC50 value indicates greater activity. Ascorbic acid was used as positive
controls, with IC50 values of 70 µg/mL respectively.
Table 5 Free radical scavenging activity of extracts from the leaves of Phoebe grandis (Nees) Merr using
1,1-Diphenyl—picryl hydrazyl radical (DDPH)
Concentration in
assay (ug/ml)
Percentage scaveging of
DPPH radical (%) (± SEM)
Ascorbic Acid
1000
500
250
125
62.5
31.25
15.63
87.33396 ±0.0007
85.66274 ±0.0041
87.11137±0.0149
84.16026 ±0.0149
69.40834 ±0.1367
50.52237 ±0.1342
21.89991 ±0.0124
70
2
Dichloromethane
Extracts
1000
500
250
125
62.5
31.25
17.74604 ± 0.0178
11.35761 ± 0.0091
4.802523 ± 0.0112
4.763099 ± 0.0173
5.827539 ±0.0448
7.300552 ± 0.1250
>1000
3
Alkaloids
Extracts
1000
500
250
125
62.5
31.25
15.63
46.78768 ± 0.0326
36.54565 ± 0.1026
30.38263 ± 0.1266
17.74159 ± 0.1510
13.41342 ± 0.1701
7.866168 ± 0.2606
3.933084 ± 0.0313
>1000
S/N
Sample
1
IC 50 (ug/ml)
All values are expressed as mean ± SEM for three tests.
Figure 1 Effect of Dichloromethane
and alkaloids extracts of the leaves of
Phoebe grandis on DPPH assay; all
values are expressed as mean ± SEM
for three tests.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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3.5.2: Ferric reducing antioxidant power (FRAP) assay
The Ferric Reducing Antioxidant Power (FRAP) assay measures the reducing ability of antioxidants
3+
against oxidative effects of reactive oxygen species. The potential of the plant extracts to reduce Fe
2+
to Fe by electron transfer is in indication of their antioxidant ability. Electron donating anti-oxidants can
be described as reductants and inactivation of oxidants by reductants can be described as redox
reactions.
3+
2+
This assay is based on the ability of antioxidants to reduce Fe
to Fe
in the presence of
2+
tripyridyltriazine [TPTZ] forming an intense blue Fe –TPTZ complex with an absorbance maximum at
593 nm [26]. Increasing absorbance indicates an increase in reductive ability. The FRAP values of the
studied fractions were calculated and results are shown in Table 3.5.2. Three difference standards such
as Quarcetin, Trolox and Ascorbic acid were used to compare the FRAP value with Hexane,
Dichloromethane, Methanol and Alkaloid extracts of the leaves of Phoebe grandis (Nees) Merr. Alkaloid
extracts showed highest FRAP value (1293.75 ± 0.0773)unit. Otherwise of methanol extracts and hexane
extracts were found to be poor sources. High FRAP values obtained for more polar extracts may be
ascribed partially to the presence of phenolic and flavonoid contents. The ferric reducing activity values of
the extracts and the standard drugs used had been arranged in decrease order: Alkaloids > Quarcetin >
Trolox > Dichloromethane > Hexane > Methanol > Ascorbic Acid.
Table 6 Ferric reducing antioxidant potential (FRAP) assay of the various extracts of the leaves of Phoebe
grandis
Samples
Quarcetin
Trolox
Ascorbic acid
Hexane Extracts
Dichloromethane (DCM) Extracts
FRAP (mM Fe (II)/mg extract)
948.2917 ±0.0694
557.125 ± 0.0589
14.7083 ± 0.0006
91.8333 ±0.0047
484.3333 ± 0.0181
Methanol Extracts
70.1666 ± 0.0026
Alkaloid Extracts
1293.75 ± 0.0773
All values are expressed as mean ± SEM for three tests
4. CONCLUSIONS
The present study shows the scavenging activity of the hexane, dichloromethane, methanol and alkaloid
extracts from the whole leaves of Phoebe grandis (Nees) Merr in. The activity was high in alkaloids and
dichloromethane extracts, but low in hexane extract and metanol extract, indicating their antioxidant
potential. Most antioxidant activities depend on the amount of the phytochemical present in the plants.
Although the content of the most phytochemicals evaluated are not very high but synergistically boost the
antioxidant activity of the whole leaves extracts of Phoebe grandis (Nees) Merr. Plants provide a great
resource for medicine, and alkaloids are very helpful in our strive to overcome the health problems.
Aknowledgement
We gratefully acknowledge the financial support provided by University of Malaya (PV091/2011B).
REFERENCES
[1]
[2]
Ng, F.S.P. Tree Flora of Malaya, A Manual for Foresters. Vol. 4. 1989: Longman Malaysia Sdn.
Bhd. 109-117.
Mukhtar, M.R., Thérése Martin, M., Michele, D., Pais, M., A. Hamid A. Hadi, K. Awang,
Phytochemistry, 45, 1543 (1997).
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6 Ed., John Wiley & Sons, New York, p. 71-143.
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Duddley, H.W. and Ian, F. (1989). Spectroscopic Methods in Organic Chemistry, 4 Ed., McGrawHill Book Company, England, p. 29-62.
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SIZE CONTROLLED SYNTHESIS OF CELLULOSE NANOPARTICLES
Fiona Beragai Jimmy*, Chin Suk Fun and Pang Suh Cem
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak,
94300 Kota Samarahan, Sarawak (MALAYSIA).
*Email: fioberagai@yahoo.com
Abstract
Cellulose nanoparticles were synthesized from commercial facial cotton through nanoprecipitation method.
Controlled precipitation was done through drop-wise addition of dissolved facial cotton to excess absolute
ethanol to obtain cellulose nanoparticles. The effects of synthesis conditions such as concentration of
facial cotton, duration of dialysis, ratio of solvent-nonsolvent, water/oil (microemulsion system) and ratio of
present surfactant on the particle size and size distribution were investigated. Other than that, dialysis
and water/oil emulsion method were used to produce homogeneous cellulose nanoparticles. Cellulose
nanoparticles with particle size ranged from 206 to 683 nm were synthesized under various synthesis
conditions. On the other hand, controlled precipitation with dialysis resulted in more homogeneous
distribution of spherical cellulose nanoparticle as compared to direct precipitation method. Our studies
shows dialysis can produce more spherical cellulose nanoparticles compared to precipitation and water/oil
emulsion methods. The smallest cellulose nanoparticles with a size of 94 nm have been prepared by
using water/oil microemulsion system. Our studies demonstrated that the cellulose nanoparticles can be
produced by a simple nanoprecipitation. Furthermore, the particle sizes can be controlled by varying the
synthesis conditions.
Keywords: Cellulose, facial cotton, nanoparticles, precipitation, microemulsion
1. INTRODUCTION
Natural biopolymers or polysaccharides (e.g starch, cellulose, gelatin) are gaining popularity as the
precursors for target specific therapy, controlled release, carrier action and increase drug solubility as they
are commercially available at low cost, non-toxic, biodegradable and biocompatible (Onofre et al., 2009,
and Hoa et al., 2009). Nano-sized polysaccharides materials served as good precursor materials for the
preparation of bionanocomposites due to their abundance, high strength and stiffness, low weight and
biodegradability (Dufresne, 2010).
Recently, cellulose-based polysaccharides nanoparticles are getting more attention due to cellulose being
the most abundant material in nature and also renewable (Zhang et al., 2010). Cellulose-based product
can be obtain easily since they are available commercially. Facial cotton which are used widely and a
popular household product is known to contain a considerably high amount of cellulose present in the
product. Facial cotton, just like any other cellulose-based product, i.e: facial tissue, cotton linter, filter
paper, linen; are being wasted and thrown away easily usually remained unprocessed to undergo
recycling process (Sun & Cheng, 2002).
Cellulose molecules contained abundant hydrogen bonds which can deter the particles distribution during
synthesizing (Lu & Hsieh, 2010) which leads to the formation of aggregates or bundles of cellulose
nanocrystals during the synthesis of cellulose nanoparticles. Thus, hydrolysis and drying process were
introduce to overcome this problem. Acid hydrolysis of cellulose in sulfuric acid is a process to minimize
hydrogen bonding and is one of the most common method chosen to reduce whilst eliminating
aggregation of cellulose nanocrystals produced from native cellulose. It is also mention in their study that
hydrolysis with sulphuric acid detached amorphous cellulose thus introducing sulfate groups on the
surface of the newly isolated cellulose crystal. This conversely produced a negatively charged cellulose
nanocrystals with repulsion effect and quick freezing with liquid nitrogen can prevent aggregation caused
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by hydrogen bonds. Lu and Hsieh (2010) reported their studies on properties of cellulose nanocrystals
through acid hydrolysis of cotton cellulose. Hydrolysis with sulfuric acid during this study permitting a
complete dispersion of cellulose in aqueous and other medias. Zhang et al. (2007) produced cellulose
nanospheres with size ranging from 60 to 570 nm through hydrolysis of cellulose sample and acid
sonication treatment.
However, this method has to use strong acid (H2SO4) which makes the method not very attractive and not
environmentally friendly. Thus, Jin and coworkers (2007) reported the usage of NaOH/urea/thiourea
solvent system that have high soluble capability of cellulose and has low toxicity where each chemical
component mutually react in improving the dissolution of cellulose.
Nanoprecipitation technique, also known as solvent displacement method, was developed by Fessi et al.,
has been explored for nanoparticles synthesis due to their simplicity and reproducibility (Chin et al., 2011
and Rao & Geckeler, 2011), in addition to its advantages such as minimum toxicity level and external
energy sources required to proceed (Tan et al., 2009). Other than that, desired size distribution of
submicron particles can be controlled through the synthesis parameters (Tan et al., 2009).
Microemulsion technique is one of the effective way for preparing nanosized polymer particles (Rao &
Geckeler, 2011). Water-in-oil (w/o) emulsion is well defined and possess simple processing condition
thus, meets the term needed in producing nanoparticles from hydrophilic natural materials such as
cellulose, starch, gelatin and their derivatives (Shi et al., 2011). Shi and co-workers (2011) reported
nanoparticles produced using high-pressure homogenization combined with water-in-oil (w/o)
miniemulsion technique, showed small size distribution and homogeneous spherical shaped distribution
In this study, we report on the synthesis of cellulose nanoparticles from facial cotton using the precipitation
and microemulsion technique. Cellulose nanoparticles of controllable particle size and shape were
obtained through further optimizing the precipitation conditions such as volume of solvent, varying the
concentration of the cellulose, duration of dialysis, water/oil ratio (in microemulsion) and the use of
surfactants in microemulsion system.
2. MATERIAL AND METHODS
2.1 Materials
Two different cellulosic samples were used which are facial cotton (Watson, Carefeel Cotton Industries
(M) Sdn. Bhd.) and reagent grade Whatman CF11 Fibrous Cellulose Powder (Whatman CF11). All
chemicals used are sodium hydroxide (NaOH, Merck), hydrogen chloride (37%, Hamburg Chemicals),
ethanol absolute (C2H2OH, 99.84%, Hamburg Chemicals), thiourea (CH4N2S, >97%, Fluka), urea
(H2NCONH2, 99.5%, UNI-CHEM), Tween 80 (Merck), and Span 60 (C24H46O6, MERCK-Schuchardt).
Ultrapure water (∼18.2 εΏ) was generated by the Water Purifying System (EδGA, εodel Ultra Genetic).
The dialysis tubes used were molecular porous membrane tubing (Spectra/Por–MWCO: 6000–8000).
2.2 Pretreatment
Pretreament of cellulose sample are of significant in order to remove lignin and hemicellulose, reduce
cellulose crystallinity, and increase the porosity of the materials (Sun & Cheng, 2002). The raw samples
of cellulosic materials were cut into pieces to underwent mechanical pretreatment in advance. The
samples were soaked in NaOH (12 w/v %) for 2h followed by ultrasonification (Tatsumi et al., 2000; Ying,
2008). Later, the slurry undergone acid treatment with 1M HCl for 1.5h, followed by alkaline treatment
with NaOH (2 w/v %) for 2h (Wang, Sain & Oksman, 2007) and finally filtered with a vacuum pump and
thoroughly washed with ultrapure water and followed by acetone. The pretreated samples were dried and
kept in the dessicator.
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2.3 Preparation of celluose solutions
The facial cotton was dissolved by using the aqueous-based solvent system reported by Jin, Zha & Gu
(2007). The optimized composition of the solvent system is NaOH (4 w/v %): thiourea (5.5 w/v %): urea (6
w/v %). 1.0 g of the cellulosic samples were dispersed and underwent sonication for 15 minutes in 100
mL of solvent into NTU solvent systems respectively. Subsequently, the suspensions were then cooled to
-20°C in a freezer for 24h until they became a solid frozen mass and was allowed to thaw at room
temperature. The frozen mass was thawed and became clear solution with no suspension and solid
fragments systems observed.
2.4 Synthesis of cellulose nanoparticles
2.4.1 Effect of concentration of facial cotton
Different concentrations (0.001, 0.005, 0.01, 0.03 and 0.1 w/v %) of facial cotton solution mixture (1 mL)
were added drop-wise to a fixed quantity of absolute ethanol (20 mL) while undergo sonication. Cloudy
preciptation formed instantaneously indicating the formation of cellulose nanoparticles. It is important to
maintain sonication throughout the process to obtain homogeneous mixture. The suspension was later
centrifuged and rinsed 3 times with absolute ethanol to remove NaOH, urea and thiourea.
2.4.2 Effect of ratio of cellulose solution to ethanol on particle size
Dissolved cellulosic facial cotton sample (1 mL) was added drop-wise into a 3 different volume of absolute
ethanol (20 mL, 40 mL, 60 mL) under sonication. The white precipitate formed was then centrifuged and
rinsed 3 times with absolute ethanol to remove NaOH, urea and thiourea.
2.4.3 Water in oil (W/O) Microemulsion Method
1, 3, 5 and 7 w/v % of surfactants (Span-60 & Tween-80) were added into a mixture of different ratios (1:1,
1:3, 1:5 and 1:7) of cyclohexane (oil) against absolute ethanol (co-surfactant). The oil/surfactant/cosurfactant mixture was stirred for 1h until no clumps of surfactants was observed and the mixture has
completely homogenize. The aqueous solution (water phase) utilized was 0.01 w/v % facial cotton with 1
mL was added drop-wise into the microemulsion system and stirred continuously for another 1h. The
resulting suspension was centrifuged and rinsed 3 times with absolute ethanol then dried in 60°C oven
until further testing.
2.4.4 Effect of dialysis duration on particle morphology
Dialysis process was used to control the precipitation rate of the cellulose nanoparticles. An amount of 20
mL of cellulose solution dissolved from facial cotton (0.01 wt%) was introduced into a dialysis tube and
dialysed against 400 mL of absolute ethanol and stirred continually for 24 hours. Samples were taken at
fixed timeline (2 hr, 4 hr, 6 hr, and 24 hr) for further analysis.
2.4.5 Characterization of cellulose nanoparticles
The morphology of cellulose nanoparticles was observed using a scanning electron microscope (SEM)
(JEOL Model JSM 6390LA). The average particle size of the cellulose nanoparticles was determined by
randomly measuring particles using the “SmileView” software.
3. RESULTS AND DISCUSSION
Ethanol were used as non-solvents due to their relatively low dielectric constant such as ethanol (24.6)
and propanol (20.3) which will decrease the chance for the non-solvent to dissolve the hydrophilic
compounds (Bilati, Allemann & Doelker, 2004).
3.1 Effect of concentration of facial cotton
The ratios of facial cotton concentrations to vome of ethanol used in precipitation were observed to have a
significant impact on the particles size. There is a significant increase in the average diameter size of the
resulting particles. Figure 1 shows the obtained average particles size.
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Figure 1 Mean diameter sizes of
particles obtained from different facial
cotton concentration (w/v %) into fixed
(20mL) absolute ethanol.
As observed from figure (Figure 1), lower concentration of facial cotton gives smaller diameter of particle
size. The mean diameter size of cellulose nanoparticles increased as the facial cotton concentration
increased and more aggregates formed. Aggregation as discussed by Tan et al. (2009), molecules
dispersed better in lower concentration and can be separated into monodomains better. The interaction of
the hydrogen bonds between coagulated cellulose nanoparticle and the dissolved cellulose may play an
important role in the stability of cellulose nanoparticle in the suspension (Chang et al., 2010).
3.2 Effect of ratio of facial cotton solution to ethanol on particle size
Figure 2 SEM micrographs of
cellulose nanoparticles by addition of 1
mL of 0.01% facial cotton solution into
(a) 10 mL, (b) 20 mL, (c) 40 mL and
(d) 60 mL of absolute ethanol.
Figure 2a shows the morphology of precipitated cellulose when the ratio of facial cotton solution to ethanol
1:10 (v/v) was used. The precipitated cellulose appeared to form agglomeration and no visible particle
formed. As the volume ratio of ethanol increase to 1: 20 (Figure 2b), the sample was observed to form a
mixture of agglomerates and spherical particles with sizes ranges from 300 to 500 nm. However, when
the volume ratio of facial cotton to ethanol increase to 1: 40 (Figure 2c), the sample showed mainly
spherical shaped cellulose nanoparticles with sizes ranged between 60 to 90 nm. As the volume ratio
increased further to 1: 60 (Figure 2d), the precipitated cellulose particles showed evenly distributed and
individual particles with sizes ranged from 70 to 100 nm. It is shown that higher volume of ethanol
provides better reaction medium for the cellulose nanoparticle to form evenly distributed and smaller
particle size.
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3.3 Water in oil (W/O) Microemulsion Method
a
b
c
d
Figure 3 Particle size comparison in respect of the variable in concentration of the surfactants (Span-60
& Tween-80) and ratio of oil:co-surfactant (a) 1:1, (b) 1:3, (c) 1:5 and (d) 1:7.
Unlike nanoprecipitation that was done earlier, these particles are completely covered with surfactant and
the particle sizes relies on the surfactant concentration, which in turn possess an interfacial tension at the
oil/water interface (Rao et al., 2011).
3.3.1 Effect of surfactant concentration
Various surfactants concentrations (2, 4, 6 & 8 w/v %) were manipulated in order to study the effect of
surfactant concentration on the particle size and morphology. Both surfactants used are non-ionic
surfactants. From Figure 3, in exception of Span-60 with 1:1 (oil:co-surfactant), all of the other condition
tested shows an increasing trend in particle size as the surfactant concentration increases. Cellulose
nanoparticles synthesized in the presence of Tween 80 shows smaller of particle size compared to when
Span-60 was used as surfactant with particle size ranges from 116 to 252 nm and 122 to 317 nm
respectively.
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3.3.2 Effect of type of surfactants
Figure 4 SEM images of cellulose nanoparticles produced from 1:1 ratio of oil to co-surfactant with 2 w/v
% of (a) Span-60 and (b) Tween-80.
Cellulose nanoparticles produced through mixture of 1:1 ratio of oil to co-surfactant with addition of 2 w/v
% of Span-60 and Tween-80 shows different morphology although their particle size diameter are almost
equal, 148 nm and 133 nm respectively. SEM images in Figure 4 showed that, the usage of Tween-80 as
the surfactant produced spherical, individual and homogeneously distributed cellulose nanoparticles.
Span-60 shows aggregation activity between the cellulose nanoparticles probably due to lypophilic
property of Span-60 molecules in the microemulsion system which are weak in facilitating the nucleation
of cellulose nanoparticles in order to form separated and individual particles.
3.4 Effect of dialysis duration on particle morphology
Figure 5 SEM micrographs of various morpholgy for cellulose nanoparticles (0.01 w/v % facial cotton)
prepared by dialysis against excess absolute ethanol taken at (a) 2h, (b) 4h, (c) 6h and (d) 24h.
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Dialysis procedure was performed for 24h in order to control the cellulose nanoparticles precipitation rate.
Figure 5 shows that the spherical morphology started to appear after 2h of dialysis and the spherical
shape started to develop perfectly after 4h. After 6h of dialysis, the particles started to coagulate but the
spherical shapes still maintain visible. The size of the particles ranged from 140 to 240 nm. The particles
lost it spherical form after 24h into dialysis.
4. CONCLUSIONS
In this study, cellulose nanoparticles were being synthesized by precipitating dissolved cellulose solution
in absolute ethanol under controlled conditions. The synthesis method used was simple, fast and easy to
perform. The mean sizes of cellulose nanoparticles could be adjusted by the synthesis parameters such
as the use of an appropriate surfactants and adjusting ratio of oil and aqueous phase in the microemulsion
during the synthesis process.
REFERENCES
Bilati, U., Allenman, E. & Doelker, E. (2004). Development of nanoprecipitation method intended for the
entrapment of hydrophilic drugs into nanoparticles. Carbohydrate Polymers, 30 (4), 1211-1232.
Chang, C., Zhang, L., Zhou, J., Zhang, L. & Kennedy, J. F. (2010). Structure and properties of hydrogels
prepared from cellulose in NaOH/urea aqueous solutions. Carbohydrate Polymers, 82, 122-127.
Chin, S. F., Pang, S. C. & Tay, S. H. (2011). Size controlled synthesis of starch nanoparticles by a
simple nanoprecipitation method. Carbohydrate Polymers, 86 (4), 1817-1819.
Dufresne, A. (2010). Processing of polymer nanocomposites reinforced with polysaccharide nanocrystals.
Molecules, 15, 4111-4128.
Errico, C., Bartoli, C., Chiellini, F. & Chiellini, E. (2009). Poly(hydroxyalkanoates)-Based Polymeric
Nanoparticles for Drug Delivery. Journal of Biomedicine and Biotechnology, 2009, 571702.
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Jin, H. J., Zha, C. X. & Gu, L. X. (2007). Direct dissolution of cellulose in NaOH/thiourea/urea aqueous
solution. Carbohydrate Research, 86(6), 851-858.
Hoa, L. T. M., Chi, N. T., Triet, N. M., Nhan, L. N. T. and Chien, D. M. (2009). Preparation of drug
nanoparticles by emulsion evaporation method. Journal of Physics: Conference Series, 187, 1- 4.
Lu, P. & Hsieh, Y. L. (2010). Preparation and properties of cellulose nanocrystals: Rods, spheres, and
net work. Carbohydrate Polymers, 82, 329-336.
Onofre, F., Wang, Y. & Mauromoustakos, A. (2009). Effects of structure and modification on sustained
properties of starches. Carbohydrate Polymers, 76, 541-547.
Rao, J. P. & Geckeler, K. E. (2011) Polymer nanoparticles: Preparation techniques and size-control
parameters. Progress in Polymer Science, 36(7), 887-913.
Shi, A., Li, D., Wang, L., Li, B. & Adhikari, B. (2011). Preparation of starch-based nanoparticles through
high-pressure homogenization and miniemulsion cross-linking: Influence of various process
parameters on particle size and stability. Carbohydrate Polymers, 83, 1604-1610.
Sun, Y. & Cheng, J. (2002). Hydrolysis of lignocellulosic materials for ethanol production: a review.
Bioresource Technology, 83, 1-11.
Tan, Y., Xu, K., Li, L., Liu, C., Song, C. & Wang, P. (2009). Fabrication of Size-Controlled Starch-Based
Nanospheres by Nanoprecipitation. Applied Materials and Interfaces, 4(1), 956-959.
Tatsumi, D., Higashihara, T., Kawamura, S. & Matsumoto, T. (2000). Ultrasonic treatment to improve the
quality of recycled pulp fiber. Journal of Wood Science, 46(5), 405-409.
Wang, B., Sain, M. 7 Oksman, K. (2007). Study of Structural Morphology of Hemp Fiber from micro to
nanoscale. Applied Composite Materials, 14(2), 89-103.
Ying, W. (2008). Cellulose fiber dissolution in Sodium Hydroxide solution at low temperature: Dissolution
kinetics and solubility improvement. Georgia Institute of Technology.
Zhang, J., Elder, T. J., Pu, Y. & Ragauskas, A. J. (2007). Facile synthesis of spherical cellulose
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Zhang, S., Li, F. X., Yu, J. Y. & Hsieh, Y. L. (2010). Dissolution behaviour and solubility of cellulose in
NaOH complex solution. Carbohydrate Polymers, 81, 668-674.
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EFFECT OF EXTRACTIVES CONTENT ON WOOD DENSITY AND DURABILITY OF
Tristaniopsis whiteana
1
1
Farawahida Abu Zaharin *, Ismail Jusoh & Zaini B. Assim
1
2
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak.
2
Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak,
94300 Kota Samarahan, Sarawak.
*Email: azfarawahida@gmail.com
Abstract
Wood extractives are also known to be one of the most important parameters that contribute to wood
durability. However, there is no information available about the influence of extractives in Tristaniopsis
whiteana. Thus, the objective of this study was to determine the effect of extractives of T. whiteana on its
wood density and durability. Extraction was carried out by removing the extractives by using five organic
solvents of increasing polarity sequentially. Extraction was performed firstly by n-hexane followed by
dichloromethane, ethyl acetate, methanol and distilled water. Wood densities values were determined and
compared before and after extraction. Extracted and unextracted wood blocks were exposed to white rot
fungus namely Trametes versicolor and Pycnoporus sanguineus for durability test. Mean density value
3
3
before extraction for T. whiteana was 1085 kg/m while after extraction the density was 1011 kg/m . The
amount of extractive was similar between inner, middle and outer wood. However, amount of extractives
did not show any consistent trend between height levels. T-test showed that extracted wood samples
were significantly reduced while the correlation coefficient between extractives content and air-dry density
was strong with r=0.77. Further analysis using regression showed that the relationship was strong with
2
r =0.66. This indicated that extractive contribute to the air-dry density of T. whiteana. Strong inverse
relationship was observed between extractives content and weight loss. This showed that the higher the
extractives content the smaller the weight loss which suggests that extractives of T. whiteana are toxic to
T. versicolor and P. sanguineus.
Keywords: Tristaniopsis whiteana, wood extractives, wood density, wood durability
1. INTRODUCTION
Extractives are secondary constituent present in plant and it can be removed by using organic solvents
such as benzene-alcohol, methanol, ethyl acetate and cold or hot water. They are unevenly distributed
(Singleton et al., 2003) and deposited during the heartwood transformation and secondary wall
maturation. These organic compounds are known to influence the wood properties and quality.
Wood density was long recognized as the key indicator to wood quality especially for wood utilization
(Hillis, 1978). Presence of the extraneous compounds strongly affects wood density therefore this can
caused error in the estimation of wood properties especially wood mechanical and pulp properties (Chafe,
3
1987; Lee, 1986). According to Mitchell (1963), a difference in 0.02 g/cm after a wood being extracted
2
2
can change the modulus of rupture (MOR) by about 1000 lbs/in (70.4 kg/cm ). Apart from that, it can
also change the yield of pulp from a cord of pulpwood by 50 lbs (22.7kg) (Zobel & Buijtenen, 1989). Thus
effect of extractives on wood density is very important to be investigated. Since density varies within trees
(Swenson & Enquist, 2008) and so is the extractives (Ona et al., 1995a,b), investigation on effect of
extractives on wood density should be based on within tree variation (Ona et al., 1997).
Another parameter that contributes to wood quality influenced by extractives is the wood durability (Zabel
and Morrell, 1992). Research on the durability of Malaysian hardwoods by Yamamoto and Hong (1994)
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found that among of all parameters that contribute to durability, extractives content is the most important
for all species studied. Extractives can be categorized into several groups. Common groups are alcohol,
phenols, terpenes and terpenoids, polyphenols, tropolones and primary metabolites such as aliphatic
compounds (Hacks & Slansky, 1987). Some of these extractives were reported to be toxic to fungi (Fengel
& Wegener, 1983), invertebrates (Goldstein, 1991) and bacteria. According to Suttie and Orsler, (1996),
the search of natural compounds that are able to inhibit decay was extensively done by researcher
nowadays in the effort to replace the hazardous chemical preservatives used for timber treatment. Thus,
Antwi-Boasiako et al, (2010) suggested that evaluation of the effect of extractives content on durability
should be carried out especially among the tropical timbers. This is because research on role of
extractives content on the durability of hardwood species is less documented.
Tristaniopsis whiteana is one of the durable hardwoods that are less investigated for its properties in
Malaysia. Belongs to the Myrtacea family, it is the third most abundant plant family in Borneo after
Dipterocarpaceae and Euphorbiceae (Slik et al., 2003). In countries abroad such as Hawaii, they already
mixed *Tristania conferta from Australia, which is durable with Eucalyptus saligna wood to make pulp
chips, bridges, mullets and flooring and pallets for construction (Elbert et al., 2003). However, the used of
Tristaniopsis species in Malaysia was less documented. Tristaniopsis whiteana which is also known as
durable is widely exploit as firewood by the local peoples in Sarawak (Latiff et al., 1999 ) and Indonesia
(Tawaraya et al., 2003). Thus, this study was carried out to investigate the potential of Tristaniopsis
whiteana especially in term of the effect of extractives on the wood density and durability.
2. MATERIALS AND METHOD
2.1 Samples Preparation and Wood Density Determination.
Planks were cut from three different height level of Tristaniopsis whiteana. In this study, within tree
variations of density was investigated in term of heights and radial positions of the tree. The level were
labelled as L1, L2 and L3 where samples from L1 was cut at DBH, L2 at approximately 2m from L1 while
3
L3 at 2m from L2. From each level, 120 wood blocks in size of 0.5 x 0.5 x 1.0 cm were taken from the
outer part (sapwood), middle part (heartwood) and inner part (heartwood). The blocks were extracted
sequentially in solvents of increasing polarity. The extraction process started with n-hexane followed by
DCM, ethyl acetate (EtoAc) and methanol (MeOH) to ensure maximum amount of extractives were
removed. Densities of wood blocks were calculated by using calculation as follow:
Volume was determined based on water displacement method.
D1 = Density of conditioned samples before extraction
D2 = Density of conditioned samples after extraction
2.2 Decay Test for White Rot Fungi.
Extracted and unextracted wood blocks were exposed to two white rot fungi namely Trametes versicolor
and Pycnoporus sanguineus based on soil block test method describe by American Society for Testing
and Materials: D 2017-71 (ASTM D 2017-17). The blocks were then ovendried at 103°C for 48 hours
before subjected to weight loss determination. The formula is as follows:
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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W1 = mass of wood cubes before exposed with fungi
W2 = mass of wood cubes after treatment and conditioned in 103 °C
Durability classes were given to the unextracted and extracted woodblocks that have been exposed to
decay test by comparing their weight losses against Trametes versicolor and Pycnoporus sanguineus.
2.3
Data Analysis
Paired T-test was performed to see the difference between extracted and unextracted samples while oneway ANOVA was conducted to see the significant difference within the tree variations. Relationship
between extractives content on durability was investigated by using regression analysis. All analysis was
conducted by using SPSS 18.0.
3. RESULTS AND DISCUSSION
3.1 Effect of Extractives on Wood Density
3
The air-dry density of Tristaniopsis whiteana was 1085 kg/m . However, after extraction process, the
mean of air-dry density of extracted samples from Tristaniopsis whiteana wood were reduced to 1011
3
kg/m (Figure 1). Apart from that, paired t-test also proved that in term of within tree variation, unextracted
density was significantly reduced after extraction process across the radials and between the tree height
level when p= < 0.01 (Table 1). Compared to other studies on Tristaniopsis species, Reyes et al., (1992)
3
reported that basic density for Tristania species was about 810 kg/m . The value obtained from this study
was slightly higher compared to Reyes et al., (1992) because basic density is always lower than airdry
density. Moreover, variation on wood density can be influenced by different type of species, site variation
and genetic sources (Bowyer et al., 2003). As for the effect of extraction, Antwi-Boasiako (2004) and
Singleton et al., (2003) observed similar result where wood density drastically reduced after extractives
were removed by using organic solvents.
3
Figure 1 Density (kg/m ) of
T. whiteana before and after
extraction process.
Mean values of density before and after extraction, the percentage of weight change, significant reduction
between unextracted and extracted density, density reduction, extractives content percentage and
reduction percentage between L1, L2 and L3 according to radial position of T. whiteana tree was shown
as in Table 1. Radial variation of density was similar between height level. Heartwood (inner and middle
part) was found significantly higher than the sapwood (outer part). There was no specific trend observed
on the distribution of the extractives content of T. whiteana. Extractives content generally were similar
across the radial of L1and L3 where densities across the radial of each tree level were not significant from
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one another. Nevertheless, in L2, the density of inner part was significantly higher than the outer and
middle part with 4.74%, 10.14% and 10.55% respectively.
Table 1 Mean values of density before and after extraction, the percentage of weight change, significant
reduction between unextracted and extracted density, density reduction, extractives content percentage
and reduction percentage between L1, L2 and L3 according to radial position of T. whiteana tree.
n-Hexane+DCM+EtoAc+MeOH
Height
L1
Parts
Unextracted
Air-dry
density
3
(kg/m )
Extracted
air-dry
density
3
(kg/m )
Density
reduction
3
(kg/m )
Extractives
content
(%)
Reduction
percentage
(%)
Significant
of
reduction
Inner
1126a*
1060a
66
5.85a
6.03a
**
Middle
1110ab
1054ab
56
5.05a
5.18a
**
Outer
1095b
1027b
67
6.11a
6.32a
**
1110
1047
63
5.67
5.84
**
Inner
1061a
1011b
50
4.74a
4.86a
**
Middle
1060a
953a
107
10.14b
10.96b
**
Outer
1077a
963a
114
10.55b
11.39b
**
1066
977
90
8.28
8.85
**
Inner
1073a
1003a
70
6.51a
6.73a
**
Middle
1080a
1002a
78
7.2a
7.47a
**
Outer
1082a
1018a
64
5.95a
6.14a
**
1079
1008
71
6.55
6.77
**
Average
L2
Average
L3
Average
* Mean values with different letter within column (between parts of each level) are significant at α = 0.05
** Significant of reduction between unextracted and extracted samples are significant at 1% level by using paired Ttest
Height level variation density of L1 (DBH) was significantly higher than L2 and L3 before and after
extraction (Figure 2). In term of extractives content, L2 significantly contained more extractives than L1
and L3 (Figure 3). Thus, between the height levels, there were no trends observed. This may due to the
distribution of the extractives content itself.
Correlation coefficient and regression analysis were conducted to see the relationship between the two
variables namely extractives content and air-dry density (Figure 4). Both variables were positively
correlated with high correlation coefficient, r = 0.73.This mean that linear trend may exist between
extractive content and air-dry density. Further analysis on regression between the two parameters also
2
show that the relationship was strong with r = 0.66 when the ρ = <0.01. The graph was linear and
inversed where the higher the extractives content, the higher the air-dry density.
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3
Figure 2 Density (kg/m ) of T.whiteana according to tree heights after extraction. Different letter within
heights are significant at 0.05 level.
b
a*
a
Figure 3 Extractives content percentage (%) of T. whiteana according to tree heights after extraction.
Different letters within heights are significant at 0.05 level.
Figure 3. Relationship between extractives content and air-dry density of T. whiteana.
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3.2 Effect of Extractives on Wood Durability
Weight loss caused by Trametes versicolor and Pycnoporus sanguineus on extracted Tristaniopsis
whiteana was significantly higher than on the unextracted samples (Table 3). This shows that, extracted
wood blocks were susceptible to decay fungi. After extraction process, T. whiteana was classified as
moderately durable against T. versicolor and P. sanguineus according to Findlay (1985) classification.
Table 3 Total extractives content and durability classes of wood blocks from T. whiteana and T. beccarii
exposed to T. versicolor and P. sanguineus.
Extracted T.whiteana
Trametes versicolor
Mean
Mean
extractives
weight
content
loss
(%)
(%)
a
9.32
8.02
Unextracted
T. whiteana
-
Wood species
3
Pycnoporus sanguineus
Mean
Mean
extractives
weight
content
loss
(%)
(%)
a
6.17
6.67
2
-
Findlay **
b
3.8
b
1.69
Findlay
(1985)
**
3
2
* εean values with different letter within column are significant at ρ = <0.05. ** 1 Very durableν 2 Durableν
3 Moderately durable; 4 Non durable; 5 Perishable (Findlay, 1985).
Relationship between extractives content and weight loss due to decay for both fungi were significant at
0.01 level (Figure 4). All relationships were power-curve fit and the relationship was classified as strong
and inversed on exposure to T. versicolor and P. sanguineus. Antwi-Boasiako et al., (2010) also observed
inverse relationship between weight loss and total extractives content upon exposure to T. versicolor
which mean that, higher amount of extractives content resulting to smaller weight loss (higher durability).
a
b
Trametes versicolor
16
y=
12
16
R² = 0.72
14
Weight loss (%)
Weight loss (%)
14
Pycnoporus sanguineus
37.019x-0.778
10
8
6
4
2
y = 27.907x-0.901
R² = 0.64
12
10
8
6
4
2
0
0
5
10
15
0
20
0
5
10
15
c
b
c
T. versicolor & P. sanguineus altogether
16
y = 39.875x-0.877
R² = 0.75
Weight loss (%)
14
12
10
8
6
4
2
0
0
5
10
15
Extractives content (%)
20
Figure 4 Relationship between total extractives content and weight loss for T. whiteana exposed to: a) T.
versicolor b) P. sanguineus c) T. versicolor and P. sanguineus altogether.
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4. CONCLUSION
There was no consistent trend observed between density and extractives content neither between height
levels nor across the radial of the tree. Significant amount of extractives present in Tristaniopsis whiteana
and the relationship between extractives content and air-dry density was high. It can be concluded that
density was significantly affected by extractives content. Thus removing the extractives to accurately
estimate the density of T. whiteana is crucial especially when investigating the Tristaniopsis species
qualities and abilities for future utilization. Apart from that, extractives content found to be a good predictor
for density as proven by regression analysis.
Extractives were variedly distributed within tree. It is considerable to say that extractives present in T.
whiteana were able to control T. versicolor and C. globosum. Thus, the durability of T. whiteana might
highly caused by its extractives content. Further research on type of compound present in T. whiteana
should be carried out for the determination of the specific compound that contributes to the toxicity
characteristic. Research on T. whiteana properties and wood quality should be investigated to enable
T.whiteana to be utilized as a new resource in wood industry.
REFFERENCES
Antwi-Boasiako, C. 2004. Assessment of Anatomy, Durability and Treatability of Two Tropical LesserUtilized Species and Two Related Species from Ghana. PhD Thesis (2004) submitted to The
University of Reading, Reading, England (UK). 319pp
ASTM. 2007. Standard Test Method for Preparation of Extractive-Free Wood, ASTM Standard D1105-96,
(2007). ASTM International, West Conshohocken, PA, USA.
Bowyer, J.L., Schmulsky, R. and Haygreen, J.G. 2003. Forest Products and Wood Science: An
th
Intoduction. 4 ed. Iowa State Press. Iowa. Pp 554
Chafe, S. C. 1987. Collapse, Volumetric Shrinkage, Specific Gravity and Extractives in Eucalyptus and
other Species. Part2: The Influence of Wood Extractives. Wood Science and Technology, 21:2741.
Elbert, L. Little, Jr. and Roger, G. S. 2003. Agriculture Handbook. College of Tropical Agriculture and
Human Resources, University of Hawai’i, εanoa. 377p.
Goldstein, I.S. 1991. Overview of the Chemical Composition of Wood cited in Lewin, M. And Goldstein,
st
I.S. (Eds). Wood Structure and Composition. 1 ed. International Fibre Science and Technology.
Marcell Dekker, New York. 488p.
Hacks, R.A. and Slansky, F.J. 1987. Nutritional Ecology of Wood-Feeding Coleoptera, Leptidoptera, and
Hymenoptera 15 cited in Slansky, F.J. and Rodrigues, J.G. (Eds). Nutritional Ecology of Insects ,
Mites, Spiders and Related Invertebrates. Wiley, New York. 449-486 pp.
Hillis, W.E. 1978. Wood Quality and Utilization. Hillis, W. E and Brown, A.G. (Eds.). Australia, CSIRO.
259-289 pp.
Lee, C. H. 1986. A Note on the Effect of Alcohol-Benzene Extractives on Juvenile Wood Specific Gravity
in Red Pine, Wood Fiber Sci. 18: 376-381
Mitchell, H.L. 1963. Specific Gravity Variation in North American Conifers. IUFRO Sect. 41 Committee on
Fiber Characteristics. Melbourne, Australia. 2:15pp
Ona, T., Sonoda, T., Ito, K. and Shibata, M. 1997. Relationship between Various Extracted Basic
Densities and Wood Chemical Components in Eucalyptus camaldulensis. Journal of Wood
Science and Technology, 31: 205-216.
Singleton, R., DeBell, D.S. and Gartnert, B. L. 2003. Effect of extraction on wood density of Western
Hemlock (Tsuga heterophylla (Raf.) Sarg.). Wood and Fiber Science, 35(3) : 363-369
Slik, J. W. F., Poulsen, A. D., Ashton, P. S., Cannon, C. H., Eichhorn, K. A. O., Kartawinata, K., Lanniari,
I., Nagamasu, H., Nakagawa, M., van Nieuwstadt, M.G.L., Payne, J., Purwaningsih, Saridan, A.,
Sidiyasa, K., Verburg, R.W., Webb, C.O., and Wilkie, P. A Floristic Analysis of the Lowland
Dipterocarp Forests of Borneo. Journal of Biogeography, 30: 1517–1531
Tawaraya, K., Takaya, Y., Turjaman, M., Tuah, S.J., Limin, S.H., Tamai, Y., Cha, J.Y., Wagatsuma, T.,
Osaki, M. 2003. Arbuscular Mycorrhizal Colonization of Tree Species Grown in Peat Swamp
Forests of Central Kalimantan, Indonesia. Forest Ecology and Management, 182: 381–386.
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st
Zabel, R.A and Morrell, J.J. 1992. Wood Microbiology: Decay and Its Prevention. 1 ed. Academic Press,
London. 47p.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
AMPLIFICATION PROFILE FOR AVIAN INFLUENZA A VIRUS (AIV A) DETECTION BASED ON
REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT PCR)
1*
1
Zahirunisa Abd Rahim , Mustafa Abdul Rahman & Ismail Ahmad
1
2
2
Department of Zoology, Department of Molecular Biology,Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak,
Abstract
Immunocapture technique was inherited to enhance the possibility of detecting AIV A from 402 samples
isolated from wild birds. The immunocapture plates were pre-coated with antibody of selected
hemagglutinin subtypes of H5, H6, H7 and H9 that may attract the targeted antigen to bind to the bottom
of the plate. Several modifications were made to the samples to maximise the detection efforts. The
immunocapture PCR was conducted based on four mixture protocols with annealing step carried out at
seven different temperatures ranging from 48 °C to 54 °C. However, all samples show negative infection.
The optimisation of the detection technique was further conducted using the positive strain obtained from
an infected duck. The strains were treated and amplified using nucleoprotein (NP), H5, H6, H7 and H9
primers. The samples were first synthesised into complementary deoxyribonucleic acid (cDNA) with the
use of M-MuLV reverse transcriptase. The cDNA products were amplified in the PCR initiated by Taq
polymerase. The manipulative materials were the RT PCR mixture, the RT PCR cycling condition, type of
primers annealed, the temperature used in the protocol and also the concentration of the product use in
gel electrophoresis. The annealing temperature starts from as low as 60 °C to 75 °C. However, the
products amplified using H5 primer showed a promising result when annealed at the given temperature
ranging from 65-70 °C. The products amplified using NP primer was obviously unspecific as the range of
molecular weight given was quite wide with approximately 1497 bp. Thus, NP primer may not be essential
in the current study survey.
Keywords: immunocapture, avian influenza A viruses, reverse transcriptase, polymerase chain reaction,
optimisation
**Please contact the corresponding authors for further details of this paper.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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APPLICATIONS OF GENOMICS TO PLANTATION FORESTRY WITH KELAMPAYAN IN SARAWAK
1*
2
1
1
1
1
1
3
Ho, W.S. , Pang, S.L. , Tchin, B.L. , Lai, P.S. , Tiong, S.Y. , Phui, S.L. , Liew, K.S. , Ismail, J. &
2
Julaihi, A
1
Forest Genomics and Informatics Laboratory (fGiL), Department of Molecular Biology
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan,
Sarawak
2
Applied Forest Science and Industry Development (AFSID), Sarawak Forestry Corporation, 93250
Kuching, Sarawak
3
Department of Plant Science and Environmental Ecology
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan,
Sarawak
*Email: wsho@frst.unimas.my
Abstract
Wood-based industries in Sarawak are increasingly encouraged to adapt to “new wood” from planted
forests composed of fast-growing species such as Kelampayan with short rotation cycle (6-8 years). The
3
rationale is that natural forests at the most produce about 3m /ha/yr of commercial timber, whereas
3
3
plantations can produce annually from 10m /ha to 30m /ha of commercial timber. It is estimated at least
30 million seedlings are required for annual planting or reforestation programmes to meet the increasing
global demand for raw materials. To date, several molecular genetics studies have been completed for
Kelampayan. These include genetic diversity of Kelampayan, genetic marker (SSRs) development,
‘Touch-incubate-PCR’ approach for preparing plant tissues for high throughput genotyping, and
transcriptomics and bioinformatics on wood formation of Kelampayan. This information provides a useful
resource for genomic selection of Kelampayan aiming at the production of high value forests for maximum
returns.
Keywords: genomics, genetic diversity, plantation forestry, Neolamarckia cadamba, genomic selection
INTRODUCTION
Rapid socio-economic changes in the world are having profound impacts on all sectors, including forestry.
While wood products demand is increasing, so is the demand for environmental services of forests.
However, these slow growing trees from natural forests with long generation intervals are unable to meet
current global demand for wood, resulting in the loss and degradation of natural forests by logging. Woodbased industries are increasingly encouraged to adapt to “new wood” from planted forests composed of
fast-growing species with short rotation cycle (6-8 years). Planted forests are more efficient in producing
commercial timber needs on a long-term basis than natural forests. For instance, natural forests at the
3
most produce about 3 m /ha/yr of commercial timber, whereas planted forests can produce annually from
3
3
10 m /ha of hardwoods to 30 m /ha of softwoods (Krishnapillay and Razak, 2001). Furthermore, planted
forests are easier to manage due to the mono or double species mix compared to very diverse natural
forest stands. Hence, plantations development will serve as a strategy for maintaining a sustainable
supply of timber and at the same time, reducing the logging pressure on natural forests for wood
production to an acceptable level.
3
3
The global demand for wood products is projected to increase from 3.5 billion m in 1990 to 6.4 billion m
in 2020. Apart from that, the demand for environmental services of forest is also increasing whereby more
natural forests will be excluded from wood production, and recently the bioenergy policy, the use of
biomass, including wood is increasingly encouraged. Therefore, there is a need to invest more in the
research and development (R&D) of high-yielding, faster growth and short-rotation planted forests to
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increase the competitiveness of the forest-based sector in Sarawak. In Sarawak, timber and timber
products continue to be the third largest export revenue after natural gas and petroleum as it contributed
about 35% or RM 7.68 billion to the overall annual export earnings in Malaysia which stood at RM 21.94
billion, as of 2005-2009. It is also the largest producer of plywood in Malaysia and the largest exporter of
tropical plywood for the country.
Why Kelampayan is Chosen?
Neolamarkia cadamba (Roxb.) Bosser, locally known as Kelampayan belongs to the family of Rubiaceae,
has been selected as one of the fast growing plantation species for planted forest development in
Sarawak (Figure 1). It is praised as a “miracle tree” in China due to its fast growing characteristics and an
ideal tree species to study genetic functions related to tree growth and cell wall development (Li et al.,
2011). In Sarawak, the state government has introduced the Forest (Planted Forest) Rules (1997) to
encourage the development of commercial forest plantations and has set a target of 1.0 million hectares
for forest plantations to be established by 2020. It is estimated that 30 million of high quality seedlings are
required for the annual planting programme. As of May 2010, total area planted with Kelampayan was
18,851 ha (8% out of 240,075 total area planted) in Sarawak, which is approximately about 9 million trees
planted with planting distance of 4 x 4 meters.
Figure 1: The current and other/future uses of Kelampayan
Kelampayan is a large, deciduous and fast growing tree species, thus with characteristics which
guarantee early economic returns within 8 to 10 years. Under normal conditions, it reaches a height of 17
m and diameter of 25 cm at breast height (dbh) within 9 years. A mature tree can attain 20 to 30 m in
height and 50 to 100 cm dbh. Growth statistics of Kelampayan or locally known as Kadam in India showed
that Kadam when planted at 5 by 5 m spacing under good growth conditions on a rotation period of 10
3
years would grow to an average of 0.4 m peelable timber per tree. On a very conservative estimate, yield
3
of 0.4 m wood can be easily obtained from a Kadam tree under rotation of 10 years by which time each
tree will have a girth varying from 90 cm to 1 m.
3
Kelampayan is a lightweight hardwood with a density of 290-560 kg/m at 15% moisture content. It has a
fine to medium texture; straight grain; low luster and has no characteristic odour or taste (Joker, 2000). It
is easy to work with hand and machine tools, cuts cleanly, gives a very good surface and is easy to nail.
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Thus, it is one of the best sources of raw material for the plywood industry, besides pulp and paper
production. It can also be used as a shade tree for dipterocarp line planting, whilst its leaves and bark
have medical application. The dried bark can be used to relieve fever and as a tonic, whereas a leaf
extract can serve as a mouth wash. It also has high potential to be utilized as one of the renewable
resource of raw materials for bioenergy production such as cellulosic biofuels in the near future. Due to its
multipurpose function and utility, the species is favoured in tree plantation programmes.
Despite the high economic value of Kelampayan, little is known about this species and none of the DNAbased markers have been developed to date. In this paper, we highlighted the projects that have been
conducted with special focused on molecular genetic studies. Among others are 1) genetic diversity of
Kelampayan in natural and planted forests in Sarawak; 2) development of simple sequence repeat DNA
markers specific for genotyping Kelampayan, 3) the one step ‘Touch-incubate-PCR’ approach for
preparing plant tissues for high throughput genotyping of Kelampayan, and 4) transcriptomics and
bioinformatics on wood formation of Kelampayan.
Genetic Diversity of Kelampayan in Natural and Planted Forests via ISSR
Genetic diversity is important for the maintenance of the viability and the adaptive potential of populations
and species. This information will be a basis for establishing tree improvement programme and for
management or conservation of natural communities. It has been reported elsewhere that populations
with little genetic variation are more vulnerable to the arrival of new pests or diseases, pollution, changes
in climate and habitat destruction due to human activities or other catastrophic events. In the present
study, ISSR markers have been used to determine the genetic diversity of Kelampayan in Sarawak. ISSR
technique is a PCR-based method to amplify DNA fragments between two closely spaced and oppositely
oriented SSRs (Moreno et al., 1998).
A total of 236 Kelampayan trees were collected from two planted forests (Kanowit and Song) and six
natural forests (i.e. Similajau (Bintulu), Lawas, Matang, Simunjan, Mukah Hill and Niah). Three ISSR
primers i.e., (GTG)6, (AC)10 and (AG)10 were used and generated a total of 138 loci, of which 32.6% to
59.4% (Table 1), with an average of 45.1% of the loci were polymorphic. The mean Shannon’s diversity
indices ranging from 0.1399 to 0.2354 and this indicates that Kelampayan are genetically less diverse
among the populations. Low percentage of polymorphic loci also indicates that Kelampayan is relatively
less diverse compared to other plant species, such as teak (Tectona grandis) plus tree that has 95.5%
polymorphic loci (Narayanan et al., 2007) and Asparagus acutifolius L. with 100% polymorphic loci (Sica,
et al., 2005). Among the two planted forests studied, Kelampayan in Kanowit’s planted forest are most
closely related (I = 0.13λλ) compared to Song’s planted forest that has a higher level of genetic diversity (I
= 0.1597). This phenomenon might be due to the seed sources or planting materials were originated from
a few selected mother trees.
Table 1 Summary of mean Shannon’s diversity index
trees in 6 natural and 2 planted forests in Sarawak
ISSR
Forest type
Populations
n
Natural forest
Bintulu
29
Lawas
29
Matang
29
Niah
31
Simunjan
30
Mukah Hill
28
Planted forest
Kanowit
30
Song
30
and percentage of polymorphic loci of Kelampayan
I
0.2354
0.1863
0.1681
0.1555
0.1565
0.1543
0.1399
0.1597
P (%)
59.42
53.62
47.10
45.65
43.38
41.30
32.61
37.68
Note: n = number of samples; I = Mean Shannon’s diversity index; P = percentage of polymorphic loci
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The coefficient of population differentiation (G st) was higher for natural forests (0.2013) compared to
planted forests (0.0871). However, the Gst value of Kelampayan trees was considered low in comparision
to other species such as; Ceriops tagal (Gst = 0.529) (Ge and Sun, 2001), Ceriops decandra (0.882) (Tan
et al., 2005), Hagenia abyssinica (Gst = 0.25) (Feyissa et al., 2007) and Taxus fauna (Gst = 0.5842) (Shah
et al., 2008). The genetic relationship of natural and planted forests based on shared allele distance (DSA)
(Chakraborty and Jin, 1993). had divided these forests into two obvious clusters (Figure 2). Natural forests
were grouped in one cluster while planted forests were grouped in another cluster. This indicates that
most of the Kelampayan trees are closely related to each other according to their forest types.
C-
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Fig. 2: Neighbour-joining tree of 236 Kelampayan trees in eight forests performed using shared allele
distance, DSA [11] based on proportion of shared alleles from 3 ISSR markers. Each tip represents a
single individual.
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Development of Simple Sequence Repeat DNA markers Specific for Genotyping Kelampayan
The development of an ideal molecular DNA marker system which is genetically co-dominant and multiallelic is becoming a major concern due to the genetic complexity of breeder’s populations and high levels
of heterozygosity in individual genotypes (Elizabeth et al., 2003). As such simple sequence repeat (SSR)
marker is the ideal marker system of choice for the tree breeders. In the present study, we used ISSRSuppression PCR method in developing the SSR markers for Kelampayan. In total, 31 SSR primer pairs
were designed to flank the targeted SSR in the Kelampayan genome. Among the 31 SSR loci, 7 (22.6%)
SSRs were classified as the perfect type, 11 (35.5%) as the imperfect type and 13 (41.9%) with the
compound type. The most abundant dinucleotide motif found in the Kelampayan genome was TG/AC and
CT/GA repeats.
A total of 15 SSR primers were successfully validated and characterized, resulting in 48.4% primer-tomarker conversion ratio. Considerable allelic amplifications were obtained for these SSR markers across
the tested genotypes. A total of 66 alleles were identified and the mean number of alleles per locus was
4.4. Most of the SSR loci analyzed showed high polymorphism, as indicated by the number of alleles
detected at particular loci and by the PIC value, which was above 0.5. The most polymorphic among the
15 loci were: AC11 (9 alleles, PIC=0.849), AC12 (5 alleles, PIC=0.722) and AG01 (6 alleles, PIC=0.712).
SSR markers developed in this study opens a new perspective for generation of baseline genetic
information for effective selection of plus trees, provenance trials and establishment of forest seed
production areas (SPAs) of Kelampayan in the selected forest reserves for planted forest development
and tree improvement activities.
One Step ‘Touch-incubate-PCR’ Approach for Preparing Plant Tissues for High Throughput
Genotyping
The fasTiP-X approach is a rapid extraction method which allows direct amplification without going
through conventional CTAB extraction. In the same time, it allows DNA extraction without contacting any
harmful chemicals and liquid nitrogen. This method offers a great advantage whereby it requires only
approximately 20 minutes for DNA preparation before PCR amplification thus increases the possibility for
high-throughput genotyping. Apart from that, the requirement of small amount of plant material is greatly
suitable for samples with limited quantity.
The fasTiP-X approach was tested by using the 5S rRNA primers via PCR on 4 different species, namely
Neolamarckia cadamba (Roxb.) Bosser (Kelampayan), Duabanga moluccana (Sawih), Durio zibelthinus
(Durian) and Dimocarpus longan Lour. (Longan). The amplification of DNA template obtained from the
fasTiP-X was comparable to the positive control which extracted using conventional CTAB method (Fig.
3). The PCR analysis using DNA template isolated by the fasTiP-X approach for each species was
repeated 3 times to prove the reliability and reproducibility of this method. This result shows that fasTiP-X
approach was more suitable for high-throughput genotyping compared to the conventional DNA extraction
considering its rapidity, simplicity and cost-effective features.
M
P
I
P
II
P
III
P
IV
Figure 3: PCR amplification using 5S rRNA primers with template obtained from fasTiP-X.
M: 100bp marker; P: positive control; I: Neolamarckia cadamba; II: Duabanga moluccana; III: Durio
zibelthinus; and IV: Dimocarpus longan
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Transcriptomics and Bioinformatics on Wood Formation of Kelampayan
Despite the high economic value of tropical wood, little is known about the genetic control of wood
formation or xylogenesis in Kelampayan. No Kelampayan EST information is available in the NCBI
GenBank to date. Thus, we applied genomics approaches to explore the molecular basis of wood
formation in Kelampayan. Here we report the generation and analysis of a genomic resource (10,368
expressed sequence tags, ESTs) for wood formation in Kelampayan via high-throughput DNA sequencing
of cDNA clones derived from a 2-year old developing xylem tissues.
Assembly of 6,622 high quality ESTs from 5’ end sequences generated 4,728 xylogenesis unigenes with
an average length of 672 bp. The analysis formed 2,100 consensus contigs sequences (representing
3,994 or 60.3% of the total high quality ESTs), with a length ranging from 132 bp to 2706 bp and an
average length of 621bp. The remaining 2,628 (representing 39.7 % of the total high quality ESTs) were
singletons which ranged from 104 to 839 bp, with an average length of 723 bp. About 59.3% of the ESTs
were assigned with putative identifications whereas 40.7% of the sequences showed no significant
similarity to any sequences in the GenBank. Assembly analysis revealed a redundancy level of 28.5% in
the Kelampayan EST database. By comparison, the EST redundancy in the kelampayan EST database is
comparable to the estimated redundancy of 28% in Populus (Aspeborg et al., 2005) and the 28.8% in
Pinus radiate (Li et al., 2009).
The most abundant protein in the ESTs whose putative function was inferred from sequence comparison
was 60s ribosomal protein with 92 ESTs, followed by 40s ribosomal protein with 42 ESTs. Interestingly,
most genes involved in lignin biosynthesis were present in the kelampayan EST database with 1 to 21
ESTs. These include phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), coumarate 3hydroxylase (ρ-coumaryl shikimate/quinate 3-hydroxylase) (C3H), caffeic acid O-methyltransferase
(COMT), caffeoyl-CoA-3-O-methyltransferase (CCoAOMT), 4-coumarate:CoA reductase (4CL), ferulate 5hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), hydroxycinnamoyl-CoA: shikimate/quinate
hydroxycinnamoyl transferase (HCT) and cinnamoyl-CoA reductase (CCR). COMT, CCoAOMT and C3H
are in the 30 highly abundant genes with 18 to 21 ESTs. Also, several ESTs exhibiting homologies to cell
wall biosynthesis genes were also identified in the Kelampayan EST database. The most highly abundant
cell wall genes are tubulin (42 ESTs), arabinogalactan protein (AGPs) (30 ESTs) and cellulose synthase
(CesA) (13 ESTs). Other cell wall related genes including sucrose synthase (SuSy), expansin, UDPglucose dehydrogenase (UGDH), xyloglucan endotransglycosylase/hydrolase (XET/XTH) and pectate
lyase are moderately abundant with 2 to 11 ESTs in the Kelampayan EST database.
The NcdbEST was also employed to predict the full-length cDNA of several important genes involved in
wood formation of Kelampayan through contig mapping approach. These include cinnamate 4hydroxylase (C4H), cinnamyl alcohol dehydrogenase (CAD), xyloglucan endotransglycosylase (XET) and
cellulose synthase (CesA). Cinnamate 4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid and
lignin biosynthesis pathway. The full-length C4H cDNA, designated as NcC4H was 1,651 bp long with a
1,518 bp open reading frame encoding a protein of 505 amino acids (εWμ 58.28 kDa), a 18 bp 5’untranslated region and a 115 bp 3’-untranslated region. The NCBI blastn indicated that the NcC4H cDNA
displayed several similarities to the C4H genes from other plants species. By comparing the deduced
amino acid sequence of NcC4H with other sequences reported previously, the NcC4H showed higher
identity with the class I C4Hs, which is preferentially involved in phenylpropanoid pathway. Three domains
were also found within the open reading frame of NcC4H that are proline-rich region, threonine-containing
binding pocket for the oxygen molecule and a heme-binding region.
Cinnamyl alcohol dehydrogenase (CAD) catalyzes the reduction of cinnamaldehydes to ρ-coumaryl,
coniferyl and sinapyl alcohols during the final stage of lignin biosynthesis pathway. Sequence analysis of
the full-length CAD cDNA, designated as NcCAD showed that it was 1,240 bp long with a 1,086 bp open
reading frame encoding a protein of 361 amino acids (εWμ 38.563 kDa), a 68 bp 5’- untranslated region
and a 86 bp 3’- untranslated region. Blastn search revealed that the NcCAD shares high sequence identity
of 72 % with CAD/SAD of Populus trichocarpa and P. tremuloides. A zinc-containing alcohol
dehydrogenases signature (GHEIVGEVTEVGSKV) was detected in the NcCAD amino acid sequence
from position 72 to 86.
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Xyloglucan endotransglycosylase/hydrolase (XTH) is a key enzyme that plays an important role in plant
cell wall remodeling which later determines the wood quality. The full-length nucleotide sequence, named
as Nc-XTH1 and Nc-XTH2 were 893 and 1,024 bp long with 858 and 915 bp open reading frame,
respectively. Blasting results supported these two genes as xyloglucan endotransglycosylase family with
the identity value more than 75% with populus and some other species. Nc-Nc-XTH1 and Nc-XTH2 were
predicted to be classified in different XTH family gene with the pairwise alignment similarity of nucleotide
and amino acid sequences scored at 64.0 and 45.0, respectively. This also supported by the phylogenetic
analysis which grouped Nc-XTH1 and Nc-XTH2 into two distinct clusters. Nc-XTH1 encoding 286 amino
acids contained a typical catalytic DEIDFEFLG motif whereas Nc-XTH2 encoding 305 amino acids has a
slightly different catalytic sequence (NEFDFEFLG). However, both enzymes possess the two catalytic
glutamates and the stabilizing aspartate (underlined amino acids). Both Nc-XTH1 and Nc-XTH2 also have
putative N-glycosylation site (ADDWATR/QGGL/R I/VKTDW) with three nucleotide differences as
underlined. The active and catalytic side of Nc-XTH1 and Nc-XTH2 with the conserved domain cd02176
from NCBI database supported they were GH16_XET, a member of xyloglucan endotransglycosylase in
glycosyl hydrolase family 16.
Cellulose is the key component of cell walls which is proposed to be synthesized by a large membranebound protein complex, cellulose synthase (CesA). The predicted full-length cDNA was 3,472 bp with
3,126 bp open reading frame encoding 1,042 amino acids. A highly conserved zinc binding domain
(Cx2Cx12FxACx2Cx2PxCx2CxEx5Gx3Cx2C, where x is any amino acid) at amino acid 37 to 82 was found
towards the N-terminal which corresponding to cell microfibril structures. The presence of D, D, D, QxxRW
motif suggested that the hypothetical NcCesA1 involves in glycosyltransferase activity, whereas the plantconserved region (CR-P) is suggested to be implicated in the cellulose biosynthesis at “rosette”
complexes that consist of multiple catalytic subunits formed by CesA gene embedded in plasma
membrane of plant cells.
As a conclusion, the present study has generated a useful resource for genomic selection of Kelampayan.
The identified genes will be candidates for association genetic studies in Kelampayan (Fig. 4) aiming at
the production of high value forests (Thumma et al., 1995).
Figure 4 Detailed layout of genomics-assisted breeding (association mapping) of Kelampayan. The most
immediate impact would be a great increase in the speed of the breeding cycle, thereby reducing the
breeding and production costs and accelerating the production of elite genotypes or clones into market.
This project should also facilitate germplasm exchange and increase the probability of selecting useful
germplasm.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
For instance, in our proof-of-concept study on gene-associated single nucleotide polymorphism (SNP) in
C4H and CAD genes from Acacia mangium Superbulk trees revealed an association between SNPs in
CAD gene with the wood density, specific gravity and cell wall thickness (p<0.05) (Tchin et al., 2011).
Thickening of cell wall is affected by the arrangement of biopolymer aggregates which comprise of
cellulose, hemicellulose and lignin. While the concentration of lignin is as a proportion of the cell wall and
decrease when come closer to the lumen (Haygreen and Bowyer, 1996). Single nucleotide mutation in
CAD gene might alter the lignin biosynthesis and thus, lead to changes in phenotypic characteristics of the
trees. Thus, the outcome that will produce through this strategy is new clones for plantation and also the
crosses between these clones to produce hybrid, with double yields as well as enhance pest and
environmental tolerance. Such new clone or hydrid has great economic potential.
Acknowledgements
The authors would like to thank all the lab assistants and foresters involved in this project for their
excellent field assistance in species identification and sample collection. This work is part of the joint
Industry-University Partnership Programme, a research programme funded by the Sarawak Forestry
Corporation (SFC) and UNIMAS.
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Haygreen, J. G. & Bowyer, J. L. (1996). Forest Products and Wood Science, 3 edition. IOWA State
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Li, X., Wu, H.X., Dillon, S.K. & Southerton, S.G. (2009). Generation and analysis of expressed sequence
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Narayanan, C., Wali, S.A., Shukla, R., Kumar, R., Mandal A.K. & Ansari S.A. (2007). RAPD and ISSR
markers for molecular charazterization of teak (Tectona grandis) plus trees. Journal of Tropical
Science 19(4): 218-225.
Shah, A., Li, D.Z., Gao, L.M., Li, H.T. & Moller, M. (2008). Genetic diversity within and among populations
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Sica, M., Gamba, G., Montieri, S., Gaudio, L. & Aceto, S. (2005). ISSR markers show differentiation
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Tan, F.X., Huang, Y.L., Ge, X.J., Su, G.H., Ni, X.W. & Shi, S.H. (2005). Population genetic structure and
conservation implications of Ceriops decandra in Malay Peninsula and North Australia. Aquatic
Botany 81: 175-188.
Tchin, B.L., Ho, W.S., Pang, S.L. & Ismail, J. (2011). Gene-associated single nucleotide polymorphism
(SNP) in cinnamate 4-hydroxylase (C4H) and cinnamy alcohol dehydrogenase (CAD) genes from
Acacia mangium superbulk trees. Biotechnology 10(4): 303-315.
Thumma, B.R., Nolan, M.F., Evans, R. & Moran, G.F. (1995). Polymorphisms in Cinnamoyl CoA
reductase (CCR) are associated with variation in microfibril angle in Eucalyptus spp. Genetics 171:
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o
EFFECTS OF LENGTH OF SOAKING IN 100 C WATER AND EMS ON GERMINATION OF
Neolamarckia cadamba AND Leucaena leucocephala SEEDS.
1*
1*
2
3
Zayed, M.Z., Ho, W.S., Fasihuddin, B. A. and Pang, S.L.
1
Forest Genomics and Informatics Laboratory (fGiL), Department of Molecular Biology
Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia
Sarawak, 94300, Kota Samarahan, Sarawak
3
Applied Forest Science and Industry Development (AFSID), Sarawak Forestry Corporation,
93250 Kuching, Sarawak
2
*Emails: zaky_tree@yahoo.com, wsho@frst.unimas.my.
Abstract
o
A study was conducted to determine the effects of length of soaking in 100 C water and EMS on the
germination of Neolamarckia cadamba and Leucaena leucocephala seeds. The seeds were exposed to
o
one of the three treatments: (1) untreated (control); (2) soaking in 100 C water for 20 s followed by
soaking in water for 24 hours and 48 hours at room temperature to assess the dormancy period (CDP),
cumulative germination (%) (CGP), mean daily germination (%) (MDG) and co-efficient velocity of
o
germination (CVG) in N. cadamba and L. leucocephala. Results showed that soaking in 100 C water for
20 s and then soaked in water for 48 hours had the highest speed of germination, higher cumulative
germination (%) (CGP) and shortened period of complete dormancy over soaking duration of 24 hours or
no soaking of seeds before planting. The germination speed of seeds increased with increasing soaking
for the durations of 0, 24 and 48 hours. Production of the first true leaf was earliest with 48 hours soaking
and least with the seeds that were not pre-soaked in water. For EMS study, three different concentrations
(i.e., 0.1, 0.3 and 0.6%) of ethyl methane sulphonate (EMS) were used to treat N. cadamba and L.
leucocephala seeds to assess seed germination percentage, lethality, seedling height and survival
percentage after 6 months of planting. It was noted that the germination percentage, survivability and
seedling height were decreased; whereas lethality increased with increasing mutagenic doses. Higher
lethality over control was observed at 0.6% EMS for N. cadamba (57.1%) and L.leucocephala (75.6%).
So, the effect of chemical mutagenesis on seedling with 0.6% EMS treatment was much more beneficial
as compared to 0.1% and 0.3% EMS. The effectiveness of the three treatments on N. cadamba and L.
leucocephala was ranked as 0.6>0.3>0.1.
Keywords: Neolamarckia cadamba, Leucaena leucocephala, Germination speed, EMS, Survival
1. INTRODUCTION
Neolamarckia cadamba or locally known as Kelampayan belongs to family, Rubiaceae. It is a fast growing
tree and suitable for reforestation in watersheds and eroded areas and for windbreaks in agroforestry
systems. It is stated to be one of the most frequently planted trees in the tropics and suitable for
ornamental use and agroforestry practices. Germination takes place after 2-3 weeks and when the
seedlings are 8-12 weeks old, they are transplanted to nursery beds or plastic bags. Leucaena
leucocephala Lam or locally known as lamtoro belongs to family Leguminosae It is a fast growing tree.
The common type is widespread and shrubby. It is also known as white lead tree, leucaena. It is a
perennial leguminous tree native to Central America with a wide distribution in the tropics and subtropics,
and successfully suitable for growing in marginal and submrginal lands with a wide assortment of uses. It
is cultivated for multipurpose, e.g. forage or fodder (Lefroy et al. 1992), lumber, Germination rates are
commonly 50 to 98% for fresh seeds (Daguma et al, 1988; NAS 1984). Scarified seeds germinate 6 to 10
days after sowing; unscarified seeds germinate 6 to 60 days after sowing (Parrotta 1992).
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Mutation breeding has been widely used for the improvement of plant characters in various species. It is a
powerful and effective tool in the hands of plant breeders having narrow genetic base (Micke, 1988). The
prime strategy in mutation breeding has been to upgrade the well-adapted plant varieties by altering one
or two major agronomic metrical traits which limit their productivity or enhance their quality. Many chemical
mutagens have been employed for obtaining useful mutants in various crop species (Singh and Singh,
2001). However the various workers emphasizes that artificial induction of mutation by ethyl methane
sulphonate (EMS) provide tool to overcome the limitations of variability in plants especially Carnation and
induces specific improvement without disturbing their better attributes (Mensah and Obadoni, 2007; Islam,
2010; Roychowdhury and Tah, 2011). It might be considered that, this chemical induced growth
abnormalities were mainly due to cell death and suppression of mitosis at different exposures. EMS is a
common alkylating agent; however, this chemical has also proven as mutagens to induce genetic
variability.
Thus, it becomes an important tool to enhance agronomic traits of crop plants. The role of mutation
breeding in increasing the genetic variability for the desired traits in various crop plants have been proven
beyond doubt by a number of scientists (Tah, 2006; Adamu and Aliyu, 2007; Khan and Goyal, 2009;
Kozgar et al., 2011; Mostafa, 2011). Several factors such as properties of mutagens, duration of
treatment, pH, pre and post- treatment, temperature and oxygen concentrations and etc. influence the
effect of mutagens.
The present works was aimed at finding a cost effective, easily administrable and suitable methods for
improving, germination speed and to obtain practical knowledge about the effectiveness of EMS such as
to estimate the mutagen doses effective to reduce the growth in Neolamarckia cadamba and Leucaena
leucocephala.
2. MATERIALS AND METHODS
2.1 Plant materials
Leucaena leucocephala (Lam.) de Wit and Neolamarckia cadamba (Roxb.) Miq. were highest used in this
study. Leucaena leucocephala and Neolamarckia cadamba seeds were obtained from the nursery of
Forestry and Wood Technology Department, Faculty of Agriculture, Alexandria University, Egypt and the
Bank, Sarawak Forestry Corporation, Sarawak, respectively.
2.2 Experimental design
For EMS study, the completely randomized design containing four replicates was used each replicate
o
contained four treatments and 120 seeds. For length of soaking in 100 C water study the completely
randomized design containing three replicates was used each replicate contained three treatments and 30
seeds for each species.
The germination test was carried out according to the methods outlined in the "International Rules for
Seed Testing Rules " published by the International Seed Testing Association ISTA. The seeds were
o
exposed to one of three treatments: (1) untreated (control); (2) soaking in 100 C water for 20 s followed by
soaking in water for 24 hours and 48 hours at room temperature. Three replicates were used each with 30
seeds. 30 seeds were soaked in water for one day, 30 seeds were soaked in water for 2 days and 30
seeds for the control were not given any treatment for each species after that the treatments were planted
in trays of 50 holes and contained sand and compost (3:1). The trays were then watered for 25 days.
Records consisted of daily count of germinated seeds; complete dormancy period, (CDP), mean daily
germination, (MDG). Seed were scored as having germinated when the radical length is 1.5 mm long.
Other parameters recorded were Days to the appearance of first true leaf and coefficient velocity of
germination (CVG) which was calculated using the formula outlined by Kotowski (1978) as stated below.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Co-efficient velocity of germination (CVG)
1
CVG=__________________________________________
N1T1+N2T2+N3T3+---------NXTX
Where:
N= are the number of seed germinating within consecutive interval of time.
T= the time between the beginning and the end of the particular interval of measurement
(Kotowski 1978).
CDP= number of days from sowing to the start of germination.
MDG= final germination period divided by number of days required to attain the value.
CGP= Cumulative germination percentage until 9 days after planting
2.3 The mutagenic treatments
For breeding purposes, it seems necessary to induce artificially new genetic variability by means of the
very effective mutagens, such as ethyl methanesulphonate (EMS), Seeds were pre-soak in distilled water
for 1 hours, air dried and then soaked in the different EMS concentrations in Petri-dishes for 3 hours.
o
Laboratory temperature during the treatment was 21 C, whereas the relative humidity is about 59 %.
Three different concentrations of EMS were used, i.e.: 0 (distilled water), 0.1, 0.3 and 0.6%. After the
o
mutagen treatment, seeds were washed in distilled water for 15 min; air dried and then soaked 100 C
o
water for 20 sec followed by soaking for 24 – 48 hours in 25 C water. The treated seeds were then plantd
in seed beds for recording the germination behavior such as germination percentage, survival after
germination and maturation, and lethality over control (LOC). The germination percentage per treatment
st
with four replicates was counted and recorded on 21 day after seed sowing. Percent inhibition or
stimulation over control (lethality over control, LOC) were calculated as [Control-Treated/Control] X 100.
2.4 Plant height
Plant height was measured in cms from the soil surface to the top of the longest branch. Measurements
were taken after 6 months after planting.
3. RESULTS & DISCUSSION
o
3.1 Effects of length of soaking in 100 C water at room temperature in N. cadamba and L.
leucocephala.
The results obtained from the complete dormancy period, the cumulative germination percentage until
nine days after planting, the mean daily germination, the co-efficient velocity of germination, the days to
o
50% germination and the Days to first true leaf of these seeds which soaked in 100 C water for 20 s and
then for durations of 0, 24 and 48 hours are summarized in Tables (1 and 2). The results showed that the
parameters were significantly differed and affected. The complete dormancy period, (CDP) of these seeds
which soaked for durations of 0, 24 and 48 hours was 19.6%, 14.3% and 6.3% for N. cadamba and
12.3%, 10.3% and 4.3% for L. leucocephala, respectively. The cumulative germination percentage until
nine days after planting was 0%, 0% and 92.22% for N. cadamba and 0%, 0% and 95.9% for L.
leucocephala, respectively. The different treatments showed substantial variation in germination
percentage at nine days after sowing (Tables 1 and 2). Seeds soaked for duration of 48 hours showed
germination percentage of 92.22% and 95.9% (Figure 1), while seeds soaked for duration of 24 hours and
the control both had no germination at 9 days after sowing for N. cadamba and L. leucocephala,
respectively. While various pretreatment methods have been advocated to reduce dormancy and hasten
germination, no single pretreatment technique has been found to be equally effective for all seeds in both
o
species. The soaking in 100 C water for 20 sec and then soaked in water for duration of 48 hours had the
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highest speed of germination, higher cumulative germination (%) (CGP) and shortened period of complete
dormancy over soaking duration of 24 hours. or no soaking of seeds before planting (Tables 1 and 2) for
N. cadamba and L. leucocephala, so, the germination speed of seeds increased with increasing soaking
for durations of 0, 24 and 48 hours.
Table 1 Effects of length of soaking the seeds in water at room temperature on the dormancy period
(CDP), cumulative germination (%) (CGP), mean daily germination (%) (MDG) and co-efficient velocity of
germination (CVG) in Neolamarkia cadamba.
Duration
of
soaking in water
(hours)
0
CDP
CGP
MDG
CVG
Days to 50%
germination.
19.6
0
1.04
0.05
22.7
Days to
true leaf.
50%
31.3
24
14.3
0
1.33
0.06
17
19.7
48
6.3
92.22
2.67
0.11
8
13.7
first
Table 2 Effects of length of soaking the seeds in water at room temperature on the dormancy period
(CDP), cumulative germination (%) (CGP), mean daily germination (%) (MDG) and co-efficient velocity of
germination (CVG) in Leucaena leucocephala.
Duration
of
soaking in water
(hours)
0
CDP
CGP
MDG
CVG
Days to 50%
germination.
Days to
true leaf.
12.3
0
1
0.05
20.5
28.5
24
10.3
0
0.8
0.06
15.5
19
48
4.3
95.9
0.5
0.13
7
13.5
Germination of Leucaena leucocephala seeds
first
Germination of Neolamarckia cadamba seeds
Figure 1 Germination of Leucaena leucocephala and Neolamarckia cadamba seeds.
Production of the first true leaf was earliest with 48 hours soaking and least with the seeds that were not
presoaked. This result was not in agreement for Neolamarkia cadamba as reported by (Soerianegara and
Lemmens, 1993). They found that the germination of Neolamarckia cadamba seed takes place after 2-3
weeks, because of their small size, the seeds are mixed with fine sand (1:10) and sown in seedbeds but
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they did not use pre-soaking in water. In contrast, Argel and Paton (1999) reported that L. leucocephala
o
o
seeds were successfully scarified and then immersed in 60 C water for 15 – 30 min or at 80 – 100 C for 1
– 3 min without affecting seed viability and seedling vigor, but this was not as effective as the procedure
o
reported by Gosling et al. (1995), which entailed soaking L. leucocephala seeds in 100 C water for 4 s,
o
resulting in 82% germination. Maher et al. (2011) showed that soaking the seeds in 70 C water was the
most effective pre-sowing treatment in L. leucocephala compared to the control or the sandpaper
o
scarification and the use of 70 C water for 12 – 24 min increased seed germination of L. leucocephala to
66–68%, but this was not as effective as the procedure reported by Gosling et al. (1995).
Hot water treatments have been reported to enhance germination of hard coated seeds by elevating water
and O2 permeability of the testa by Aydin and Uzun (2001). The treatment time exerts significant effect on
seed germination according to Awodola (1994). Duguma et al. (1988) observed higher germination
percentage in the seeds treated with hot water for 5 min in Leucaena leucocephala and Acacia nilotica.
This result was in agreements with the report of Tomlinson et al. (2000) that seed dormancy resulting from
an impermeable seed coat may be overcome by peeling off the coat. Germination must have occurred as
a result of the partial exposure of the cotyledons of the seeds which permits the process of hydrolysis
whereby hormones such as auxins and ethylene which could increase nucleic acid metabolism and
protein synthesis are released (Uwaegbute, 1996) Similar to other forage legume species (Van Assche et
al. 2003), the hard seed coats of many forest species have evolved to withstand unfavorable conditions
such as intense heat from sunlight, dispersing animals, severe drought, and physical damage.
Germination requires rupture of the seed coat and subsequent absorption of water by the seed (Freas and
Kemp 1983; Baskin and Baskin 1998; Silvertown 1999).
3.2 Effects of EMS treatments in N. cadamba and L. leucocephala.
The seed germination in control was 80.83% and 50% for N. cadamba and L.leucocephala, respectively
(Figure 2) (Tables 3 and 4). It decreased with an increase in the concentration of EMS. The germination
percentage recorded was the highest (75%) in 0.1%EMS and the lowest (36.66%) in 0.6%EMS for N.
cadamba and also a gradual decrease in germination percentage was observed with an increase in the
concentration of EMS for L.leucocephala. It was maximum (18.33%) in 0.1%EMS and minimum (9.16%)
in 0.6% EMS concentration. Decrease in the germination percentage with EMS was recorded in Plantago
ovata by Dube (1981) and Sareen and Koul (1999). Sen (1978) attributed the decrease in germination
percentage of Psoralia cordifolia to the genetic damage like chromosomal aberrations. Cheema and Atta
(2003) suggested that reduction in germination percentageof rice might be due to the sterility of seeds
induced by mutagens. In N. cadamba and L.leucocephala it seems that the cell cycle arrested by higher
doses of physical and chemical mutagens might have been resulted in the decrease in seed germination.
Table 3 Germination percentage of the treated seeds for N. Cadamba
Treatments
Total
seed
Survival
seedling
after
germination
Germination
percentage
(%)
Survival
seedling
after 6
months
Survival
percentage
(%)
Seedling
height(cm)
Lethality
over
control
(%)
Control
120
97
80.83
42
35
80
0
0.1
120
90
75
35
29.16
64.79
16.66
0.3
120
70
58.33
25
20.83
51.27
40.47
0.6
120
44
36.66
18
15
37.5
57.14
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Table 4 Germination percentage of the treated seeds for L.leucocephala
Treatments
Total
seed
Survival
seedling
after
germination
Germination
percentage
(%)
Survival
seedling
after
6
months
Survival
percentage
(%)
Seedling
height(cm)
Lethality
over
control
(%)
Control
120
60
50
45
37.50
149
0
0.1
120
22
18.33
22
18.33
138
51.11
0.3
120
15
12.50
15
12.50
127.72
66.66
0.6
120
11
9.16
11
9.16
126.36
75.55
Seedlings of Leucaena leucocephala
Seedlings of Neolamarckia cadamba
Figure 2 Seedlings of Leucaena leucocephala and Neolamarkia cadamba
The seedling height in control plants was 80 cm and 149 cm for N. cadamba and L.leucocephala,
respectively (Tables 3 and 4). It was reduced with the corresponding increase in the concentration of
EMS, being highest (64.79 cm) in 0.1% EMS and lowest (37.5 cm) in 0.6% EMS for N. cadamba and also
the gradual decrease in seedling height was recorded with an increase in the concentration of EMS for
L.leucocephala. The highest seedling height (138 cm) was observed in 0.1% EMS while the lowest
(126.36 cm) was noted in 0.6% EMS. Khalatkar and Bhatia (1976) in Barley and Katosh and Massar
(1992) and Wang et al. (1995) in rice have reported decrease in seedling height due to the action of
gamma rays or EMS. Cheema and Atta (2003) have stated that phytochromes responsible for normal
growth might have affected pre-synthetic level of DNA-RNA and reduced the seedling height in rice. In the
present investigation it seems probable that possible interference of irradiation and chemical mutagen
causing damage in the synthesis of new DNA may have lead to the inhibition of seedling height in N.
cadamba and L.leucocephala.
The survival of plants in control was 35.0% and 37.50% for N. cadamba and L.leucocephala, respectively
(Tables 3and 4). However, it was decreased with the increasing doses of EMS. It was highest (29.16%
and 18.33%) in 0.1% EMS and lowest (15.0% and 9.16%) in 0.6% EMS for N. Cadamba and
L.leucocephala, respectively. The lethality was highest (57.14 and 75.55) in 0.6% EMS and lowest
(16.66% and 51.11%) in 0.1% EMS for N. cadamba and L.leucocephala, respectively. Dube (2011)
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observed reduction in the survival percentage induced by gamma rays was less as compared to that by
EMS in alone treatments. However, the drastic reduction in the survival percentage was recorded in
gamma rays followed by EMS combination treatments in Cyamopsis tetragonoloba L. variety Sharada.
Sree Ramulu (1970) also observed more drastic reduction in the percentage of germination and survival in
Sorghum in gamma rays followed by EMS combination treatments than their alone treatments. Sayed et
al. (1973) reported increased lethality with EMS treatment in Hordeum sp. Din et al (2003) observed high
reduction in the mean values of survival percentage in the treated seeds of three wheat varieties with
various doses of gamma irradiation. In N. cadamba and L.leucocephala, it seems that, the genetic
damages like chromosomal aberrations and the physiological imbalance in the cells caused by the
mutagens might have decreased the survival percentage.
4. CONCLUSION
o
To overcome seed dormancy in L. leucocephala and N. cadamba seeds. The soaking in 100 C water for
20 sec and then soaked for 48 hours is recommended. Our results will assist forestry nurseries to reduce
the time and labor needed to overcome seed dormancy, especially for these two species. Knowledge of
the most effective and lowest-cost pre-sowing treatment can be used to increase seedling viability and
enhance field establishment for greater production and profit as well. It is advocated that the effect of EMS
treatments on germination, seedling height and survivability with 0.6% EMS treatment was much more
beneficial as compared to 0.1%and 0.3% EMS., The effectiveness of the three treatments on N. cadamba
and L. leucocephala was ranked as 0.6>0.3>0.1. Hence, these treatments could be used as a means of
improving the genetic background of N. cadamba and L. leucocephala with desirable alleles for further
improvement in the near future.
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EFFECTS OF DIFFERENT INOCULATION CONCENTRATION OF CHLORELLA SP. ON SEA BASS,
LATES CALCARIFER WASTEWATER PHYTOREMEDIATION
Nora’aini Ali , Siti Hajar Abdul Hamid , Fathurrahman Lananan ,
1,2
Ahmad Jusoh
1*
1
1
1
Department of Engineering Science, Faculty of Science and Technology, Universiti Malaysia
Terengganu, 21030 Terengganu (MALAYSIA)
2
Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Terengganu (MALAYSIA)
*E-mail: noraaini@umt.edu.m
Abstract
Phytoremediation is a process that utilized phytoplankton such as microalgae to treat the nutrient pollution
in the aquaculture wastewater. Microalgae has high potential to absorb inorganic nutrients from
wastewater makes them a useful phytoremediation tool in wastewater treatment process. Microalgae
genus Chlorella sp. at different ratio of microalgae to wastewater in terms of inoculation volume is used to
determine its kinetic growth at the same time their nutrient removal efficiency specifically total ammonia
nitrogen (TAN) and total phosphorus (TP). Silver Barramundi, Lates calcarifer wastewater was inoculated
with microalgal culture with different concentration of 10%, 15%, 20%, 25%, 35% and 40% (v/v) in 14days batch treatment period at room temperature (25 ± 2 °C). Results indicated that the best nutrient
removal for TAN and TP was in the range of 15 - 20% (v/v) Chlorella sp. concentration yielding over 90%
nutrient removal with final effluent of 0.08 mg/L and 0.01mg/L, respectively. Microalgal growth kinetics
fitting the Monod models and the nutrient removal fitted with the first order kinetics. Microalgae are highly
potential to treat aquaculture wastewater.
Keywords: Chlorella, batch phytoremediation, total ammonia nitrogen, total phosphorus
1. INTRODUCTION
Phytoremediation is a biological treatment utilizing marine microalgae Chlorella sp. Microalgae are one of
the most important bioresource that are currently receiving a lot of attention due to a multiplicity of
reactions (Rawat et al., 2010). The use of microalgae is desirable since they are able to serve a multiple
role such as bioremediation as well as generating biomass for biofuel production with concomitant carbon
sequestration (Mulbry et al., 2008; Olguin, 2003). In addition, wastewater remediation by microalgae is an
eco-friendly process with no secondary pollution as long as the biomass produced is reused and allows
efficient nutrient recycling (Mulbry et al., 2008; Pizarro et al., 2006; Munoz and Guieysse, 2008). The
release of agricultural, industrial and municipal wastewater poses serious environmental challenges to the
receiving water bodies (Arora and Saxena, 2005; de-Bashan and Bashan, 2010). This research is
motivated by the increasing interest in application of biotechnology and the implementation of
environmentally friendly tools in treating wastewater (Concas et al., 2010; Sato et al., 2010; Yoshimoto et
al., 2005).
Chlorella sp. was classified as a biological tool in wastewater treatment to reduce the present of nutrient
content (Sydney et al., 2010). At the same time, biomass of Chlorella sp. which is the by-product of the
treatment process could be marketed as high-value products (Brennan and Owende, 2010). In addition,
treatment of phytoremediation could also help in the uptake of various heavy metals and greenhouse
gasses present in the wastewater (Wang et al., 2009). This would contribute to overcome environmentally
related problem of domestic wastewater reported around the world (Listowski et al., 2011; de Godos et al.,
2009; Rawat et al., 2010).
The aim of this study was to determine the possibilities of microalgae Chlorella sp. in treating aquaculture
wastewater. This was implemented through monitoring of TP and TAN removal performance in
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aquaculture wastewater by phytoremediation using Chlorella sp.. The most suitable inoculation
concentration in batch mode and the growth kinetics of Chlorella sp. throughout the treatment period were
investigated.
2. MATERIALS AND METHODS
2.1 Cultivation of Chlorella sp.
The pure strain of green algae Chlorella sp. was supplied by Institute of Tropical Aquaculture
(AKUATROP) of University Malaysia Terengganu. It was cultivated for 8 days to produce secondary
culture for up-scaling. The medium used for culture was Conway media with autoclaved and filtered
seawater. Normal air filtered with 40 µm air filter was provided as sterile aeration to prevent any bacterial
contamination. Microalgae cultures were maintained at room temperature of about 25 ± 2 ˚C under a light
intensity of 4100 lux from white fluorescent light for 24 h photoperiod.
2.2 Maintenance of fish culture
The aquaculture wastewater was supplied from the culture of Lates calcarifer also known as Barramundi
or Silver Sea bass as livestock. Lates calcarifer was chosen because of their ability to survive in water at
various salinity levels, ranging from 0 ppt up to 30 ppt. In this study, 30 fishes were reared in a tank under
controlled temperature of 27 ± 2 °C with continuous aeration.
The same type of feed was given based on 3% of body weight to control phosphorus and nitrogen content
in the effluent. Nutrients content in the effluent were considered maximum on the fourth day of rearing
period. At this time, the wastewater produced was channelled for phytoremediation treatment by
microalgae.
2.3 Maintenance of fish culture
The aquaculture wastewater was supplied from the culture of Lates calcarifer also known as Barramundi
or Silver Sea bass as livestock. Lates calcarifer was chosen because of their ability to survive in water at
various salinity levels, ranging from 0 ppt up to 30 ppt. In this study, 30 fishes were reared in a tank under
controlled temperature of 27 ± 2 °C with continuous aeration.
The same type of feed was given based on 3% of body weight to control phosphorus and nitrogen content
in the effluent. Nutrients content in the effluent were considered maximum on the fourth day of rearing
period. At this time, the wastewater produced was channelled for phytoremediation treatment by
microalgae.
2.4 Microalgae phytoremediation
The treatment of wastewater was conducted in batch mode using 5000 ml flasks. This treatment
commenced with the inoculation of Chlorella sp. culture into the wastewater. Six samples with different
concentration of Chlorella sp. cultures i.e., 10%, 15%, 20%, 25%, 35%, and 40% (v/v), were examined in
this study. Illumination was provided continuously from the top of the flask. The experiments were
conducted at controlled room temperature of 25 ± 2 °C for 14 days.
2.5 Nutrient and growth analysis
Three parameters of total phosphate (TP), total ammonia nitrogen (TAN) and biomass of Chlorella sp.
concentrations were analyzed. The changes of concentrations in the nutrient content and Chlorella sp.
biomass were monitored daily for a period of 14 days. Nutrient concentrations (phosphate and nitrogen)
were the main concerned for evaluating removal efficiency in accordance to the growth of Chlorella sp.
biomass as kinetic study.
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2.5.1 Chlorella sp. biomass concentration analysis
Analysis of Chlorella sp. biomass concentration was performed simultaneously with nutrient
concentrations. Daily samples withdrawn from flasks were centrifuged at 4 °C to separate algae biomass
from water. Two-hundred-milliliters sample from each flask were poured into the four 50 ml centrifuge
tubes and then they were centrifuged at 5000-6000 rpm for about 30 min to separate the Chlorella sp.
cells from water. Then, 10 mL sample of algal suspension was again centrifuged at 3000 rpm and the
supernatant was discarded. The algae were suspended in 3 mL methanol and heated for about 5 min in a
water bath. The samples were cooled to room temperature and then the volume was made up to 5 mL by
adding methanol. The Chlorophyll a concentration in the extract was determined using the Equation 1
(Becker, 1994) with the reading of absorption (A) of the pigment extract in a spectrophotometer at the
given wavelength, (650nm and 665nm) against a solvent blank.
Chlorophyll a (mg/L) = (16.5 × A665) – (8.3 × A650)
…… (1)
2.5.2 TAN and TP concentration analysis
Analysis of TP and TAN concentrations were carried out by using HACH DR2400 kit. A 200 mL samples
were daily withdrawn from the flasks. Then, the samples were centrifuged at 5000-6000 rpm to separate
algae in order to obtain a clear supernatant. Measurements of TP and TAN were executed in clear
supernatant by colorimetric methods i.e., Ascorbic Acid Method and Salicylic Acid Method, respectively.
These methods were adapted from Standard Method for Examination of Water and Wastewater (APHA,
2005). They were equivalent to USEPA Method 365.2 and Standard Method 4500-PE for wastewater.
3. RESULTS AND DISCUSSION
3.1 Comparative study of TP and TAN removal at different Chlorella sp. Inoculation concentrations.
The use of several species of microalgae as a tertiary wastewater treatment was proposed over a decade
ago, and various potential treatments continue to be evaluated today (de-Bashan et al., 2002). The
underlying assumption is that the microalgae will transform some of the contaminants to non-hazardous
materials and then the treated water can be reused or safely discharged (Oswald, 1992). Figure 1 shows
the growth kinetics of Chlorella sp., TP and TAN concentrations were plotted for comparative studies
between different inoculation concentrations used. The growth kinetics of Chlorella sp. suited the Monod
models in line with the growth phases such as lag, log, stationary and declining phases. The TP and TAN
reductions followed the First Order Kinetics where removal percentage continued to increase
exponentially until reaching an asymptote where the removal percentage was higher than 99%. Once the
TP and TAN concentrations mostly depleted, Chlorella sp. growth began to decline. This was also
supported by Droop-based model suggested by Bougaran et al. (2010). The interaction of TP and TAN
concentrations on the growth of microalgae should be considered at the acquisition level rather than at the
assembly level. Nutrient present in the wastewater (extracellular region) affect the Chlorella sp. growth
more than those in the intracellular region. Thus, this phenomenon indicated that the present of these
nutrients in wastewater contributed significantly on the growth of Chlorella sp.
In order to further investigate the effect of different Chlorella sp. inoculation concentrations on TP and
TAN removal, correlation and regression analysis were performed specifically on Day 7 of the treatment
period since the most obvious reduction occurred at this point. Figure 2 shows the remaining TP and TAN
concentrations logarithmically decreased with the increased of Chlorella sp. inoculation concentration.
Therefore, a higher inoculation concentration of Chlorella sp. would reduce the remaining nutrient
concentrations.
Figure 3 shows the correlation between treatment periods required to achieve the maximum reduction and
different inoculation concentrations of Chlorella sp.. The increased of Chlorella sp. inoculation
concentration contributed to a shorter treatment period required to reach the maximum TP and TAN
2
removal. A very strong negatively linear relationship with R of 0.9742 was established for TP, whereas a
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2
similar pattern of strong relationship with R of 0.7423 for TAN removal with the increase of Chlorella sp.
inoculation concentration. The higher inoculation concentration contributed to a higher readily available
biomass which produces higher initial nutrient reduction. After that, Chlorella sp. would undergo
subsequent mitotic cell division of growth to increase its biomass concentration by utilizing the remaining
nutrients exist in the wastewater (de-Bashan and Bashan, 2010).
(a)
(b)
(c)
(d)
(e)
(f)
Figure 1 Growth performance of Chlorella sp., TP and TAN reduction for 14 days treatment period for (a)
10% , (b) 15%, (c) 20%, (d) 25%, (e) 35% and (f) 40% (v/v) biomass concentration. ( - Chlorella sp.
biomass, - total phosphorus and - total ammonia nitrogen)
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Figure 2 TAN and TP remaining concentrations after Day 7 of the treatment period at different Chlorella
sp. inoculation concentration.
Figure 3 Treatment period required to reach maximum TAN and TP reduction at different Chlorella sp.
inoculation concentration.
3.2 Microalgal growth
Microalgae can be effectively used to remove high amount of nutrient because they require large quantity
of nitrogen and phosphorus for protein (45-60% of microalgae dry weight), nucleic acid and phospholipids
synthesis (Rawat et al., 2011). Batch phytoremediation on aquaculture wastewater utilizing Chlorella sp.
was successfully performed. During the study, the concentrations of Chlorella sp. used for
phytoremediation was varied at 10%, 15%, 20%, 25%, 35% and 40% (v/v). All samples were treated for a
period of 14 days after Chlorella sp. inoculation.
Different concentrations of Chlorella sp. have shown a quite similar growth pattern and suited the Monod
Model. As shown in Table 1, the maximum growth rate constant and half saturation constant of TP show
reduction with the increase of inoculation concentrations. However, there was no clear trend between
kinetics coefficients of TAN and different Chlorella sp. inoculation concentrations. After the inoculation,
reduction of microalgae growth occurred within a short period of about 2 days known as lag phase. In this
phase, the reduction of growth occurred because microalgae were still adapting with the new
environment. When microalgae culture inoculated into the aquaculture wastewater, other pollutants
present may also contribute to the reduction in its growth.
The microalgae growth rate rebounded back once they were successfully adapted to the new
environment. This stage was known as log phase. This is indicated by the rapid increase in the biomass
as depicted with exponential growth of microalgae. Thus, microalgae utilized the TP and TAN that present
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Table 1 Kinetics coefficient of Chlorella sp. growth using Monod Model for TP and TAN removal at
different inoculation size.
Kinetics coefficient (TP)
Chlorella sp.
(% v/v)
10
15
20
25
35
40
Kinetics coefficient (TAN)
Max. growth rate
constant, µm
(1/day)
Half saturation
constant, Ks
(mg/L)
Max. growth rate
constant, µm
(1/day)
Half saturation
constant, Ks
(mg/L)
0.521
0.525
0.560
0.481
0.480
0.471
0.354
0.252
0.227
0.191
0.166
0.164
0.5048
0.4789
0.4814
0.4774
0.5147
0.4164
1.23 x 10
0.0751
0.703
0.0653
0.0617
0.0622
-6
in aquaculture wastewater as source of food contributing to the reduction of nutrient. As shown in Table 2,
the characteristics of log phase were accurately quantified in accordance to the First Order Kinetics.
Reduction rate of TP was recorded as the highest within the range of 15 – 35% (v/v) whereas for TAN at
the range of 10 – 20 % (v/v) inoculation concentrations. Thus, the most suitable concentration of Chlorella
sp. inoculation that produced the optimum reduction for both nutrients was determined in the range of 1520% (v/v).
Table 2 First order kinetics equation and reduction rate of TP and TAN for different inoculation
concentration of Chlorella sp.
Total Phosphorus (TP)
Chlorella sp.
(% v/v)
10
15
20
25
35
40
First order kinetics
equation
-kt
C = C0 e
-0.3x
2.1359e
-0.4x
4.0063 e
-0.355x
1.6260 e
-0.379x
3.2414 e
-0.408x
2.359 e
-0.377x
2.079 e
Reduction rate, k
-1 -1
(mg L d )
0.3
0.4
0.355
0.379
0.408
0.377
Total Ammonia Nitrogen (TAN)
First order kinetics
equation
-kt
C = C0 e
-0.357x
0.8262 e
-0.361x
0.7871 e
-0.354x
0.6837 e
-0.347x
0.6289 e
-0.339x
0.5152 e
-0.321x
0.4351 e
Reduction rate, k
-1 -1
(mg L d )
0.357
0.361
0.354
0.347
0.339
0.321
-
3.3 Total phosphorus removal (PO₄³ -P)
The initial concentrations of TP for all treatments were maintained at 2.50 ± 0.05 mg/L orthophosphate,
PO₄³¯ -P in order to determine the percentage of TP removal for a period of 14 days. The reading for TP
concentration was taken daily to observe its reduction in the effluent.
From Table 3, the TP concentration in each treatment decreased sharply at early period of Chlorella sp.
growth. This is due to the rapid increase of Chlorella sp. biomass as indicated in log phase. All samples
achieved the high percentage of TP removal, 99.6% which indicate that the effluent contain 0.01 mg/L
PO43- except for sample of 10% (v/v) Chlorella sp.. The concentration of 0.01 mg/L PO43- in effluent is
being considered as a safe level for effluent to be released to water body and being used for water
recycled in aquaculture. The earliest sample that achieved the maximum removal percentage of more
than 99% of TP is the sample with 35% (v/v) Chlorella sp., followed by the samples with 20% and 40%
(v/v) Chlorella sp., respectively. Next is the sample with 25% (v/v) Chlorella sp. and the last one is the
sample with 15% (v/v) of Chlorella sp..
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Ino. (% v/v)
Table 3 Percentages of TP removal over 14 days of treatment.
10
15
20
25
35
40
1
53.0
12.2
43.5
26.5
20.9
18.2
2
55.7
28.1
59.7
39.5
31.6
33.2
3
64.8
42.7
70.8
55.7
72.7
69.6
4
71.5
60.7
90.1
64.0
78.3
83.0
Percentage of TP reduction in effluent (%)
Treatment period (D)
5
6
7
8
9
10
84.2 88.5 90.9 91.7 90.5 95.3
68.8 82.6 87.4 96.0 95.3 95.7
91.7 94.5 94.9 96.0 97.2 99.6
71.5
83
87.7 91.3 96.4 98.4
82.2 91.3 95.7 96.4 99.6 99.6
87.0 91.7 96.0 96.8 98.4 99.6
11
96.4
97.2
98.8
99.6
99.2
98.8
12
98.8
99.6
98.8
98.0
98.8
98.0
13
98.8
99.2
99.2
98.8
99.6
99.2
14
99.2
99.2
98.8
98.8
98
97.6
TP in the energy and reaccumulation of phosphate into polyphosphate. The energy generated in this
transformation process was used by Chlorella sp. for its growth. However, phosphorus concentration
above 6 mg/L will lead to explosive growth of algae. It has become a global problem where ponds and
lakes gradually turn into marshes. Algae are considered to be a biological means of phosphorus removal
which helps in detecting a potential eutrophication. Since phosphorus is ecologically significant in algal
productivity, its removal from aquatic bodies is essential to prevent the occurrence of eutrophication
problems.
+
¯
3.4 Total ammonia nitrogen removal (NH3, NH4 , NO2 )
During the experiment, it was found that microalgae started to degrade themselves after all substrate had
been used. When degradation of microalgae occurred, the microalgae would release the nutrients that it
had absorbed. Due to this phenomenon, the removal percentage of TAN had decreased after achieving its
maximum value. Initial TAN concentrations for all 6 aquaculture wastewater samples were approximately
0.80 ± 0.05 mg/L before being inoculated with Chlorella sp. Once the treatment of phytoremediation
completed, the final TAN concentration with 10% (v/v) Chlorella sp. was reduced to 0.12mg/L. As shown
in Table 4, for 15%, 20%, 25%, 35%, 40% (v/v) Chlorella sp., the final TAN concentrations was reduced to
0.11 mg/L, 0.08 mg/L, 0.14 mg/L, 0.2 mg/L and 0.23 mg/L, respectively. Therefore, the optimum
percentage of microalgae for phytoremediation at specified treatment period was found to be in the range
of 15% - 25% (v/v) Chlorella sp. The determination of proper inoculation concentration of Chlorella sp. is
crucially important for the implementation on the real scale aquaculture wastewater treatment.
Ino. (% v/v)
Table 4 The percentages of TAN removal over 14 days of treatment.
10
15
20
25
35
40
1
33.3
27.5
28.1
11.1
10.8
11.9
2
48.6
36.2
50.0
25.9
24.5
56.2
3
55.6
53.6
56.3
50.0
54.4
57.1
4
61.1
68.1
64.1
64.8
70.3
71.4
Percentage of TAN reduction in effluent (%)
Treatment period (D)
5
6
7
8
9
10
73.6 80.6 88.9 94.4 95.8 98.6
71.0 82.6 91.3 95.7 97.1 98.6
76.6 79.7 89.1 95.3 98.5 95.3
72.2 83.3 90.7 96.3 96.3 98.1
80.2 86.1 92.2 96.3 99.9 86.7
76.2 81.0 95.2 85.7 76.2 66.7
11
93.1
92.8
92.1
88.9
78.1
61.9
12
90.3
88.4
89.1
83.3
70.2
45.2
13
83.3
84.1
87.5
74.1
60.3
45.2
14
80.4
82.7
85.5
72.1
58.3
43.5
The final TAN concentration has been reduced to 0.08 mg/L after undergoing the full treatment period. In
the real application of phytoremediation, this value would represent the actual concentration of TAN
before it is disposed into the water body such as river or sea. As recommended by the USEPA Standard,
effluent TAN concentration released must be less than 0.2 mg/L to be considered as environmentally safe
since the effluent discharged did not exert significant impact on the flora and fauna. Thus, this indicated
that the phytoremediation treatment of aquaculture wastewater was an excellent method in removing
nutrient especially TAN and TP.
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4. CONCLUSION
It was shown that the used of microalgae Chlorella sp. in reducing harmful nutrients in the aquaculture
wastewater was suitable and effective. Based on the kinetic coefficients using Monod Models and First
Order Kinetics on the removal of both TAN and TP, the optimum concentration of Chlorella sp. biomass
occurred in the range of 15 – 20% (v/v) that contributed over 99% nutrient removal with final effluent of
0.08 mg/L and 0.01mg/L, respectively. Microalgae Chlorella sp. did exhibit a normal growth patterns with
clear distinction of growth phases. Aquaculture wastewater treatment using microalgae Chlorella sp. also
known as phytoremediation could be considered as a novel innovation of wastewater treatment
technology.
REFERENCES
APHA, 2005. Standard method for examination of water and wastewater. 21st Edition, New York, NY
American Public Health Association.
Arora, A., Saxena, S. (2005). Cultivation of Azolla microphylla biomass on secondary-treated Delhi
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Becker, E.W. (1994). Microalgae Biotechnology and Biotechnology. Cambridge University Press.
Bougaran, G., Bernard, O., Sciandra, A. (2010). Modeling continuous cultures of microalgae colimited by
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Brennan, L., Owende, P. (2010). Biofuels from microalgae – A review of technologies for production,
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Concas, A., Pisu, M., Cao, G. (2010). Novel stimulation model of the solar collector of BIOCOIL
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de Godos, I, Blanco, S., García-Encina, P.A., Becares, E., Munoz, R. (2009). Long-term operation of high
rate algal ponds for the bioremediation of piggery wastewater at high loading rate. Bioresource
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de-Bashan, L.E., Bashan, Y. (2010). Immobilized microalgae for removing pollutants: review of practical
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de-Bashan, L.E., Moreno, M., Hernandez, J-P, Bashan, Y. (2002). Removal of ammonium and
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Listowski, A., Ngo, H.H., Guo, W.S., Vigneswaran, S., Shin, H.S., Moon, H. (2011) Greenhouse gas
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Mulbry, W., Kondrad, S. Pizarro, C., Kebede-Westhead, E. (2008). Treatment of dairy manure effluent
using freshwater algae: algal productivity and recovery of manure nutrient using pilot-scale algal
turf scrubbers. Bioresource Technology 99: 8137-8142.
Munoz, R., Guieysse, B. (2008). Algal-bacterial processes for the treatment of hazardous contaminants: a
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Olguin, E.J. (2003). Phycoremediation: key issues for cost-effective nutrient removal processes.
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Oswald, W.J. (1992). Micro-algae and waste-water treatment. In: Borowitzka, M.A., Borowitzka, L.J.
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Pizarro, C., Mulbry, W., Blersch, D., Kangas, P. (2006). An economic assessment of algal turf scrubber
technology for treatment of dairy manure effluent. Ecological Engineering 26: 321-327.
Rawat, I., Ranjith Kumar, R., Mutanda, T., Bux, F. (2011). Dual role of microalgae: Phycoremediation of
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Sydney, E.B., da Silva, T.E., Tokarski, A., Noval, A.C., de Carvalho, J.C., Woiciecohwski, A.L., Larroche,
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Wang, L., Min, M., Li, Y., Chen, P., Chen, Y., Liu, Y. (2009). Cultivation of green algae Chlorella sp. in
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A RAPID COMPUTER-ASSISTED FTIR METHOD FOR ANALYSIS OF LIGNOCELLULOSIC BIOMASS
Siong Fong Sim*, Murtedza Mohamed, Nurul Aida Lu Mohd Irwan Lu,
Noor Safitri P. Sarman and Siti Nor Sihariddh Samsudin
Universiti Malaysia Sarawak, Faculty of Resource Science and Technology, 94300 Kota Samarahan,
Sarawak, Malaysia;
*Email: sfsim@frst.unimas.my
Abstract
This paper introduces a rapid computer assisted Fourier Transform Infrared (FTIR) method for analysis of
lignocellulosic biomass. Conventionally, the functional features of lignocellulosic components are
characterized using FTIR. This technique is well known for its simplicity in sample preparation and speed
of analysis. Very often, an analyst overlays the spectra for visual interpretation however when there are
too many spectra, it is unlikely to deduce comprehensible inferences. For this reason, the application of
FTIR for lignocellulosic studies typically involves spectra comparisons of 1-3 biomass materials with
limited number of samples. In this paper, we employed the automated peak detection approach for
analysis of 15 different biomasses. This approach allows efficient and simultaneous comparisons of
lignocellulosic components present in various biomass.
Keywords: Lignocellulosic biomass; Fourier Transform Infrared; Peak detection and matching; Principal
Component Analysis
1. INTRODUCTION
Fourier Transform Infrared (FTIR) is commonly used to study the functional characteristics of
lignocellulosic biomass and the changes caused due to different treatments. The spectra offer qualitative
and semi-quantitative information suggesting the presence and absence of lignocellulosic compounds,
and whether the adsorption band due to a specific compound/functional group has increased or reduced
after a treatment process (Li et al. 2010). This technique is well known for its simplicity in sample
preparation and speed of the analysis (Davis and Mauer 2010); one could easily generate hundreds of
spectra within a short period of time, nevertheless the spectra integration process could turn out to be
complicated and exhaustive. For this reason, the application of FTIR for lignocellulosic studies typically
involves spectra comparisons of 1-3 biomass materials with a limited number of samples (Davis and
Mauer 2010; Daffalla et al. 2010; Ding et al. 2012). Very often, the analyst overlays the spectra for visual
interpretation; however, when there are too many spectra, it is unlikely to deduce comprehensible
information. In this paper, we examine the lignocellulosic properties of 15 biomass using FTIR (i.e., 5 raw
biomass and 10 acid and alkali treated biomass), where the computational approach was employed to
analyze the spectra. The adsorption peaks were de-convoluted automatically generating a peak table that
allowed straightforward and efficient comparisons across various lignocellulosic biomasses, and in
addition, it was readily available for multivariate analysis.
2. EXPERIMENTAL
2.1 Sample preparation
The agriculture biomass selected included coconut husk, banana trunks, sago hampas, rice husk, and oil
palm empty fruit bunches. The biomass was washed extensively with running tap water to remove dirt and
cut into smaller pieces (1-2 cm). They were then oven dried at 105 °C for 24 hrs, ground, and stored in
desiccators.
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2.2 Treatment processes
The ground biomass was treated with acid and alkali, respectively. For the acid pretreatment process, the
ground biomass was mixed with 97% of concentrated sulfuric acid (H 2SO4) in a 1:1 ratio, and placed in an
oven at 200 C for 24 hrs. The samples were allowed to cool to room temperature, washed with distilled
water, and soaked in 1% NaHCO3 solution for 1 hr to remove the remaining acid. The samples were then
washed with distilled water and the pH was adjusted to 6.5 with a 1% sodium bicarbonate solution
(NaHCO3). The samples were placed in an oven at 105 C for 24 hrs and were stored in airtight containers
(Fernando et al. 2009).
For alkali pretreatment, the ground biomass was mixed with 0.25 M of sodium hydroxide (NaOH) in a 1:10
ratio and left for 1 hr. The samples were neutralized with hydrochloric acid (HCl) and washed thoroughly
with distilled water. The washed materials were dried at 105 C in an oven for 24 hrs. The products were
stored in airtight containers (Fernando et al. 2009).
2.3 Infrared spectroscopic studies
The functional groups were characterized using FTIR. All spectra were obtained on a Perkin Elmer FTIR
system using the potassium bromide (KBr) disc method with a 2 mg sample in 100 mg KBr. The scanning
range was 4000cm1.
2.4 Data analysis
For each biomass, FTIR spectra of 5 to 6 replicates were obtained. A fully automated approach was used
to handle the large amount of FTIR spectra and to capture the FTIR pattern of various untreated and
treated biomass to demonstrate their qualitative and quantitative variations (Sim and Ting 2012). The
analysis process involved the conversion of FTIR spectra in SP format to Matlab version 7.8. Each
spectrum was a vector of dimensions (3601 × 1) with a scanning rate of 1 cm1. The spectrum was
baseline corrected using asymmetric least squares and subjected to the peak detection and matching
algorithm (Sim and Ting 2012; Boelens et al. 2004) yielding a peak table (N × M) with rows corresponding
to samples and columns to variables (in wavenumber/ (cm1)). Fundamentally, the computational
approach identifies peaks and measures the corresponding peak area automatically where the peaks are
later matched across samples to produce a peak table for multivariate analysis.
2.5 Multivariate Analysis
The peak table was square rooted and standardised prior to multivariate analysis. Square-rooting aims to
reduce the influence of large variables and standardisation ensure each variable (represents an
absorption peak) had a similar influence. The preprocessed peak table was subjected to Principal
Component Analysis (PCA) (Esbensen 1998).
3. RESULTS AND DISCUSSION
The FTIR spectra of various untreated and treated biomass are shown in Figure 1. The spectra of raw
biomass are hardly distinguishable by eyeballing, while the treated biomass demonstrates greater
intensities at regions 3300 cm 1, 1700-1500 cm1, and 1200-1000 cm1. Several common adsorption
bands were found in the raw materials at 1157 cm 1, 1329 cm1, 1370 cm1, 1423 cm1, and 2923 cm1.
These adsorption bands are associated with the presence of lignocellulosic components i.e., cellulose,
hemicelluloses, and lignin.
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EFB(H2SO4)
RH(H2SO4)
SW(H2SO4)
CH(NaOH)
BT
(H2SO4)
Abs
CH(H2SO4)
EFB
(NaOH)
RH (NaOH)
CH(NaOH)
SW(NaOH)
BT(NaOH)
CH(NaOH)
EFB
RH
3501
3001
2501
2001
1501
1001
SW
BT
501 CH
Wavenumber(cm-1)
Figure 1 The FTIR of various treated and untreated agriculture biomass (CH: coconut husk, BT: banana
trunk; SW: sago hampas; RH: rice husk; EFB: empty fruit bunch)
Table 1 summarizes the important adsorption bands found in the spectra of various agriculture biomass
and the corresponding functional groups (Ding et al. 2012). The adsorption bands primarily explain the
structural features of lignocellulosic compounds, for example the adsorption bands at 1157 cm 1 as well
as those at 1423 cm1, 1370 cm1, and 1335 cm1 are attributable to common glucosidal bonds (C-O-C
ring vibrational stretching) and carbohydrates, respectively. The adsorption band at 2923 cm 1 however is
assigned to the C-H stretching of alkanes (Taherzadeh and Karimi 2008).
Table 1 Adsorption peaks detected and the corresponding functional groups
Wavenumber, cm1
Functional groups
1031
1043
1060
1157
1329,1370, 1423
1457
1552
1615, 1511, 1266, 1238
1640, 1730
2855, 2890, 2923
3392, 3412 (broad)
C-O, C=C, C-C-O vibrational stretching
C-O, C-C and C-OH stretching vibrations
Si-O-Si
C-O-C ring vibrational stretching
Characteristic of structural carbohydrates
The aliphatic part of lignin
C=C stretching of alkenes and aromatic
C=C stretching vibrations of the aromatic rings of lignin
C=O stretching of aromatic
C-H stretching of alkanes
Hydrogen bonded OH bond
In this study, the algorithm produced a peak table of dimensions (87 × 60). The preprocessed peak table
was subjected to PCA yielding scores, T and loadings, P to describe the underlying relationships between
samples and variables. Figures 2 and 3 show the scores plots of PC 2 against PC 1, with samples labeled
according to treatments and biomass, respectively. The scores plot in Fig. 2 indicates that untreated ()
and treated biomass (NaOH () and H2SO4 ()) are distinguishable as respective groups of samples
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clustered accordingly, nevertheless some NaOH treated samples appear to be mixed with the acid treated
samples. Figure 3 further indicates that the confused NaOH treated samples are coconut husk, implying
that acid and alkali treated coconut husk are similar in their functional properties. The distributions of
some important lignocellulosic components and the corresponding spectral region are shown in Figure 4.
4
2
PC 2
0
-2
-4
NaOH
H2SO4
-6
Untreated
-8
-6
-4
-2
0
PC 1
2
4
6
4
6
Figure 2 The PCA scores plot according to treatments
4
2
PC 2
0
-2
-4
-6
-8
-6
Coconut husk
Banana trunk
Sago hampas
Rice husk
Empty fruit bunch
-4
-2
0
PC 1
2
Coconut husk NaOH
Banana trunk NaOH
Sago hampas NaOH
Rice husk NaOH
Empty fruit bunch NaOH
Coconut husk H2SO4
Banana trunk H2SO4
Sago hampas H2SO4
Rice husk H2SO4
Empty fruit bunch H2SO4
Figure 3 The PCA scores plot according to agricultural biomass coconut husk: CH(-); banana trunk BT: (-); sago hampas
133
4
3
2
1
0
1157 cm
Abs
Square rooted peak area
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1
CH
BT
SW
RH
EFB
1176
1171
1166
1161
1156
1151
1146
1141
1136
1131
3
2.5
2
1.5
1
Abs
Square rooted peak area
Wavenumber / (cm-1)
0.5
0
Square rooted peak area
1370 cm1
CH
BT
SW
RH
EFB
2.5
1371
1361
1351
1341
1331
1321
1311
1301
Wavenumber / (cm-1)
2
1.5
1
0.5
0
CH
BT
SW
RH
EFB
BT
SW
RH
EFB
5
4
3
2
Abs
Square rooted peak area
1320 cm1
1
0
1612-1650 cm1
CH
1661
1651
1641
1631
1621
1611
1601
1.5
1
0.5
Abs
Square rooted peak area
Wavenumber / (cm-1)
0
1511 cm
1
CH
BT
SW
RH
EFB
1519
1517
1515
1513
1511
1509
1507
1505
1503
1501
Wavenumber / (cm-1)
Figure 4 The distribution of some important lignocellulosic components in raw biomass and the
corresponding spectral region
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The adsorption bands at 1423 cm 1, 1329 cm1, and 1370 cm1 (characteristics of carbohydrates) are
relatively more abundant in sago hampas and banana trunk, suggesting they are richer in cellulose and
hemicellulose content. The adsorption band corresponding to lignin at 1511 cm 1 further indicates a higher
abundance in coconut husk, while sago hampas exhibits a shoulder band that is too weak to be identified
as a peak.
The prevailing lignin content in coconut husk has been reported previously (Hernandez et al. 2007); this
feature may result in a disruption of the adsorption process, as it prevents the accessibility of adsorbate to
the active sites (Rawangkul et al. 2010). The hemicelluloses dominated by the carboxyl group is
characterised by the adsorption at 1640 cm 1 where the adsorption band is shifted to a lower wave
number in the coconut husk (1612 cm 1), whilst for rice husk, the peak is moved to a higher wave number
at 1650 cm1. The distribution suggests that banana trunk has a relatively higher amount of hemicellulose
compared to other biomass; this observation corroborates the findings of Li et al. (2010).
After the treatment processes, changes caused can be rapidly monitored using the peak table. Figure 5
illustrates the distribution of some lignocellulosic components in treated and untreated biomass. At 1157
cm1, alkali treated biomass demonstrated similar distribution patterns as all of the raw materials except
the adsorption band is completely missing in the alkali treated coconut husk. The peak is also absent in all
of the acid treated biomass, suggesting that the NaOH treatment is milder than the concentrated H 2SO4
with the coconut husk susceptible to both treatments. The missing adsorption band at 1157 cm 1 in acid
and alkali treated coconut husk further explains the earlier observation of PCA, that coconut husk treated
with both treatments are comparable.
The disappearance of the adsorption band is likewise observed at 2923 cm 1, indicating disintegration of
aliphatic fraction (He et al. 2008). Other important changes due to treatments are noticed at 1650-1600
cm1 and 1511 cm1. The peak found at 1650-1600 cm1, designated to the carboxyl group, has apparently
increased after the treatment, suggesting decomposition of the cellulose and hemicellulose. The
adsorption band due to lignin at 1511 cm 1, on the other hand, shows an increase in some alkali treated
biomass, whilst absent in other samples. This implies that the NaOH treatment has caused breakage of
linkages between lignin units, transforming the original three-dimensional network structure to a linear
structure in some biomass and the disappearance suggests removal of lignin (He et al. 2008).
Square rooted peak area
10
8
6
4
2
0
CH
BT
SW
RH
EFB
CHNaOH BTNaOH SWNaOH RHNaOHEFBNaOHCHH2SO4 BTH2SO4SWH2SO4RHH2SO4
EFBH2SO4
CH
BT
SW
RH
EFB
CHNaOH BTNaOH SWNaOH RHNaOHEFBNaOHCHH2SO4 BTH2SO4SWH2SO4RHH2SO4
EFBH2SO4
1157 cm1
Square rooted peak area
3
2.5
2
1.5
1
0.5
0
2923 cm1
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Square rooted peak area
14
12
10
8
6
4
2
0
CH
BT
SW
RH
EFB
CHNaOH BTNaOH SWNaOH RHNaOHEFBNaOHCHH2SO4 BTH2SO4SWH2SO4RHH2SO4
EFBH2SO4
CH
BT
SW
RH
EFB
CHNaOH BTNaOH SWNaOH RHNaOHEFBNaOHCHH2SO4 BTH2SO4SWH2SO4RHH2SO4
EFBH2SO4
1612-1650 cm1
Square rooted peak area
2.5
2
1.5
1
0.5
0
1511 cm1
Figure 5 The distribution of some important lignocellulosic components in treated and untreated biomass
4. CONCLUSIONS
As a whole, agriculture biomass types are primarily characterized by the presence of lignocellulosic
components with diverse compositions. The distribution indicates that sago hampas and banana trunk are
predominated with cellulose and hemicellulose. The computer-assisted analysis of FTIR also provides
information on the changes due to different treatments. NaOH treatment appears to have milder effects
than concentrated H2SO4 treatment, which is not unexpected; in addition, the solubilisation of cellulose
and hemicellulose as well as lignin removal is experienced in various agriculture biomass. The information
derived allows rapid monitoring of the lignocellulosic compositions of numerous biomass.
REFERENCES
Boelens, H. F. M., Dijkstra, R. J., Eilers, P. H. C., Fitzpatrick, F. & Westerhuis, J. A. (2004). New
background correction method for liquid chromatography with diode array detection, infrared
spectroscopic detection and Raman spectroscopic detection. J. Chromatogr. A 1057(1-2), 21-30.
Daffalla, S. B., Mukhtar, H. & Shaharun, M. S. (2010). Characterisation of adsorbent developed from rice
husk: Effect of functional group on phenol adsorption. J. Appl. Sci. 10 (12), 1060-1067.
Davis, R. & Mauer, L. J. (2010). Current research, technology and education topics in applied
microbiology and microbial biotechnolog. Badajoz: Formatex.
Ding, T. Y., Hii, S. L. & Ong, L. G. A. (2012). Comparison of pretreatment strategies for conversion of
coconut husk fiber to fermentable sugars. BioResources 7(2), 1540-1547.
rd
Esbensen, K. (1998) Multivariate Analysis in Practice (3 ed.). Oslo: CAMO.
Fernando, A., Monteiro, S., Pinto, F. & Mendes, B. (2009). Production of biosorbents from waste olive
2+
cake and its characteristics for Zn ion. Sustainability 1(2), 277-297.
He, Y., Pang, Y., Liu, Y., Li, X. & Wang, K. (2008). Physicochemical characterization of rice straw
pretreated with sodium hydroxide in the solid state for enhancing biogass production. Energy Fuels
22(4), 2775-2781.
Hernandez, J. R., Capareda, S. C. & Aquino, F. L. (2007). Activated carbon production from pyrolysis and
steam activation of cotton gin trash. Proceedings of the Beltwide Cotton Conferences, New Orleans.
Li, C., Knierim, B., Manisseri, C., Arora, R., Scheller, H. V., Auer, M., Vogel, K. P., Simmons, B. A. &
Singh, S. (2010). Comparison of dilute acid and ionic liquid of switchgrass: Biomass recalcitrance,
delignification and enzymatic saccharification. Bioresource Technol. 101(13), 4900-4906.
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Li, K., Fu, S., Zhan, H., Zhan, Y. & Lucia, L. A. (2010). Analysis of the chemical composition and
morphological structure of banana pseudostem. BioResources 5(2), 576-585.
Rawangkul, R., Khedari, J., Hirunlabh, J. & Zeghmati, B. (2010). Characteristics and performance analysis
of a natural dessicant prepared from coconut coir. ScienceAsia 36, 216-222.
Sim, S. F. & Ting, W. (2012). An automated approach for analysis of Fourier Transform Infrared Spectra
(FTIR) of edible oils. Talanta 88, 537-543.
Taherzadeh, M. H. & Karimi, K. (2008). Pretreatment of lignocellulosic wastes to improve ethanol and
biogas production: A review. Int. J. Mol. Sci. 9, 1621-1651.
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SYNTHESIS AND CHARACTERIZATION OF CELLULOSE AEROGEL WITH TUNABLE
MORPHOLOGY
Ain Nadirah Romainor*, Chin Suk Fun and Pang Suh Cem
Department of Chemistry
Faculty Resource Science and Technology,Universiti Malaysia Sarawak,
94300 Kota Samarahan, Sarawak.
*Email: nadirahain@yaho.com
Abstract
Highly porous and light weighted properties of cellulose aerogels have been successfully synthesized
from fibrous native cellulose through simple and green method of (i) cellulose dissolution in sodium
hydroxide (NaOH)/thiourea/urea (NTU) solvent system, (ii) regeneration of cellulose in ethanol by
immersed precipitation method and (iii) removal of the liquid filled in the polymer pores via lower surface
tension drying techniques. We found that porous structure of cellulose aerogel can be tuned by
manipulating the synthesis conditions of cellulose concentrations, regeneration temperatures and drying
effects of supercritical drying and dehydration followed by freeze drying. Nitrogen adsorption-desorption
isotherm of all the regenerated cellulose aerogels showed type III characteristic, indicated the
mesoporous (2-50 nm pores size) class of pores structure. The specific surface area and pore volumes of
the produced cellulose aerogel determined from Barret-Jorner-Halenda (BJH) method showed that more
porous structure was generated at lower cellulose concentration and lower regenerating temperature of
25°C. Both of supercritical drying and freeze drying techniques gave rise to thread like- and rougher
porous structure respectively. However, supercritical drying technique produced higher specific surface
area and pore volume compared to freeze drying technique.
Keywords: cellulose aerogel, regeneration, synthesis conditions, drying effect
1. INTRODUCTION
Cellulose is an inexhaustible and sustainable natural biopolymer that derived from plants, animal and
bacterial (Sescousse et al., 2011, Siro and Plackett, 2010, and Wang et al., 2008). Cellulose biopolymer
composed of ß-(1→4) glycosidic bonds in their structure, thus have linear and strong intra and inter
molecular hydrogen bonds interaction (Dogan and Hilmioglu, 2009 and Kadokawa et al., 2008). Due to
this reason, it is hard to dissolve cellulose in water and organic solvent (Sehaqui, 2011; Zhao et al., 2007).
Recently, the utilization of cellulose in aerogel production has gain more attention to replace petroleum
based materials (Sehaqui, 2011, Almeida et al., 2010) due to (i) sustainable and renewable cellulose
sources and (ii) uniqueness of highly porous and light weighted properties, low thermal conductivity and
low sound velocity of aerogel (Sehaqui, 2011 and Tsioptsias et al., 2009). The intriguing cellulose aerogel
material offering wide ranges of potential applications such as heat and sound insulators, catalyst carrier,
precursor for carbon aerogels, medical and cosmetic (Sehaqui, 2011, Sehaqui et al., 2011,Sescousse et
al., 2011, Aaltonen and Jauhiainen, 2009 and Gavillon and Budtova, 2008).
Cellulose aerogel can be prepared from (i) dissolution of cellulose, (ii) regeneration through immerse
precipitation technique and (iii) drying. Many dissolution methods such as viscose and cuprammonium
technology have been made to break the strong intra and inter molecular cellulose hydrogen bond in order
to dissolve the cellulose. These dissolution technology is no longer used because its requires the use of
harmful chemical and generates heavy metals (Jin et al., 2007).To date, N-methyl-morpholine-N-oxide
(NMMO)/ water system has been introduced as the most powerful solvent to dissolve the cellulose.
Although it poses a good solvent, their high cost (expensive solvent) has limited their applications
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(Gavillon, 2007 and Jin et al., 2007). Most recently, sodium hydroxide (NaOH) alkaline based system has
been developed to completely dissolve the cellulose. Among the alkaline solvent used (instead of
NaOH/thiourea, and NaOH/urea), NaOH/thiourea/urea (NTU) aqueous system was identified to be the
most powerful and stable solvent to completely dissolve the cellulose (Zhang et al., 2010 and Jin et al.,
2007). NaOH break the intra and inter molecular hydrogen bond to dissolve the cellulose (Almeida et al.,
2010). Urea and thiourea act as hydrogen bond donor and acceptor to prevent the reformation or reassociation of the hydrogen bonds in cellulose molecular chain (prevent the cellulose from undissolve
again) (Zhang et al., 2010 and Jin et al., 2007). We proposed to use NTU solvent system to dissolve the
natural cellulosic fibres throughout the study.
One of the effective ways of making highly porous membrane is the regeneration of cellulose through
facile phase inversion process of immersed precipitation technique (Liu and Zhang, 2009, Kim et al.,
2006, and Di Luccio et al., 2000). Rationales of immersed precipitation technique is the solvent exchange
or diffusion between solvent and non solvent that occurred on the cellulose polymer to formed liquid-filled
pores as the precursor of cellulose porous structure (Sescousse et al., 2011, Liu and Zhang, 2009,Kim et
al., 2006 and Gavillon, 2007).
Finally liquid-filled pores in the regenerated cellulose aerogel were extracted out by using lower surface
tension drying conditions, such as supercritical drying and freeze drying without collapsing the pores
structure (Sehaqui, 2011, Aaltonen and Jauhiainen, 2009, Gavillon and Budtova, 2008, Hoepfner et al.,
2008 and Innerlohinger et al., 2006). The supercritical drying method is based on exposing the
regenerated cellulosic material at above supercritical fluid critical pressure and temperature, where at this
drying condition no capillary stress occur to destroy the porous network (Alnaeif, 2011 and Gavillon,
2007). On the other hand, freeze drying used sublimation technique to maintain the porous structure by
which the liquid in the wet regenerated cellulose gel is frozen and sublimated (Sehaqui, 2011). Apart from
that, porous structure of cellulose aerogel also can be preserved by solvent exchange (dehydration
process) of lower surface tension solvent and followed by freeze drying (Sehaqui, 2011 and Sehaqui et
al., 2011).
In this study, we prepare cellulose aerogel material by dissolving the natural cellulosic material in stable
NTU solvent, regenerated it through immersed precipitation by which ethanol was used as a non solvent
to form liquid-filled pores of cellulose gel. Our studies also focus on the effects cellulose polymer
concentrations, regeneration temperatures and the drying effect between supercritical and dehydration
followed by freeze drying on cellulose aerogel structure. Liquid filled pores in regenerated cellulose have
been removed through supercritical drying of supercritical fluid carbon dioxide because it poses lower
critical temperature and pressure (31.1°C and 7.38 MPa), inexpensive, non toxic and non flammable
solvent (Tsioptsias et al., 2009 and Yu et al., 2008). In the present study, liquid filled pores in regenerated
cellulose also have been extracted out through dehydration with gradient acetone (lower surface tension
solvent) and followed by freezing at -21°C as the simple technique to preserve pores structure.
2. MATERIALS AND METHODS
2.1 Materials
Commercial cellulose Fibrous Cellulose Powder CF11 was purchased from Whatman. Absolute ethanol
and acetone were purchased from HmbG Chemicals. Absolute ethanol and acetone was used without
purification. Ultrapure water (UPW) was obtained in our laboratory by a Water Purifying System (ELGA,
Model Ultra Genetic) and was used throughout the study.
2.2 Methods
2.2.1 Preparation of Cellulose Dissolution
Aqueous mixture with the ratio of 8wt%: 6.5wt%: 8wt% of NaOH: thiourea: urea was prepared and used
as solvent system. Fibrous cellulose powder corresponding to the final concentration of 1%, 3%, 7% and
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9% (w/v%) was added in NTU solvent and cooled down in freezer at -21°C for 12h. The frozen cellulose
was thawed at room temperature to obtain cellulose solution.
2.2.2 Preparation of Cellulose aerogel
The prepared cellulose solutions were poured into ethanol regeneration bath ( ratio of 10ml: 20ml of
cellulose solution to ethanol) and kept at 25°C for 4h to allow the solvent exchange (between cellulose
solution and ethanol) and complete the precipitation reaction. Regenerated cellulose was then washed
with ultrapure water (UPW) several times to remove the excessive salts.
2.2.2.1 Variable of Regeneration Temperatures
Ethanol regeneration bath was heated up to 50°C and 70°C. Cellulose solution (3% cellulose solution
concentration) was poured into the ethanol regeneration bath and kept at desired temperatures for 4h.
Regeneration and washing step was repeated as in method 2.2.2.
2.2.3 Drying Approach Preparation
2.2.3.1 Critical Point Drying
The washed regenerated cellulose was wrapped in filter paper and soaked in acetone for 30 minutes to
remove the water and to have acetone liquid in the cellulose network pores (Sesccousse and Budtova,
2009). The wrapped sample was then placed in CPD chamber that filled with acetone (half of the
chamber) and closed. Supercritical fluid carbon dioxide was introduced into the chamber in order to get
the compatible mixture of acetone and supercritical fluid. This step was repeated 6 to7 times in order to
wash the sample. Finally the supercritical fluid carbon dioxide was pressurized and heated above its
critical pressure and temperature (80 bar and 40°C) to dry the regenerated cellulose into cellulose
aerogel.
2.2.3.2 Freeze Drying
The washed regenerated cellulose sample was treated in gradient acetone (30%, 50%, 70%,80%,90%
and 99% for 15 minutes each time and three times in 100% acetone for 30 minutes each time).
Dehydration technique is crucial in order to get rid of UPW in the regenerated sample and replaced it by
acetone. After complete dehydrating, acetone was removed from the samples through sublimation
process by placing the sample in freezer at -21°C. This drying approach was applied to dry the
regenerated cellulose at 9% cellulose concentration.
3. RESULTS AND DISCUSSION
3.1 Effects of Cellulose Concentrations on Cellulose Aerogel
Figure 1 shows the Scanning Electron Microscopy (SEM) images of cellulose aerogel regenerated at
1%,3%,7% and 9% (w/v%) of cellulose concentration. Cellulose aerogel synthesized was highly porous in
nature compared to fibrous native cellulose as shown in Figure 1 (a) and (b) to (e) respectively. The
nitrogen adsorption-desorption isotherms of the regenerated cellulose aerogel was measured in all porous
samples and the representative isotherm is presented in Figure 2. All the samples showed type III
characteristic of mesoporous (2-50nm) pores classification (Gavillon, 2007 and Sing et al., 1986).
The porosity characteristics of all samples are summarized in Table 1. Specific surface area and pore
volume of regenerated cellulose aerogel was decreased as the increasing of cellulose concentration as
can be seen in Table 1. Regenerated cellulose at 1% cellulose concentration resulted in the most porous
2
structure with the specific surface area of 400.760 m /g and pore volume of 0.895cc/g. The surface area
2
2
and pore volume was dramatically drop to 54.708 m /g and 0.385cc/g , 19.755 m /g and 0.166 cc/g and
2
2.325 m /g and 0.025 cc/g as the cellulose solution was increased from 3%, 7% and 9% respectively.
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(a)
(b)
(c)
(d)
Figure 1 SEM images of (a)
native cellulose and regenerated
cellulose at (b) 1%, (c) 3%,(d)
7(e)
9%
(w/v%)
cellulose
concentrations.
(e)
Figure 2 Nitrogen adsorptiondesorptionof regenerated cellulose
aerogel
at
3%
cellulose
concentaetion
Table 1 Pore volume and surface area regenerated cellulose aerogel at various concentrations
Cellulose aerogel
concentration
(wt/v)%
1
3
7
9
Cumulative pore
volume (cc/g)
Specific surface
2
area (m /g)
0.895
0.385
0.166
0.025
400.760
54.708
19.755
2.325
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Formation of porous structure was favoured at lower cellulose concentration. When lower cellulose
solution was immersed in ethanol bath, the NTU solvent was diffused out from the cellulose solution and
ethanol was diffused into cellulose polymer (solvent exchange) to form more porous structure.
Coagulation rate of higher cellulose concentration was slow and resulted in denser and less porous
structure (Sescousse et al., 2011, Liu and Zhang, 2009 and Wijmans et al., 1983).
3.2 Effects of Regeneration Temperatures on Cellulose Aerogel
Figure 3 shows the SEM images of cellulose aerogel regenerated at 25°C, 50°C and 70°C. The porous
structure with smaller pores size was presented in cellulose aerogel regenerated at 25°C and 70°C
compared to 50°C. Pore size distribution curve of regenerating cellulose at different temperatures in
Figure 4 indicate that the dominated pores radius size (contributes from the highest peak) of cellulose
aerogel regenerated at 25°C, 50°C and 70°C was 8.6994nm, 14.9652nm and 8.6883nm respectively.
Increasing regeneration temperatures from 25°C to 50 °C induce the precipitation to occur quickly and
produced large pores. While at higher regeneration temperature, precipitation occur faster and rapid thus
suppress the pores size (Liu and Zhang 2009 and Gavillon, 2007).
(a)
(b)
(c)
(d)
Figure 3 SEM images of cellulose aerogel
regenerating
at
different
regenerating
temperatures of (a) and (b): 25°C, (c) and (d):
50°C and (e) and (f): 70°C respectively.
(e)
(f)
Figure 4 Pore size distribution curve of cellulose
aerogel regenerated at 25°C, 50°C and 70°C.
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The cumulative pore volume and surface area of regenerating cellulose aerogel at 25°C, 50°C and 70°C
are summarized in Table 2. It can be concluded that porosity of regenerated cellulose aerogel was
decreased with the increasing of regeneration temperatures with dramatic drop of specific surface area
2
2
2
and pore volume : 54.708 m /g and 0.385 cc/g, 13.914 m /g, 0.167cc/g and 12.087 m /g and 0.087 cc/g at
25°C, 50°C and 70°C.
Table 2 Cumulative pore volume and surface area of cellulose aerogel regenerated at different
regenerating temperatures.
2
Regenerating temperatures (°C)
Cumulative pore volume (cc/g)
Specific surface area (m /g)
25
0.385
54.708
50
0.167
13.914
70
0.087
12.087
3.3 Effect of Drying Approach on Cellulose Aerogel
Figure 5 shows the SEM images resulted from drying approach of supercritical point drying and freeze
drying on the cellulose aerogel morphology. Both of the drying technique was succesfully produced
“thread-like” pores network and rougher and large pores (14λ.λ424 nm) network as shown in Figure 5 (a)
and(b) respectively.
(a)
(b)
Figure 5 SEM images of regenerated cellulose aerogel dried at (a) supercritical drying and (b) freeze
drying approaches.
Cumulative pore volume and specific surface area that are listed in Table 3 indicated that freeze drying
approach produced less porous structure due to lower cumulative pore volume and specific surface area
compared to supercritical drying. In supercritical drying approach, supercritical fluid of carbon dioxide was
heated above to its critical pressure and temperature to remove the solvent under supercritical condition
(have zero surface tension) and remain the pores structure from collapse (Tsioptsias et al., 2009, Yu et al,
2008 and Gavillon, 2007). On the other hand, freeze drying approach evoporated the solvent in the wet
regenerated cellulose aerogel through sublimation principle as it was dried at -21°C (Stadlander, 2007).
Table 3 Results of pore volume and surface area of cellulose aerogel dried at different condition.
2
Drying approach
Cumulative pore volume (cc/g)
Specific surface area (m /g)
Supercritical drying
0.025
2.325
Freeze drying
0.005
0.088
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4. CONCLUSIONS
We have successfully prepared cellulose aerogel from native cellulose. The morphology, surface area and
porosity of the cellulose aerogel were affected by the synthesis conditions such as cellulose
concentrations, regeneration temperatures and drying method.
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Almeida, E.V.R., Frollini, E., Catellan, A., and Coma,V. (2010). Chitosan, sisal cellulose, and biocomposite
chitosan/sisal cellulose films prepared from thiourea/NaOH aqueous solution. Carbohydrate
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(GTG)5-PCR ANALYSIS AND IDENTIFICATION OF BACTERIA FROM SARAWAK AQUACULTURE
ENVIRONMENT
*Kathleen Michelle Mikal, Samuel Lihan, Felecia Collick, Lesley Maurice Bilung and Kasing Apun
Department of Molecular Biology, Faculty of Resource Science and Technology,
94300 Kota Samarahan, Sarawak.
*Email: kathmichelle@hotmail.com
Abstract
(GTG)5 PCR is a type of repetitive extragenic palindromic (rep)-PCR which amplifies the (GTG)5 repetitive
element that lays throughout the bacterial genome. In this study, fifty, thirty-nine and forty-nine unknown
bacteria isolates from aquaculture in selected farms in Miri, Limbang and Lundu, respectively, were prescreened using (GTG)5-PCR. (GTG)5 PCR was used to screen for clonal diversity among the isolates
according to sampling sites. Banding profiles obtained from electrophoresed (GTG) 5 PCR products were
analyzed by RAPDistance Software to generate a dendogram of neighbor joining tree (NJT) format.
Based on the constructed dendrogram, few isolates were selected for further identification. Conserved
16S rRNA region of the selected bacteria isolates were amplified and purified DNA products were
sequenced. (GTG)5 PCR and 16S rRNA analysis revealed that the aquaculture bacteria in Sarawak
aquaculture environment are highly diverse in terms of genetic and species. The high diversity of bacteria
in aquaculture environment may be caused by contamination from various sources.
Keywords: Aquaculture environment, bacteria isolation, (GTG) 5-PCR, 16S rRNA PCR
1. INTRODUCTION
Aquaculture sector is one of the important and rapidly growing sectors in Malaysia. Large number of
bacteria from highly diverse bacterial species could be isolated from the aquaculture pond and its
environment. Presence of a large number of bacteria in the pond indicates the presence of high nutrient
level in the water. The number and species of freshwater microbiota depends on the availability of light
and oxygen (Tortora et al., 2007). Based on previous study by Apun et al. (1999), fish intestine contains
the most number of bacteria from different species. Among the commonly found bacterial species in
aquaculture pond and its environment were Aeromonas, Escherichia, Enterobacter, Klebsiella,
Pseudomonas, Vibrio, Bacillus, Listeria, Staphylococcus, Citrobacter (Apun et al., 1999) and Edwardsiella
(Seong Wei et al., 2011). The objective of this study is to determine the genetic differences to group the
bacteria and also to identify their species based on the 16S rRNA sequencing.
2. MATERIALS AND METHODS
2.1 Samples collection and processing
Sampling was carried out at aquaculture farm located at Miri, Limbang and Lundu. Three types of samples
collected were sediment, water and cultured species. The sediment and water samples were taken using
a sterile PVC pipe and 100 ml sterile Schott’s bottle, respectively (Huys, 2003). Litopenaeus vannamei or
Pacific White Shrimp (local name: udang putih) was freshly caught from the farms at Lundu and Limbang.
Pangasius pangasius or River Catfish (local name: ikan patin) was freshly caught from Miri farm. Samples
were transported to the laboratory in an ice container containing ice within 24 hours. Samples were
processed immediately upon arrival in the laboratory.
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2.2 Bacteria isolation
The sediment and water samples were homogenized before performing 10-fold serial dilution. One gram
of the fish or shrimp intestine, one ml of the homogenized water and sediment samples were diluted with 9
o
ml of 0.85% saline solution. Samples were plated on duplicate TSA and incubated at 29 C for 24 hours.
Plates containing 30 to 300 colonies were selected for bacteria isolation. Five to ten colonies were
randomly isolated and streaked onto TSA until pure isolate was obtained.
2.3 DNA Extraction
Bacterial DNA was extracted using the boiling-centrifugation method as described by Freschi et al. (2005)
with modifications. Briefly, 3 ml of the overnight culture was centrifuged at 10,000 rpm for 5 minutes. The
supernatant was discarded. The pellet was resuspended in 500 µl of sterile dH 2O and boiled for 10
minutes. Immediately, the boiled suspension was cooled in ice at 4°C for 5 minutes and centrifuged at
10,000 rpm for 10 minutes. The supernatant was used for the PCR analysis.
2.4 (GTG)5 PCR analysis
(GTG)5 PCR was conducted according to Matsheka et al. (2005) with slight modifications. Briefly, 25µl
PCR mixture containing 5X Taq Green Buffer (Promega), 25mM MgCl2, 25mM deoxyribonucleotide
phosphate (dNTPs), 25µM (GTG)5 primer (5’- GTGGTGGTGGTGGTG – 3’), sterile distilled water (dH2O),
DNA template and Taq DNA polymerase. The amplification begins with pre-denaturation at 95°C (7
minutes), followed by 4 cycles of denaturation, annealing and extension at 95°C (2 minutes), 36°C (2
minutes) and 72°C (2 minutes), respectively. This was followed by another 30 cycles of denaturation at
95°C (1 minute), annealing at 50°C (1 minute) and elongation at 72°C (1 minute). The final elongation was
carried out at 72°C for 5 minutes. Five microliter of the amplified PCR product was electrophoresed on
1.5% agarose gel pre-stained with 1 µl of 10 mg/ml EtBr in 1X Tris-Borate-EDTA (TBE) buffer at 100V for
1 hour and 30 min. The gel was then visualized under UV transilluminator. Scoring was done for
construction of dendrogram. Bands present were scored as “1”, while absence of band was scored as “0”.
The data was input in RAPDistance software and phylogenetic tree was generated by the software.
2.5 Identification by 16S rRNA PCR
The genotypic identification was done using the 16S rRNA identification method (Hutter et al., 2003) using
51λR primer (5’-GWATTACCGCGGCKGCTG-3’) and 27F primer (5’-AGAGTTTGATCMTGGCTCAG-3’)
(Ream et al., 2003). The 25µl reaction mixture consists of 5X Taq Green Buffer, 25mM MgCl2, 25mM
dNTPs, 10µM of each primer, sterile dH2O, DNA template and Taq DNA polymerase. The cycling
conditions were as followed: predenaturation at 95°C (10 minutes), denaturation at 94°C (30 seconds),
annealing at 55°C (1 minute), extension at 72°C (1.5 minutes) and final extension at 72°C (10 minutes) at
the end of 26 cycles. The amplified PCR product was purified using QIAquick PCR purification kit (Qiagen,
Germany). Four microliter of purified DNA were electrophoresed on 1.0% agarose gel pre-stained 1 µl of
10 mg/ml EtBr in 1X TBE buffer at 80V. The stained gel was then visualized under the UV transilluminator.
The leftovers of purified DNA were sent to First BASE Laboratories Sdn. Bhd. for DNA sequencing. The
DNA sequence was then compared with those in the GenBank database using the BLAST server by the
National Center for Biotechnology Information (NCBI).
3. RESULTS AND DISCUSSIONS
One hundred and thirty-eight bacteria isolates from three different aquaculture farms were pre-screened
using the (GTG)5-PCR analysis. Figure 1, 2 and 3 showed the banding profiles of the amplified bacterial
(GTG)5 elements in 1.5% agarose gel from Miri, Limbang and Sampadi, respectively. The size of the
bacterial isolates’ (GTG)5 gene from all location are within the range of 250 bp to 3,000 bp. All Limbang
isolates possess different (GTG)5 pattern. Miri (GTG)5 PCR profiles revealed that two isolates possess the
same (GTG)5 pattern. The bacterial isolates in Miri and Limbang are highly diverse. The (GTG)5 pattern for
Sampadi isolates are less heterogeneous compared to Miri and Limbang isolates. The (GTG) 5 banding
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pattern for all three locations are more obvious when represented in the form of dendrogram (Figure 4, 5
and 6).
Figure 1 Banding profiles of (GTG)5 PCR for Miri isolates. Lane M: 1kb DNA Ladder (Promega, USA), NC:
negative control, 1: MYY-F5, 2: MYY-F7, 3: MYY-F8, 4: MYY-F9, 5: MYY-F10, 6: MYY-W1, 7: MYY-W2, 8:
MYY-W3, 9: MYY-W7, 10, MYY-2F3, 11: MYY-2F4, 12: MYY-2F5, 13: MYY-2F6, 14: MYY-2F7, 15: MYY2F8, 16: MYY-2F9, 17: MYY-2F10, 18: MYY-2W1, 19: MYY-2W2, 20: MYY-2W3, 21: MYY-2W4, 22: MYY2W7, 23: MYY-F4, 24: MYY-W4, 25: MYY-W5, 26: MYY-W6, 27: MYY-28, 28: MYY-W9, 29: MYY-W10,
30: MYY-2S1, 31: MYY-2S2, 32: MYY-2S3, 33: MYY-2S4, 34: MYY-2S5, 35: MYY-2S6, 36: MYY-S1, 37:
MYY-S2, 38: MYY-S3, 39: MYY-S4, 40: MYY-S5, 41: MYY-S6, 42: MYY-S7, 43: MYY-F1, 44: MYY-F3, 45:
MYY-2S10, 46: MYY-2F1, 47: MYY-2F2, 48: MYY-S9, 49: MYY-S10, 50: MYY-F2.
Figure 2 Banding profiles of (GTG)5 PCR for Limbang isolates. Lane M: 1kb DNA Ladder (Promega, USA),
1: LBG-S4, 2: LBG-S6, 3: LBG-S7, 4: LBG-P5, 5: LBG-P7, 6: LBG-P8, 7: LBG-W2, 8: LBG-W6, 9: LBGW8, 10: LBG-P2, 11: LBG-P10, 12: LBG-2S1, 13: LBG-2S2, 14: LBG-2S3, 15: LBG-2S5, 16: LBG-2S7, 17:
LBG-2S8, 18: LBG-2S9, 19: LBG-2S10, 20: LBG-2P1, 21: LBG-2P2, 22: LBG-2P3, 23: LBG-2P4, 24:
LBG-2P5, 25: LBG-2P6, 26: LBG-2P7, 27: LBG-2P8, 28: LBG-2P9, 29: LBG-2P10, 30: LBG-2W2, 31:
LBG-2W3, 32: LBG-S2, 33: LBG-P1, 34: LBG-P6, 35: LBG-W3, 36: LBG-P4, 37: LBG-2W4, 38: LBG-2W5,
39: LBG-2W6, NC: negative control.
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Figure 3 Banding profiles of (GTG)5 PCR for Sampadi isolates. Lane M: 1kb DNA ladder (Promega, USA),
1: SPD-2S4, 2: SPD-2P1, 3: SPD-2P2, 4: SPD-2P3, 5: SPD-2P4, 6: SPD-2P5, 7: SPD-2P6, 8: SPD-2P7,
9: SPD-2P8, 10: SPD-2P9, 11: SPD-2P10, 12: SPD-2W3, 13: SPD-S1, 14: SPD-S3, 15: SPD-S5, 16:
SPD-S6, 17: SPD-S7, 18: SPD-S8, 19: SPD-S9, 20: SPD-S10, 21: SPD-P3, 22: SPD-P4, 23: SPD-P5, 24:
SPD-P6, 25: SPD-P8, 26: SPD-25, 27: SPD-2S1, 28: SPD-2S3, 29: SPD-P1, 30: SPD-P7, 31: SPD-2W1,
32: SPD-2W4, 33: SPD-2W6, 34: SPD-2W8, 35: SPD-W1, 36: SPD-W2, 37: SPD-W3, 38: SPD-W4, 39:
SPD-W6, 40: SPD-W7, 41: SPD- W9, 42: SPD-S2, 43: SPD-P10, 44: SPD-2S2, 45: SPD-2S9, 46: SPD2S5, 47: SPD-2S6, 48: SPD-2S7, 49: SPD-2S8, NC: negative control.
The objective of performing (GTG)5 PCR in this study was to draw rough estimation of genetic similarity
among the bacteria isolates based on the sampling locations. After the attempt of dendrographic analysis
on the (GTG)5 banding profiles, all the constructed dendrogram tree consists of two major clusters and
many sub-clusters. As observed in the dendrograms (Figure 4, 5 and 6), some isolates from environment
(water and sediment) samples were clustered together with the isolates from the cultured organism
indicating that these isolates are closely related with one another. This is expected as these bacteria
belong to the same niche. The cultured organism may be contaminated with the bacteria from the
sediment and the water in the aquaculture farm.
The bacteria distribution in aquaculture pond and its environment is highly diverse. There are many
factors contributing to the high bacterial diversity in the aquaculture pond and its environment such as
human activity and animal’s activity. The bacterial isolated from Sampadi aquaculture farm is more
homogeneous than other locations because the aquaculture system in Sampadi was less exposed to
human activities. The Sampadi aquaculture farm is located in a remote area. The management does not
allow individuals to enter the farms without permission and without wearing the provided boots and
transportation. This is to reduce contamination from being introduced into the aquaculture system. In the
aquaculture pond itself, nettings were installed on top of every pond to prevent birds from feeding on the
cultured shrimps. Birds may also be the carrier of bacterial contamination. The aquaculture system in
Limbang and Miri were more exposed to human and animal activities.
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Figure 4 Dendrogram of isolates from Miri
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Figure 5 Dendrogram of isolates from Limbang
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Figure 6: Dendrogram of isolates from Sampadi
Out of 138 screened bacteria isolates, only 49 isolates were selected for genotypic identification using the
16S rRNA PCR analysis and DNA sequencing. The list of isolates along with its identity was tabulated in
table 1. The genotypic identification of the bacteria partial 16S rRNA gene revealed thirteen bacterial
genera. Bacillus sp., mainly isolated from sediment samples, dominated the aquaculture environment with
41%, followed by Staphylococcus sp. (21%), Acinetobacter sp. (8%), Vibrio sp. and Aeromonas sp. (6%),
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and Serratia sp. (4%). 14% of the isolated bacteria were categorized as other genera which includes
Escherichia sp., Chryseobacterium sp., Microbacterium sp., Stenotrophomonas sp., Pseudomonas sp.,
Enterobacter sp. and Exiguobacterium sp.. The bacteria distribution was presented in a chart pie (Figure
7). Bacillus sp. was a common soil bacterium and can be found abundantly in the soil. This explains the
domination of this bacterial species in the aquaculture environment.
Table 1 Bacterial 16S rRNA genotypic identification
Isolates
Bacteria Identity
MYY-2S4
Bacillus cereus
%
similarity
99%
Isolates
Bacteria Identity
LBG-P6
LBG2W5
Bacillus pumilus
%
similarity
99%
MYY-W6
Staphylococcus sciuri
99%
Serratia marcesencs
100%
MYY-W9
Enterobacter
hormaechei
99%
LBG-W8
Bacillus vietnamensis
99%
MYY-2W4
Acinetobacter sp.
99%
LBG-W6
99%
MYY-2W7
Bacillus sp.
99%
LBG-S4
Stenotrophomonas
maltophilia
Bacillus subtilis
MYY-F9
Escherichia coli
99%
LBG-2P6
Serratia liquefaciens
97%
MYY-2F4
Aeromonas punctata
99%
LBG-2P9
Microbacterium sp.
99%
MYY-2F7
Bacillus megaterium
99%
SPD W7
Staphylococcus sp.
99%
MYY-S10
Bacillus megaterium
99%
SPD W5
Vibrio sp.
99%
MYY-2S5
Bacillus sp.
100%
Staphylococcus sp.
99%
MYY-S9
Bacillus cereus
99%
SPD W3
SPD
2W4
Bacillus cereus
99%
MYY-S3
Bacillus pumilus
99%
SPD-W1
99%
MYY-2W3
Aeromonas jandaei
99%
SPD-S8
Staphylococcus
saprophyticus
Bacillus sp.
MYY-S5
Pseudomonas sp.
99%
SPD-2S4
99%
MYY-2F8
Aeromonas jandaei
99%
SPD-S7
MYY-2F2
Staphylococcus xylosus
99%
SPD-2S3
Bacillus cereus
Exiguobacterium
profundum
Bacillus cereus
LBG-2S8
Bacillus jeotgali
99%
SPD-S6
Vibrio fischeri
99%
LBG-2W2
Chryseobacterium sp.
99%
SPD-2P1
Acinetobacter sp.
99%
LBG-2S5
Bacillus infantis
99%
SPD-2P8
Acinetobacter calcoaceticus
99%
LBG-2W3
Acinetobacter sp.
99%
SPD-P4
Bacillus cereus
99%
LBG-S7
Bacillus pumilus
99%
SPD-P7
Vibrio rotiferianus
99%
LBG-2S10
Bacillus sp.
94%
SPD-P10
Bacillus cereus
99%
LBG-2P7
Staphylococcus xylosus
99%
SPD-P1
99%
LBG-P2
Staphylococcus sp.
99%
SPD-S9
Staphylococcus sp.
Staphylococcus
saprophyticus
LBG-2P3
Staphylococcus
saprophyticus
99%
153
99%
99%
99%
99%
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Figure 7 Bacterial distribution in Miri, Limbang and Sampadi aquaculture farm and its environment
4. CONCLUSION
(GTG)5 PCR profiles and phylogenetic analysis of the (GTG) 5 PCR was conducted to screen for clonal
isolates for every location. This is to prevent sequencing of the same clonal isolates. Through (GTG) 5
PCR, it was revealed that the Limbang and Miri bacteria isolates were highly diverse. Sampadi bacteria
isolates were less diverse when compared to Limbang and Miri. This may be due to the amount of
activities introduced at the aquaculture farm. Selected bacteria isolates from the (GTG)5 PCR analysis
were successfully identified using 16S rRNA PCR and DNA sequencing. Molecular identification revealed
the thirteen bacteria genera from the Miri, Limbang and Sampadi aquaculture pond and their environment.
ACKNOWLEDGEMENT
This research project was partly funded by FRGS grant no. FRGS/01(16)/745/2010(31).
REFERENCES
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and Selective Enrichment Broths on the Detection of Salmonella Typhimurium In Swine Feces by
Polymerase Chain Reaction (PCR). Brazilian Journal of Microbiology, 36: 363-367.
Hutter, G., Schlagenhauf, U., Valenza, G., Horn, M., Burgemeister, S., Claus, H. and Vogel, U. (2003).
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putative pathogens. Microbiology, 149: 67-75.
Huys, G. (2003). Sampling and Sample Processing Procedures for the Isolation of AquacultureAssociated Bacteria. Standard Operating Procedures.
Matsheka, M. I., Lastovica, A. J., Zappe, H. and Elisha, B. G. (2005). The use of (GTG)5 oligonucleotide
as an RAPD primer to type Campylobacter concisus. Letters in Applied Microbiology, 42: 600-605.
Ream, W., Geller, B., Trempy, J. and Field, K. (2003). Molecular Microbiology Laboratory: A WritingIntesive Course. Academic Press. Elsevier Science (USA).
Seong Wei, L., Najiah, M., Tse Seng, C., Noor Azhar,M.S., Wendy, W., Nadirah, M. and Mohd. Effendy,
A.W. (2011). Antibiogram and Plasmid Profiling from Edwardsiella tarda Isolated from Freshwater
Fish in East Coast Malaysia. Journal of Sustainability Science and Management, 6: 19-27.
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Education,Inc.
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Determination of Tetrodotoxin in Puffer Fish from East Malaysia Waters
1,2
2
1
Mohd Nor Azman A.* , Samsur M. and Othman M.
1
Fisheries Research Institute 11960 Batu Maung, Penang, Malaysia
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak 94300 Kota Samarahan,
Sarawak, Malaysia.
2
*Email: mohayo01@yahoo.com
Abstract
Puffer fish, mainly from Tetraodontidae family known to possess a neurotoxin or tetrodotoxin (TTX) which
can cause a puffer fish poisoning and adverse effect to human health. In current study, the tetrodotoxin
(TTX) concentration in different tissues (liver, skin, muscle, gonad and intestine) of 14 species of puffer
fish from Sabah and Sarawak waters were analysed and determined by application of liquid
chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Overall, extracted toxin for all
specimens were shown to be toxic with result more than 0.2 µg/g as calculated based on TTX standard
curve. Among the tissues, liver were found to be highest in TTX concentration (561.64 µg/g), followed by
gonad (429.48 µg/g), muscle (279.18 µg/g), intestine (35.03 µg/g) and skin (33.83 µg/g). Moreover, TTX
concentrations among puffer fish species were significantly differences (p<0.05) with Arothron
immaculatus (275.26 µg/g) showed highest mean value, while the lowest value was detected in
Lagocephalus lunaris (5.37 µg/g). From this finding, LC-MS/MS application could be a potential tools to
determine the TTX and advisedly used as a procedure in screening of seafood for monitoring program.
Furthermore, baseline data of TTX levels in selected puffer fish from the study could be important
information and used as guideline in order to mitigate puffer fish poisoning cases especially in East
Malaysia waters.
Keywords: Puffer fish, tissues, Tetrodotoxin, LC-MS/MS, East Malaysia
1. Introduction
Tetrodotoxin (TTX) is a non protein neurotoxin that is found in many diverse animals species such as
puffer fish, some species of newts, frogs, gobies, flat worms, ribbon worms, starfish, crabs, the blueringed octopus, carnivorous gastropods, etc. (Mosher and Fuhrman, 1984; Miyazawa and Noguchi, 2001;
Pires et al., 2002). Puffer fish are thought to accumulate TTX through the food chain, which starts from
marine bacteria Vibrio alginolyticus, Shewanella sp., S. putrefaciens, Alteromonas tetraodonis and others
(Yasumoto et al., 1986; Matsui et al., 1990). TTX intoxication from the ingestion of toxic puffers probably is
the most common fish poisoning along the coasts of Asia. Outbreaks of puffer fish poisoning have been
reported in various countries including Thailand (Laobhripatr et al., 1990; Brillantes et al., 2003), Mexico
(Nunez-Vasquez et al., 2000), Hong Kong (Yu and Yu 2002), Australia (Isbister et al., 2002), Taiwan (Tsai
et al., 2004), Bangladesh (Ahasan et al., 2004) and particularly Japan (Lin and Hwang 2001).
Conventionally, the biological method, similar to the method developed for PSP toxins monitoring (AOAC,
1995), has been applied for toxicity determination among puffers. However, ethical concerns from live
animal testing, non-specific test, low sample throughput, inconsistent results and analytical inaccuracies
have led to the need for an alternative method such as the application of HPLC and more recently LCMS/MS to be developed. In addition, the bioassay requires a continuous supply of mice. The results of
toxin analysis vary depending on the mouse strain, gender and weight of animals. Furthermore, the
mouse bioassay is not suitable for quantitative statements (Micheli et al., 2002). Yasumoto et al. (1982)
and Yotsu et al. (1989) constructed a similar fluorometric TTX analyzer, by combining HPLC and a post
column reaction with a hot NaOH solution, to detect TTX and its analogues. Such chemical methodology
has also led to the discovery of TTX-producing bacteria (Yasumoto et al., 1986) and the identification of
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novel TTX analogues from puffers (Nakamura and Yasumoto, 1985). There are various methods available
for testing of these substances. More recent development is the application of LC-MS/MS in the detection
and quantification of TTX and its analogues food matrices.
Puffer fishes are very common in Malaysian waters are often caught in large numbers by trawlers or line
fishing (Simon et al., 2009). There are at least 185 species of puffer fishes which distributed in 28 genera
in the family Tetraodontidae (Oliveira et al., 2006). The most common species in Malaysia is
Lagocephalus lunaris, L. sceleratus and L. spadiceus are consumed by some locals (Kan et al., 1987). In
Sarawak, Xenopterus naritus or locally known as “ikan buntal kuning” is considered a delicacy by the local
community. Almost all puffer fish are poisonous and contain the poison (TTX) in their body parts. In
Malaysia, although people do not eat puffer fish, many food poisoning cases due to ingestion of wild puffer
fish have occurred. The latest case of fish poisoning due to ingesting puffer fish has been reported in
Johor with 34 cases (Chua et al., 2009). Most of these cases were caused by ingestion of contaminated
puffer fish species. Since there is little published information on the toxicity of other species of puffer fish,
this study is also aimed at determining the concentration of TTX from different tissues. In this study, the
toxicity of some fish specimens collected from Sabah and Sarawak waters was explored by the
application of LC-MS/MS as an alternative method of the animal assays.
2. MATERIALS AND METHODS
2.1 Sample collection
A total of 14 species of puffer fish from three different families which, consisted of two species from
Diodontidae family (Diodon holocanthus and Diodon hystrix); one species from Ostraciidae family
(Ostracion nasus) and 11 species from Tetraodontidae family (Arothron immaculatus, Arothron
manilensis, Arothron stellatus, Chelonodon patoca, Lagocephalus inermis, Lagocephalus lunaris,
Lagocephalus sceleratus, Lagocephalus spadiceus, Tetraodon nigroviridis, Torquigener pallimaculatus
and Xenopterus naritus) collected from Pulau Mandi Darah, Sabah; Batang Sadong, Sampadi and
Kuching Sarawak in October 2009 for analyses in the present study. Immediately after collection, fish
o
were kept in ice and stored at -20 C until delivered to Fisheries Research Institute, Penang for further
analyses.
2.2 Sample extraction & preparation
In the laboratory, the samples were thawed and sorted according to the species. The puffer fish
identification was done based on the morphological characteristics (FishBase and FRI, 2004). After
thawing, the fish specimens were weighed (92.0 – 756.0 g) and measured for total length (15.0 – 27.5 cm)
individually. Each specimen was dissected to remove the liver, skin, muscle, gonad and intestine. Toxin
was extracted from each fish tissue according to Diener et al. (2007) with slight modifications. Each tissue
was minced and a small portion (1 g) was extracted with 3 ml of 0.03M acetic acid using an ultrasonic
probe (OMNI-Ruptor 4000, Georgia, USA) for 1 min. The homogenate was centrifuged at 5,000 rpm for 15
min (Eppendorf 5430, Hamburg, Germany) and subsequently the supernatant was collected and
transferred to a volumetric flask of 10 ml. The extraction step is repeated and the homogenate was
centrifuged at 14,000 rpm for 15 min. After centrifugation the supernatant was transferred to a volumetric
flask and made up with 0.03M acetic acid. The sample extract was filtered through a 0.45 µm nylon
membrane filter and the filtrate was analysed by an LC-MS/MS.
2.3 Analyses by LC-MS/MS
Mass spectrometry was performed using a TSQ Quantum Discovery MAX model from Thermo Electron,
USA consisting of an MS Surveyor pump with autosampler coupled to a Mass Spectrometer equipped
with an Electro Spray Ionisation (ESI) probe. Prior to analyses, mass calibration was done using 1, 3,5
Polityrosine in both negative and positive mode. Compound optimisation was carried out in the positive
mode for the detection of the analyte based on its ionisation using a 1 ppm tuning standard solution of
TTX. Optimal ion source and interface conditions were achieved at a spray voltage of 3,800V, sheath gas
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flow of 10 units, auxillary gas flow of 3 units, Collision Energy (CE) of 18, Collision Gas Pressure of 1.5
+
mTorr and capillary temperature of 300°C. The positive ionization produced the typical [M+H] molecular
ion of TTX (m/z 320) which under SRM mode, collision by Argon molecule to give the product ions (m/z
162) as quantifier. Peak detection, data acquisition and the calibration graph plot were performed using
the Xcalibur 2.1.0 software. TTX was separated on a 5 µm, 150 mm x 2.1 mm inner diameter ZIC-HILIC
column (SeQuant, Haltern, Germany) with a guard column 20 x 2.1 mm, 5 µm (SeQuant, Haltern,
Germany) at a flow rate of 250 µL/min of Mobile Phase A (10 mM ammonium formate + 10 mM formic
acid in water) and Mobile Phase B (5 mM ammonium formate + 2 mM formic acid in 80% acetonitrile and
20% water) applying a gradient program as described by Diener et al., (2007). The gradient elution started
with 10% A and increased to 40% A within 1 min, held over 9 min, then declined back to 10% A within 5
min and stayed stable for 25 min.
2.4 Toxin standards
The TTX standard (Batch number APN09032-1-1) was purchased from Groupe Biomedix, Malaysia. Stock
solution of TTX was prepared in 0.03 M acetic acid and stored at – 20˚C. Standard solutions of TTX (5,
10, 50, 100, 500 and 1000 ng/ml) were prepared by dilution of the stock solutions with 0.03 M acetic acid.
Reference material for 4-epiTTX and 4,9-anhydroTTX were not available, therefore, the concentration for
the analogs was calculated with the calibration equation for TTX.
2.5 Statistical analysis
Data were analyzed by the statistical software of SPSS (Statistical Package for the Social Sciences)
version 16.0 for Windows. One way analysis of variance (ANOVA) test was used to compare differences
in the means of TTX of different species and tissues of puffer fish and followed by Duncan multiple range
test analysis to determine the difference between species. Means ± SD were reported and considered
different when p < 0.05.
3. RESULTS AND DISCUSSIONS
The mass chromatogram was scanned at m/z 320 and TTX (C11H17N3O8, 319.27 Da) came out at about 7
min after sample loading (Figure 1). For monitoring TTX at m/z 320 Da in the total ion current (TIC) mode,
the calibration curve was obtained using the standard TTX linear within the range of 5 – 1000 ng/ml (y = 2
12635.4 + 6251.32x, r = 0.9960) (Figure 2). The product ion m/z 162 was monitored because it had the
most abundant and stable ion. Figure 1 shows example of selected ion mass chromatogram of the liver
extract of L. lunaris. The extract gave a peak at a retention time of 7.18 min at almost consistent with that
the standard TTX. This toxin was identified as TTX, which was confirmed by comparing with the TTX
standard. TTX was detected in most all the species and tissues of the puffer fish. The mass spectrum of
TTX with the formation of the daughter ion 162 is shown in Figure 1. Extract of muscle of the same L.
lunaris also contained TTX showed the same toxin profile as that of the liver extract (data not shown).
The results of TTX concentrations in the different species of puffer fish are summarized in Table 1. All of
the species were found to contain TTX. There was significant difference in TTX concentrations for all
puffer fishes (p<0.05). In this study, the maximum amount of TTX in the family Tetraodontidae ranges
from 19.0 to 562 µg/g. The highest mean value of TTX concentration was detected in A. immaculatus
(275.26 µg/g) and the lowest was detected in L. lunaris (5.37 µg/g). The maximum amount of TTX in D.
holocanthus and D. hystrix from the family of Diodontidae was 22.67 µg/g and 20.76 µg/g respectively.
While the maximum amount of TTX in O. nasus from the family of Ostraciidae was 22.91 µg/g. From the
ANOVA, the puffer fishes can be divided into three groups according to their TTX levels. There was
significant difference in TTX concentration for A. immaculatus (275.26 µg/g) and C. patoca (261.09 µg/g)
among the puffer fishes (p<0.05) (Table 1). Among the Lagocephalus sp., L. inermis showed the highest
mean TTX concentration (62.7 µg/g), followed by L. sceleratus, L. spadiceus and L. lunaris (13.9 µg/g, 9.6
µg/g and 5.3 µg/g respectively).
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TTX
RT: 0.00 - 29.99
NL:
5.07E4
TIC MS
RDVLCMS
MS2_11062
8_TTX009
7.17
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
1.15
5
3.42 3.60
13.22 14.45
8.46
17.53 19.81
23.35 24.76 28.11
0
0
5
RT: 0.00 - 30.00
10
15
Time (min)
20
25
NL:
7.19E4
TIC MS
398LlunarisL
v(b)
7.18
100
95
7.34
90
85
80
6.95
75
7.51
70
65
60
55
50
45
40
35
30
25
8.24
20
15
10
1.33
5
9.52
9.86
3.94
4.40
10.35
11.60
0
0
5
10
398LlunarisLv(b) #1 RT: 0.01 AV: 1 NL: 2.05E2
T: + c ESI SRM ms2 320.000 [161.995-162.005]
100
14.37
15
Time (min)
19.03
27.00
22.48
20
25
162.00
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
161.996
161.998
162.000
m/z
162.002
162.004
Figure 1 LC-MS/MS of TTX standard (left) and the liver extract of L. lunaris.
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Figure 3 TTX calibration curve
Table 1 TTX concentration (µg/g) in different species of puffer fishes.
Species
DIODONTIDAE
D. holocanthus
D. hystrix
OSTRACIIDAE
O. nasus
TETRAODONTIDAE
A. immaculatus
A. manilensis
A. stellatus
C. patoca
T. pallimaculatus
L. inermis
L. sceleratus
L. lunaris
L. spadiceus
T. nigroviridis
X. naritus
Std.
Deviation
Std. Error
Minimum
Maximum
1.04 x 10
c
0.99 x 10
c
12.24
12.56
4.33
6.28
nd
nd
22.67
20.76
c
11.54
4.08
0.15
22.91
2a
196.03
112.22
10.91
32.26
233.96
71.17
14.52
6.60
11.02
113.62
44.07
9.80 x 10
3.97 x 10
3.86
1.61 x 10
8.27 x 10
2.52 x 10
5.13
1.65
2.46
2.42 x 10
8.33
97.97
10.37
nd
212.79
nd
1.26
0.39
nd
nd
nd
nd
469.91
279.18
24.01
278.65
561.64
188.41
31.30
19.54
23.64
429.49
185.11
N
Mean
8
4
8
1.11 x 10
4 2.75 x 10
b
8
9.74 x 10
c
8
0.58 x 10
2a
4 2.61 x 10
2b
8 1.45 x 10
bc
8 6.27 x 10
c
8
1.39 x 10
c
16 0.54 x 10
c
20 0.96 x 10
bc
22 6.37 x 10
c
28 2.90 x 10
Total
154 4.83 x 10
101.18
8.15
nd
561.64
Results are Means ± standard deviation of duplicates, Means with the same letter in each row are not
significantly different (p>0.05).
TTX concentrations obtained for each detected toxin in all tissue extracts of the puffer fish are shown in
Table 2. In general, liver showed the highest TTX concentration among the tissues of puffer fishes (Table
2). Among the puffer fishes, T. pallimaculatus showed highest toxic potency of 521.54 µg/g in its liver
followed by A. immaculatus (443.92 µg/g), C. patoca (254.63 µg/g), A. manilensis (101.33 µg/g) and T.
nigroviridis (100.56 µg/g). L. lunaris (6.64 µg/g) showed the lowest TTX concentration in its liver. In this
study, the gonad was available only in two species of puffer fish which is T. nigroviridis and X. naritus.
Between the species, T. nigroviridis showed higher TTX concentration (155.57 µg/g) compared to X.
naritus (30.84 µg/g). The lowest TTX concentration was detected in skin in all puffer fish. However, the
TTX concentration is not significantly different from the intestine and muscle. Among the puffer fishes, X.
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naritus showed the highest TTX concentration in skin (21.09 µg/g). TTX concentration in other puffer fish
was below 10 µg/g. However, it was not detected in the skin of D. holocanthus. Most of the muscle from
the puffer fishes showed high concentration of TTX with the highest was detected in C. patoca (276.54
µg/g) followed by A. manilensis (266.88 µg/g), L. inermis (152.54 µg/g) and A. immaculatus (106.59 µg/g).
No TTX was detected in the muscle of A. stellatus, D. hystrix and T. pallimaculatus. In all T. nigroviridis
specimens, gonad was the tissue containing the highest toxin levels (155.57 µg/g) followed by liver
(100.56 µg/g) whereas lower amounts were found in muscle (44.99 µg/g), intestines (22.37 µg/g) and skin
(4.27 µg/g). Comparison of the different tissues from X. naritus specimens, liver showed the highest toxin
amount (56.69 µg/g) followed by gonad (30.84 µg/g) while lower amounts were found in skin, muscle and
intestine (21.09 µg/g, 15.44 µg/g, 6.73 µg/g, respectively).
Table 2 TTX concentration (µg/g) in the tissues of puffer fishes.
Family/Species
Toxicity (µg/g)
Liver
Muscle
Skin
DIODONTIDAE
22.66 ± 0.02
21.09 ± 0.57
nd
D. holocanthus
20.76 ± 0.00
nd
D. hystrix
OSTRACIIDAE
22.77 ± 0.19
20.87 ± 0.27
0.27 ± 0.13
O. nasus
TETRAODONTIDAE
443.92 ± 36.74
106.59 ± 12.20
A. immaculatus
101.33 ± 18.14
266.88 ± 17.39
10.70 ± 0.39
A. manilensis
23.48 ± 0.74
nd
0.44 ± 0.12
A, stellatus
245.63 ± 46.45
276.54 ± 2.97
C. patoca
521.54 ± 56.69
nd
2.91 ± 4.20
T. pallimaculatus
92.94 ± 21.74
152.54 ± 50.71
2.63 ± 1.60
L. inermis
24.71 ± 2.33
29.99 ± 1.84
0.50 ± 0.16
L. sceleratus
6.64 ± 6.24
4.96 ± 9.75
3.15 ± 7.45
L. lunaris
10.36 ± 10.97
15.18 ± 11.73
3.06 ± 8.08
L. spadiceus
100.56 ± 187.91
44.99 ± 15.37
4.27 ± 10.55
T. nigroviridis
56.69 ± 72.93
15.44 ± 23.61
21.09 ± 3.14
X. naritus
Values are mean±s.d. of duplicates, nd = not detected, - = not tested
Intestine
Gonad
-
-
-
-
6.71 ± 4.15
22.37 ± 12.08
6.73 ± 8.93
155.57 ± 183.43
30.84 ± 19.69
The TTX concentration in different tissues of puffer fishes also showed that all the tissues were toxic and
significantly different (p<0.05) (Table 3). Comparing the maximum TTX concentration found in the different
tissues, the liver showed the highest at 561.64 µg/g followed by the gonad (429.48 µg/g), the muscle
(279.18 µg/g), the intestine (35.03 µg/g) and the skin (33.83 µg/g). Although there was no significant
difference in TTX concentration between the intestine and skin, the concentration of TTX in the skin was
the lowest among the tissues tested (Table 3).
Table 3 TTX concentration (µg/g) in different tissues of puffer fishes.
Tissues
N
Mean
Std. Deviation
Std. Error
b
Minimum
Gonad
8
9.32 x 10
137.95
4.88 x 10
10.83
a
Intestine
12
1.19 x 10
11.21
3.24
nd
b
Liver
44
9.10 x 10
149.13
2.25 x 10
nd
ab
Muscle
44
5.11 x 10
80.42
1.21 x 10
nd
a
Skin
44
0.69 x 10
10.43
1.57
nd
Total
152
4.90 x 10
101.70
8.25
nd
Results are Means ± standard deviation of duplicates.
Means with the same letter in each row are not significantly different (p>0.05).
160
Maximum
429.49
35.03
561.64
279.18
33.83
561.64
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Six point calibration curves from mass spectrometric detection were calculated for TTX. Low LOD for TTX
and a good correlation of the data for the concentration ranges tested could be observed in Table 4.
Therefore, all TTX toxins present in independent of the puffer fish could be determined. Liquid
chromatography results from the puffer tissue extracts and reference standard TTX gave the same
fragmentation products after MS/MS revealed that the puffer tissue extracts also contained TTX.
Nagashima et al. (2011) demonstrated that TTX was a major toxic principle and showed the same toxin
profile in the muscle and liver extract of L. lunaris by LC/ESI-MS. The retention time of puffer fish TTX
sometimes was not identical to standard TTX. The difference was probably due to TTX exists as a mixture
of its derivatives in puffer fish (Nakamura and Yasumoto, 1985). Generally toxins in puffer fish are thought
to be TTX, but paralytic shellfish poisoning toxins have also been detected in some puffer fish including
freshwater ones from tropical regions (Kungsuwan et al., 1997; Sato et al., 1997). Helbig and Luckas
(2010) also reported the PSP toxins detected in Takifugu poecilonotus were composed of Neo, STX and
dcSTX.
Table 4 Retention time, LOD (S/N 3:1) and equation of calibration curve for TTX analysed by LC-MS/MS
Toxin
m/z > m/z
Retention time
Calibration equation
Correlation
(min)
TTX
320 > 162
7.17
LOD
(ng on column)
Y = 6251.32x – 1263.4
0.9960
0.05
Present study showed that all of the puffer fish species collected from Sabah and Sarawak waters
contained TTX, whereas toxin distribution between either the tissues of the same fish or other species
was unequal. TTX levels in L. spadiceus, T. nigroviridis and X. naritus that collected from Kuching was
higher compared to the same species collected from Pulau Mandi Darah, Sabah, Sampadi and Batang
Sadong, Sarawak (data not shown). Different toxin levels detected in the same species could be attributed
to the fact that fish were caught in different areas. Rodriguez et al. (2012) demonstrated that toxin
distribution within the tissues of six L. sceleratus specimens was different depending on fish size, area
and season where fish were caught.
Among the 14 species examined, 11 species were belonging to the Tetraodontidae family, whereas only
two and one species was belong to the Diodontidae and Ostraciidae family respectively. From the study, it
showed that most of the species of the family Tetraodontidae are toxic while all species of the family
Diodontidae and Ostraciidae are weakly toxic. This result is in agreement with the study by Tani (1945)
and Noguchi and Arakawa (2008). Distribution of TTX in puffer fish bodies appears to be species-specific.
In all puffer fish specimens from this study showed that TTX were found the highest amount in liver, gonad
and muscle whereas intestine and skin contained lower amounts. Present study showed all of the species
were found to be toxic for human consumption as the TTX level was more than 2 µg/g (10 MU/g) (Ngy et
al., 2009).
Generally, in marine species of puffer fish, liver and ovary show the highest toxicity (more than 1000
MU/g), followed by intestines and skin (Noguchi et al., 2006) and the Japanese Ministry of Health, Labour
and Welfare has prohibited these organs from being used for food from all species of puffer fish (Arakawa
et al., 2010). However, muscles in many toxic species are regarded as edible (Mahmud et al., 2001). Even
though X. naritus (28.99 µg/g), T. nigroviridis (63.68 µg/g) and L. spadiceus (9.62 µg/g) are categorized as
safe to eat (Froese and Pauly, 2011), it could be considered unsafe for human consumption. Excessive
consumption (> 1 g) of these is harmful. The minimum lethal dose and minimum acute dose of TTX to
human (wt. 50 kg) are estimated to be around 2 mg and 0.2 mg, respectively (Katikou et al., 2009). From
this study, muscles of C. patoca showed the highest toxicity (276.54 µg/g) followed by A. manilensis
(266.88 µg/g). Kungsuwan (1993) also found highest toxicity from muscle of C. patoca followed by L.
lunaris. Ghosh et al. (2004) showed the highest level of toxicity was detected in ovaries (136 MU/g). Most
of the Lagocephalus species in present study showed the highest toxicities in muscle, although the
gonads, liver and intestine were frequently the most toxic tissues. Muscle also showed high toxicity levels
in different Lagocephalus species in other studies (El-Sayed et al., 2003; Noguchi et al., 2006; Helbig and
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Luckas, 2010). In contrast, L. spadiceus was found to be a non-toxic species (Berry and Hassan, 1973;
Brillantes et al., 2003) and this species was used as raw material to make fish balls by the local fish
processing factories in Thailand. Among the puffer fishes in this study, X. naritus or locally known as “ikan
buntal kuning” is considered a delicacy by the local community in Sarawak. It is prepared either by direct
cooking, dried, salted, smoked or fermented. There have been reports on the toxicity of X. naritus from
Malaysia (Othman Bojo et al., 2006; Mohamed et al., 2008). TTX was also found in all parts of tissue of X.
naritus from Andaman Sea (Kungsuwan, 1993).
Food poisoning was reported from different geographical regions due to puffer fish ingestion and the
lethality was dependent on the concentration of TTX present in consumed fish tissues (Chou et al., 1994).
Puffer fish poisoning is considered to be the common cause of fish poisoning along the coasts of Asia
(Chew et al., 1983). To our knowledge, this is the first report on the occurrence of TTX from Malaysian
puffer fishes. In Malaysia, puffer fish is classified as trash fish which having no market value and they are
not consumed by local people. However, some of the species have been considered edible and non-toxic.
Information on the safety of puffer fish consumption is insufficient. Therefore, their consumption or
preparation as food to prevent poisoning required special regulations (Nunez-Vaazqueza et al., 2000).
Preparing and cooking puffer fish require special technique about which most people are ignorant. During
the preparation, the liver, gonads, intestines and skin which contain the highest level of toxin should be
removed carefully and usually carried out by experienced individuals. Consumers of puffer fish should be
educated on the potential risk of eating them, the warning symptoms and signs, and when to seek medical
advice. Based on the findings of our study, it can be concluded that certain puffer species and the amount
consumed may cause serious health effect on humans. It was also showed that the occurrence of TTX in
Malaysian marine puffers caused intoxication to consumers. To ensure the safety of consumers in
Malaysia, puffer fish must be closely monitored as the TTX levels may vary due to bacteria accumulation,
geographical habitats, ecological food chain and season of the year and sex. The results also indicate that
LC-MS/MS assay is applicable for the determination of TTX and has the potential to replace the mouse
bioassay. The findings on TTX levels of the puffer fish in this study gave some information on toxicity for
Malaysian puffer fish.
ACKNOWLEDGEMENTS
The authors wish to thank Mr. Mohammed Mohidin from Fisheries Research Institute Sarawak Branch,
Bintawa, Kuching, Sarawak for the supply of puffer fish samples. Appreciation goes to the Head of
Fisheries Product and Food Safety Division, FRI, Penang, Hj. Rosly Hassan for his support and special
thanks are extended to all technical staffs for their assistance in the study.
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OCCURRENCE OF THREE ALEXANDRIUM SPECIES, A. affine, A. tamutum and A. tamiyavanichii
IN KUCHING WATERS
1
1
1
2
3
Kieng-Soon HII , Toh-Hii TAN , Chui-Pin Leaw , Hui-Chin CHAI , Yoshinobu TAKATA and
2*
Po-Teen LIM
1
Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, Kota Samarahan,
94300 Sarawak, Malaysia
2
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Kota Samarahan, 94300
Sarawak, Malaysia
3
School of Marine Bioscience, Kitasato University, Japan
*Email: ptlim@frst.unimas.my
Abstract
A field survey was carried out in Kuching waters to monitor harmful microalgae in Kuching waters.
Samples were collected fortnightly from Semariang Batu and Santubong estuaries during high tide. Live
samples were used for culture establishment, while preserved samples were processed for morphological
observation under epifluorescence microscopy. The occurrence of Alexandrium affine, A. tamutum and A.
tamiyavanichii is reported for the first time in the coastal waters of Sarawak, with A. tamutum as a new
record in Malaysian waters, which increased the number of Alexandrium species found to eight species.
They are A. affine, A. leei, A. minutum, A. peruvianum, A. tamarense, A. tamiyavanichii, A. tamutum and
A. taylori. This study has provided further information to the microalgae species inventory of the country.
Keywords: Alexandrium affine, A. tamutum, A. tamiyavanichii, Kuching.
1. INTRODUCTION
Since the first outbreak of harmful algal blooms (HABs) related shellfish poisoning event in 1976, paralytic
shellfish poisoning (PSP) has caught the attention of researchers in the county. Besides the well-known
PSP-toxin producer, Pyrodinium bahamense var. compressum, other PSP-toxin producing organisms,
particularly species in the genus Alexandrium has become the main focus in local research (Usup et al.,
2002a, b; Lim et al., 2003, 2004, 2005a, 2006, 2007; Lim and Ogata, 2005; Leaw et al., 2005). Previously
seven species of Alexandrium had been reported from the coasts of Malasyia (Usup et al. 2002b, Lim et
al. 2005). They were A. affine, Alexandrium leei, A. minutum, A. peruvianum, A. tamarense, A.
tamiyavanichii and A. taylori. Distribution of each species is scattered, and not all species reported were
present throughout the waters. Alexandrium affine was found in the northern and southern of the Straits of
Malacca. On the other hand, A. tamiyavanichii was found only in the central to southern of the Straits of
Malacca (Anton et al., 2000; Usup et al., 2002b). Two Alexandrium species were reported previously from
Kuching waters, A. peruvianum and A. taylori (Lim et al., 2005). The occurrence of these toxic species in
Sarawak will certainly pose a threat to the aquaculture industries and public health if blooms of these
species occur. In this field survey, we aim to document the occurrence of this genus and to provide
reference micrographs for country HAB monitoring purposes.
2. MATERIALS AND METHODS
2.1 Samples
Field samplings were undertaken fortnightly at two sampling sites in the estuary of Kuching, Sarawak.
Qualitative plankton samples were collected using a 20 µm mesh size plankton net. Concentrated
samples were preserved in δugol’s iodine solution. δive samples were collected for isolation and
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establishment of clonal cultures. Single cell isolation technique was used to obtain clonal cultures.
Cultures were maintained in ES-DK medium (Kokinos & Anderson, 1995) at 25°C under 12h: 12h light:
dark photoperiod.
2.2 Morphological observation
Field and cultured samples were examined under an Olympus IX51 inverted microscope (Olympus,
Japan) using normal light under 200 to 600×. For epi-fluorescence microscopy, cells were fixed in 2%
formaldehyde and the theca plates were stained with Fluorstain (Fluka, Japan) for 1 min. Samples were
transferred onto a microscope slide and covered with slips. Samples were then viewed under Olympus
IX51 epifluorescence microscope equipped with UV filter set. Digital images were captured using XC30
CCD camera (SIS, Germany). Identification was based on Balech (1995).
2.3 Toxin Analysis
Cultures at exponential phase were harvested by centrifugation (2,000×g. 5 min). Cell pellets were then
extracted using 5% acetic acid and tested on SKit ELISA for PSP (Shin Nihon Kentei Kyokai, Japan)
according to the manufacturer’s instruction.
3. RESULTS AND DISCUSSION
3.1 Alexandrium affine (Inoue and Fukuyo) Balech (1985)
3.1.1 Morphology
Alexandrium affine is a chain forming species which usually forms chains of 2-8 cells long (Figure 1A).
The cells are normally longer (29-66 m) than wide (23-65 m) and convex-pentagonal. The epitheca is
longer than the hypotheca, and is conic-convex. The first apical plate (1´) is directly connected with apical
pore plate and a small ventral pore (vp) is present toward the anterior of 1´´ along the right margin (Figure
1C). The sulcal plate of the A. affine is longer than wide, with both anterior ends well projected (Figure
1D).
The posterior sulcal plate (s.p) is wider than long (Figure 1E). The main diagnostic feature of this species
is the location of the anterior attachment pore in the APC. The anterior attachment pore of the A. affine is
located at the dorsal half of APC (Figure 1F).
3.1.2 Distribution
Alexandrium affine was previously reported in Japan, Korea, Spain, Portugal, Gulf of Thailand and
Philippines (Balech, 1995). It was also found in Vietnam (Nguyen-Ngoc, 2004). In Malaysia, the species
was previously reported from Sebatu and Pulau Aman in the Straits of Malacca (Usup et al. 2002). A.
affine was found in Semariang Batu, Kuching in this study.
3.1.3 Toxicity
Toxicity test showed undetectable value from the extract.
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Figure 1 Alexandrium affine (AaSM37, AaSM86 and AaSM94) from Semariang Batu, Sarawak. LM. (A) A
chain of vegetative cell with rounded epitheca. Scale bar = 100 m. (B) Auto-flourescence of the cell
showing the arrangement of chloroplast. Scale bar = 100 m. (C) Apical-ventral view of cell, vp: ventral
pore. 1', 4': first, forth apical plates. 1',' 5'' and 6'': first, fifth, and sixth precingular plates. (D) Anterior sulcal
plate (s.a.). (E) Cell showing the elongated posterior sulcal plate (s.p.). 2'''': second antapical plate. (F)
Cell showing the apical pore complex (APC). Scale bars= 10 µm.
3.2 Alexandrium tamutum Montresor, Beran and John (2004)
3.2.1 Morphology
Alexandrium tamutum is a solitary single cell, oval shape, relatively small, 24-35 µm long and 26-32 µm
wide (Figure 2A). The A. tamutum cells have a short and wide posterior sulcal plate (SP) (Figure 2F). The
first apical plate (1´) is directly connected with apical pore plate and a small ventral pore is present which
located toward the anterior of 1´´ along the right margin (Figure 2D-E). It also lacks of the anterior
attachment pore at the APC (Figure 2E). The kidney-shape nucleus of the cell is located at middle of the
cell (Figure 2C). The non-toxic A. tamutum cells are morphologically similar to the PSP toxin producer A.
minutum. The only distinctive feature between A. tamutum and A. minutum is the 6´´ precingular plate. A.
tamutum has a wider (widthμ length, ≈1) precingular plate (6´´) while A. minutum has a narrower (widthμ
length, ≈0.5) precingular plate (6´´) (Figure 2D).
3.2.2 Distribution
Alexandrium tamutum was first reported from Adriatic, Tyrrhenian, Mediterranean Sea (Montresor et al.
2004) , Northwest Pacific (Yoshida, 2002) and Sea of Okhotsk, Russia (Selina & Morozova, 2005). In
Malaysia, the species was first discovered from Semariang Batu, Kuching and Kudat, Malaysia. This
represents a new record of Alexandrium species occurrence in Malaysia.
3.2.3 Toxicity
No detectable value was shown in the extract of A. tamutum.
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Figure 2 Alexandrium tamutum (AuKA01) from Kuala Abai, Sabah. LM. (A) Solitary cell with relatively oval
shape. (B) Auto-flourescence of the cell showing the arrangement of chloroplast. Scale bar = 100 m. (C)
Fluorescence of kidney-shape nucleus. Scale bar = 100 m. (C) Ventral view of cell, vp: ventral pore. 1',
4': first, forth apical plates. 1'', 5'', 6'': first, fifth, sixth precingular plates. S.a: anterior sulcal plate. (E)
Apical view of the cell, APC: apical pore complex. (F) Antapical view of the cell. 2´´´, 3´´´, 4´´´: second,
third, fourth postcingular plates. 1´´´´, 2´´´´: first, second antapical plates. Sp: posterior sulcal plate. Scale
bars= 10 µm.
3.3 Alexandrium tamiyavanichii Balech (1994)
3.3.1 Morphology
Under light microscope, cells are round and heavy pigmented (Figure 3A, B). Cells in chains of 2, 4 and 8cells are frequently observed (Figure 4A). Calcofluor-stained thecal plates are easily diagnosed under a
fluorescence microscope. First apical plate (1') is rhomboidal and link directly to the apical pore complex
(APC) (Figure 4B-E). The ventral pore (vp) is present on the anterior right margin of the 1' (Figure 4B).
The apical pore (po) is oval with a large anterior attachment pore located at the anterior right margin of the
attachment pore in which this characteristic weres.aspecifically observed in certain Alexandrium species
including A. tamiyavanichii (Figure 4C). However, there is absent of anterior attachment pore in some
specimens (Figure 4D).
The long anterior sulcal plate (s.a) had a triangular to trapezoidal precingular part (p.pr.) which attached to
slightly concave posterior margin of 1'. The posterior sulcal plate (s.p.) is longer than wide (Figure 4F),
with a posterior pore connected by an irregular furrow to the right margin of the plate. The second
antapical plate (2'''') is wide (Figure 4G).
3.3.2 Distribution
Alexandrium tamiyavanichii was reported from Japan (Ogata et al. 1990; Nagai et al. 2005), Brazil
(Menezes et al. 2010) , South Africa (Ruiz Sebastian et al. 2005) , Thailand (Kodama et al. 1988) and
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peninsular Malaysia (Lim et al. 2006) . Alexandrium tamiyavanichii was reported from Semariang Batu in
this study (New record).
3.3.3 Toxicity
Extract showed positive result in the ELISA assay.
Figure 3 Alexandrium tamiyavanichii, AcSm01 from Semariang, Sarawak. LM. (A) A chain of two
vegetative cells. (B) Red autofluorescence showing the chloroplast content. Scale bar = 10 µm.
Figure 4 Alexandrium tamiyavanichii (AcSm01) from Semariang, Sarawak. (A) Chain of four vegetative
cells. (B) Apical-ventral view of cell. pr.p: precingular part. s.aμ anterior sulcal plate. There’s an oblique
posterior end of 1´ and a triangular shape of the p.pr. Apical plates 1´- 4´ and precingular plates 1´´, 2´´,
4´´ – 6´´. (C) Dorsal-apical view showing apical pore (Po) and precingular plates 2´´ – 5´´. (D) Close-up of
Po. (E) Apical view showing the ventral pore (v.p). (F) Antapical-ventral view showing postcingular plates
1´´´, 4´´´, 5´´´ and antapical plate 1´´´´. (G) Dorsal-antapical view showing postcingular plates 2´´´, 3´´´, 4´´´
and antapical plate 2´´´´. Scale bars = 10 µm.
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4. CONCLUSION
The new discovery of A. affine, A. tamiyavanichii and A. tamutum in this study has increased the known
number of Alexandrium species in Malaysian waters to eight species. Total of five species were reported
from Sarawak waters thus far, namely A. taylori, A. peruvianum (Lim et al. 2006), A. affine, A.
tamiyavanichii and A. tamutum (this study). The increasing number of toxic species in Malaysian waters
should be taken seriously by the monitoring agencies. Clonal cultures deposited in the University Malaysia
Sarawak (UNIMAS) culture collection will be useful for further study in the future.
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tamiyavanichii (Dinophyceae) to other Alexandrium species based on ribosomal RNA gene
sequences. Harmful Algae, 1(1), 59-68.
Usup, G., Leaw, C. P., Ahmad, A., & Lim, P. T. (2002b). Alexandrium (Dinophyceae) species in Malaysian
waters. Harmful Algae, 1(3), 265-275.
Yoshida, M. (2002). Alexandrium spp. (Dinophyceae) in the Western North Pacific. Fisheries Science,
68(1), 511–514.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Pteridocolous discomycetes IN BUKIT BENDERA, THE PENANG HILL, PULAU PINANG,
MALAYSIA
Hideyuki Nagao*, Muhammad Zulfa bin Mohd Razikin, and Rahmad Zakaria
School of Biological Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia
*Email: nagaoh@usm.my
Abstract
Bukit Bendera is 833m above sea level and is situated in the Northern part of Penang Island. Day time
temperature there is generally about 5°C cooler than that at sea level. The hill dipterocarp forest
dominates on Bk. Bendera and tree fern scatteredly grows at higher altitude. Six pteridocolous
Hyaloscyphaceae taxa were observed on decayed rachis of tree ferns, Cyathea spp. Lachnum lanariceps,
L. oncospermatum, Lachnum sp. 1 which was characterized by black resinous matter on hair were
observed at the frequency of 0.2 to 0.25. Two Lachnum taxa, dark red resinous matter on hair type and
finely ornamented red resinous one type, were close to L. lanariceps. These taxa were appeared on the
rachis at the frequency of 0.1 and 0.15, respectively. Lachnum lanariceps is newly recorded in Malaysia.
Black resinous matter borne Lachnum sp. 1 was newly observed other than Endau Rompin, Johor. Trident
apothecia type with bright red resinous matter was collected from Cyathea sp. and non tree fern species.
Although trident apothecia were multiplicate, its appearance was different from those of L.
oncospermatum. Ascospores of Lachnum spp. could germinate and grow on Potato sucrose agar.
Cryptodiscus sp. was only observed on a specimen of Cyathea sp.
Keywords: biodiversity, Cyathea, Lachnum, morphology, taxonomy, tree fern
1. INTORDUCTION
Tree ferns are recognized as the fern with a tall trunk-like rhizome and are distributed in tropical lowland to
submontane environments, as well as subtropical and Southern hemisphere temperate forests (Large and
Braggins, 2004; Piggott, 1988). On the decayed rachides of tree ferns, several particular discomycetes
were identified (Dennis 1958; Haines 1980, 1992; Nagao 1996, 2008; Nagao and Doi 1996; Penzig and
Saccardo, 1904; Spooner 1987; Wu et al. 1998; Wu and Wang 2000). In Southeast Asia, some new
species were recorded in Java (Penzig and Saccardo, 1904).
Bukit Bendera is 833m above sea level and is situated in the Northern part of Penang Island. Day time
temperature there is generally about 5°C cooler than that at sea level. The hill dipterocarp forest
dominates on Bk. Bendera and tree fern scatteredly grows at higher altitude. However, there was no
recognition of pteridocolous fungus from Bk. Bendera.
In this report, we described six pteridocolous Hyaloscyphaceae taxa on decayed rachides of tree ferns,
Cyathea spp.
2. MATERIALS AND METHODS
2.1 Collection cites
Decayed rachides of tree fern were collected along the jeep road in Bukit Bendera. Seven specimens on
Cyathea spp. were obtained.
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2.2 Observations of micro-morphology
Fruiting bodies on the samples were immediately observed to sort the type of apothecia. Specimens of
discomycetes were sectioned by freehand using a razor-blade. Sections were mounted in distilled water
or Shear’s solution for light microscope observation. 3% KOH-1% phloxine and εelzer’s reagent were
properly added to detect septa formation and positive iodine reaction in the ascus pore, respectively. For
the further observation, asci, ascospores, paraphyses, and the texture of apothecium and stipe were
prepared from a squashed section on the slide glass. Color names are from a mycological colour chart
(Rayner, 1970).
2.3 Germination of ascospores of Lachnum spp. and its cultivation
Ascospores from fresh apothecia were subjected to be ejected on the Potato sucrose agar medium (PSA).
Apothecium was attached on the surface of water agar cube (ca. 5 x 5 x 3 mm) inside the lid of Petri dish
for 24-48 hr. After the ascospores ejected, the apothecium and agar cube were removed from the lid.
Germinated ascospore was transferred to the new PSA and cultured at room temperature, ca. 27°C.
3. RESULTS AND DISCUSSION
3.1 Taxonomy
3.1.1 Lachnum lanariceps (Cooke & Phillips) Spooner
Apothecia minute, stipitate, heavily covered with hairs. Hairs cylindrical, obtuse, pale Buff or Straw,
septate, finely granuated and bearing red or garnet resinous matter. Asci 51.1-66.8 × 3.3-5.2 m.
Ascospores 15.0-24.2 × 1.7-2.λ m. Lachnum lanariceps is newly recorded in Malaysia.
Two Lachnum taxa, dark red resinous matter on hair type and finely ornamented red resinous one type,
were close to L. lanariceps. These taxa were appeared on the rachis at the frequency of 0.1 and 0.15,
respectively.
3.1.2 Dark red resinous matter on hair type (H32444)
Asci 45.4-49.2 × 3.4-4.8 m. Ascospores 16.3-28.2 × 1.6-3.1 m. Paraphyses 65.5-65.7 × 1.9-2.2 m.
Hair pale buff, 17.7-39.5 × 2.7-4.4 m. From the morphological observation, this dark red resinous matter
type was considered as L. lanariceps.
3.1.3 Finely ornamented red resinous matter type (H32442) – Lachnum sp. 2
Ascospores 10.0-14.2 × 1.0-1.7 m. Hair Amber, darker than those of the dark red resinous matter on hair
type and L. lanariceps, 30.1-44.8 × 2.2-4.λ m, with Pure Yellow or δuteous granules, 0.5-2.λ m. From
the morphological observation, these characteristics does not match the description of known species.
This specimen is temporally proposed as Lachnum sp. 2.
3.1.4 Lachnum oncospermatum (Berk. & Broome) M.L.Wu & J.H.Haines
Apothecia minute, stipitate, branched, deeply lobed, gregarious, covered with hairs. Hairs finely
granulated entirely, containing pale yellow to yellow pigment, with amber-coloured resinous materials.
Asci 40.1-49.1 × 3.1-3.8 m. Ascospores 11.4-17.6 × 1.4-2.0 m. This species is common on Cyathea
spp. in Cameron Highlands.
3.1.5 Lachnum sp. 1
This species is characterized by black resinous matter on hairs and were observed at the frequency of 0.2
to 0.25. Black resinous matter borne Lachnum sp. 1 was newly observed other than Endau Rompin,
Johor.
3.1.6 Trident apothecia type (32H456)
3 apothecia appeared from the same origin. Hair Orange to Luteous, finely granulated, 22.3-34.5 × 1.8-3.8
m. Examined specimens were immature then no ascospore were observed yet. This specimen is similar
to L. nudipes (Fuckel) Nannf. in terms of gregarious nature of apothecia but further examination is
required for the identification. Trident apothecia type with bright red resinous matter was collected from
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Cyathea sp. and non tree fern species. Although trident apothecia were multiplicate, its appearance was
different from those of L. oncospermatum.
3.1.7 Cryptodiscus sp.
Cryptodiscus sp. was only observed on a specimen of Cyathea sp. This fungus embedded in the rachis
and its hymenium was erumpent from the epidermal tissue of rachis.
3.2 Results of ascospore germination of Lachnum spp. and its cultivation
Ascospore germination of Lachnum spp. was generally observed after 48hr incubation. Germination was
mainly occurred from the both ends of ascospore. The colonies of Lachnum spp. grew on Potato sucrose
agar.
4. CONCLUSION
Even though the population of tree ferns in Bk. Bendera was limited, six pteridocolous Hyaloscyphaceae
taxa and Cryptodiscus sp. were identified on decayed rachides of tree ferns, Cyathea spp.
REFERENCES
Dennis RW (1958) Critical notes on some Australian Helotiales and Ostropales. Kew Bull 13: 321-358
Haines JH (1980) Studies in the Hyaloscyphaceae I: some species of Dasyscyphus on tropical ferns.
Mycotaxon 11: 189-216
Haines JH (1992) Studies in the Hyaloscyphaceae IV: The genus Lachnum (Ascomycetes) of the
Guayana Highlands. Nova Hedwigia 54: 97-112
Large MF, Braggins JE (2004) Tree ferns.pp.359, Timber Press, Inc, London
Nagao H (1996) Discomycetes on decayed tree fern. (2) Lachnum varians (Rehm) Spooner and Lachnum
sclerotii (A. L. Smith) Haines et Dumont new to Japan. Bull Natl Sci Mus 22: 105-111
Nagao H (2008) Discomycetes on decayed tree fern. (3) Lachnum lanariceps and Lachnum
oncospermatum new to Japan. Mycoscience 49: 403-406
Nagao H, Doi Y (1996) Discomycetes on decayed tree fern. (1) Lachnum pteridophyllum (Rodway)
Spooner new to Japan. Bull Natl Sci Mus 22: 19-22
Penzig O, Saccardo PA (1904) Icones fungorum javanicorum, pp. 75-83.E J Brill, Leiden
Piggott AG (1988) Ferns of Malaysia in colour. pp. 458, Tropical Press SDN. BHD., Kuala Lumpur
Spooner BM (1987) Helotiales of Australasia: Geoglossaceae, Orbiliaceae, Sclerotiniaceae,
Hyaloscyphaceae. Bibl Mycol 116: 470-474
Wu ML, Haines JH, Wang YZ (1998) New species and records of Lachnum from Taiwan. Mycotaxon
67:341-353
Wu ML, Wang YZ (2000) Mycological resources of saprophytic ascomycetes in Fushan
Forest. Fung Sci 15: 1-14.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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POPULATION GENETICS OF Macaca fascicularis (CERCOPITHECIDAE) FROM PENANG,
MALAYSIA INFERRED FROM MITOCHONDRIAL CONTROL REGION SEGMENT
Jeffrine J. Rovie-Ryan
1, 2,*
1
1
2
, Frankie T. Sitam , Zaaba Zainol Abidin , Soon Guan Tan , and M. T.
3
Abdullah
1
Wildlife Genetic Resource Bank (WGRB) Laboratory, Ex-Situ Conservation Division, Department of
Wildlife and National Parks (DWNP) Peninsular Malaysia, KM10 Cheras Road, 56100 Kuala Lumpur,
Malaysia,
2
Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia,
3
Department of Zoology, Faculty of Resource Science and Technology (FRST), Universiti Malaysia
Sarawak (UNIMAS), 94300 Kota Samarahan, Sarawak, Malaysia
Email: jeffrine@wildlife.gov.my, j_rovieamit@yahoo.com
Abstract
We examine the population genetics of the cynomolgus macaques from the Penang Island and the
surrounding mainland areas using the control region (D-loop) segment of the mitochondrial DNA. 20
haplotypes were described from 46 individuals with a single haplotype sharing between the mainland and
the island population. Nucleotide diversity was observed higher in the mainland (Pi= 0.01339) as
compared to the island population (Pi= 0.00927). The sedentary nature of females delineates the
population’s structure as expected. Comparison between the mainland populations (I-L) and the island
populations (A-H) revealed an Fst index at 0.36 which suggest very great differentiation. Phylogenetic
trees generally separates the Penang M. fascicularis haplotypes into three major groups. We hypothesize
that the first group of M. fascicularis invaded both the mainland and island of Penang at ~ 0.5 Mya.
Keywords: Macaca fascicularis, population genetics, Penang Island, control region
1. INTRODUCTION
The long-tailed macaque, Macaca fascicularis, also known as the cynomolgus macaques are widely
distributed in nature and occupies vast area of the mainland southeast Asia (Thailand, Cambodia,
Vietnam, Laos, Myanmar, the Peninsular Malaysia and Singapore) and the islands of the Greater and
Lesser Sunda Islands (Indonesia and the Malaysian Borneo) and the Philippines. They can be found
almost everywhere especially at low elevations preferring the mangrove and swampy forests, river banks
and seashore (Eudey, 2008). In Peninsular Malaysia they are very common and populate areas in
sympatric with the human populations. In Penang particularly, M. fascicularis are distributed all throughout
the islands (Penang Island and smaller adjacent islands including Jerejak Island) and the mainland side of
Penang (Seberang Perai).
The patterns of biogeographical distribution and genomic divergence of primates in their present habitats
generally reflect the historical biogeography of the area (Whitmore, 1981; Morley & Flenley, 1987).
Macaca in particular, displays an extreme level of sex-biased dispersal (Melnick & Hoelzer, 1993) where
only male disperse from one population to another while female remain sedentary in nature. The
sedentary nature of female macaques leads to geographically structured mitochondrial haplotypes where
phylogenetic tree would show structuring of populations according to historical cladogenic events (Melnick
et al., 1993; Tosi & Coke, 2007). Historically, Peninsular Malaysia was part of Sundaland which were
heavily influenced by the events during the Pliocene dan Pleistocene epoch. During the Pliocene and
Pleistocene (Quartenary Period), periods of glacial maxima caused the sea levels to fall by 120 m below
present-day levels and landmasses (Malay Peninsula, Borneo, Sumatra, Java, Bali, Palawan, the
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Mentawai Islands, and the smaller intervening islands) which are currently separated were joined and
formed the Sundaland (Harrison et al., 2006). The Penang Island, which is situated about 2 km of the
coast of Seberang Perai (the mainland portion of the Penang State) would therefore, experienced
repeated connection to the mainland Peninsular Malaysia during the Pleistocene glacial periods and
subsequently, glacial intervals separated the island from the mainland thus limiting faunal interchanges.
Therefore, Macaca is a prime candidate to be used as an animal model to investigate whether the current
population structure reflects the ancient/historical structuring especially in the Penang Island.
Very few genetic researches have been conducted on M. fascicularis making the information on their
genome sequence, gene expression arrays and also single-nucleotide polymorphism (SNP) map which
are important to understand their complex trait relationships, not available (Street et al., 2007; Kikuchi et
al., 2007). Several authors studied the intraspecies variation (Smith et al., 2007), population structure
(Lawler et al., 1995; Harihara et al., 1988; Smith & McDonough, 2005; Kawamoto et al., 2007; Kawamoto
et al., 2008; Shiina et al., 2010), phylogeography (Tosi et al., 2002; Marmi et al., 2004; Modolo et al.,
2005; Blancher et al., 2008), and demography (Melnick & Hoelzer, 1992) of M. fascicularis using
mitochondrial DNA (mtDNA). Smith et al. (2007) worked on the Malaysian population (however with
unknown locality) to characterize the genetic variation within and among five regional populations
(Indonesia, Malaysia, Mauritius, Phillippines, and Vietnam,) of M. fascicularis. In Malaysia, there are
isolated studies on cynomolgus macaques to investigate their conflict with human (DWNP, 2006),
association with disease (Cox-Singh & Singh, 2008), and distribution (Karimullah & Shahrul, 2011). This
pilot study is part of a major research initiative to comparatively examine the population genetics, and
phylogeography and biogeographical history of the M. fascicularis particularly in Malaysia (Peninsular
Malaysia, Sarawak and Sabah). In summary, this pilot study is designed to achieve these objectives; (1)
to examine the population structure of the Penang (both mainland and island) M. fascicularis and (2) to
investigate the efficiency of the control region mtDNA segment in addressing population structure of M.
fascicularis particularly in Malaysia.
2. MATERIALS AND METHODS
2.1 Sample Collection
Sampling was conducted on conflict long-tailed macaques as reported by the public both from the island
and the mainland of the Penang state and also as part of a Wildlife Disease Surveillance Programme
(WDSP) launched by the DWNP in 2011 to monitor the emergence of zoonotic diseases in wildlife
species. Table 1 provides the details of each samples used in this study. Various types of samples were
collected whenever possible/permitted such as blood (FTA card, serum, RBC, slide smear), swabs (rectal,
urinal and buccal) and biopsies (liver, lung, kidney, small and large intestines). Data such as including
sex, maturity, locality (GPS coordinates), and body condition were collected and all data were
incorporated into a database maintained by the DWNP. The sampling was conducted according to all the
rules and regulations by all the responsible authorities in Malaysia. All samples were kept at the WGRB
Laboratory, DWNP.
2.2 DNA Extraction, PCR Amplification and Sequencing
Total genomic DNA was extracted from 46 samples either from blood (preserved in lysis buffer) or from
liver samples using the QIAamp DNA Kit (QIAGEN Ag., Germany) protocol for blood and tissue samples
as provided by the manufacturer. In order to look into the population genetics information on the M.
fascicularis, we incorporate sequence data from the control region (CR) of the mitochondrial DNA
(mtDNA). A pair of primers; WGRB/MFCR/F15978 and WGRB/MFCR/R580, were designed using the
program Primer3 (Rozen & Skaletsky, 2000) as a plug-in in the Geneious program v5.6 (Drummond et al.,
2012) to cover the entire length of the CR. Amplifications were carried out in an Arktik Thermal Cycler
(Thermo Scientific), using a 15µl reaction volume consisting of 0.5µl of DNA template (~15–20ng), 0.2 µl
(0.13 µM) of each primers and 14.5 µl of GoTaq® Colorless Master Mix (Promega). Amplification was
done using the following PCR profile; a preliminary denaturation at 98C for 2 min followed by 30 cycles of
95C for 30 sec, 69C for 30 sec and 72C for 40 sec. This was followed by a final extension period of
72C for 3 min before the samples were cooled to 4C. Cycle sequencing on both primers were done as a
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st
‘proofreading’ check of the data on the ABI PRISM®377 DNA Sequencer (1 Base Laboratories Sdn.
Bhd., Selangor, Malaysia).
2.3 mtDNA Sequence Analysis
Multiple alignments of the sequences were done and ambiguous flanking regions were identified and
removed from the analysis by using the program Geneious v5.6 and later manually edited. Sequence
characterisation (variable sites, conserved sites and parsimony-informative sites) were done using MEGA
version 5 (Tamura et al., 2011). Standard genetic diversity indices including the number of haplotypes,
haplotype diversity (Hd) (Nei, 1987), and nucleotide diversity (Pi) (Nei, 1987) were calculated using the
DNA polymorphism option implemented in DnaSP v5 (Librado et al., 2009).
No
Sample Label
Location/GPS of capture
Location
in Map
Sex
Hap. No.
Table 1 Detail information of each macaque sample collected from the Penang State.
GenBank
Acc. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
WDSP/11/0037
WDSP/11/0038
WDSP/11/0039
WDSP/11/0040
WDSP/11/0041
WDSP/11/0042
WDSP/11/0043
WDSP/11/0044
WDSP/11/0045
WDSP/11/0046
WDSP/11/0047
WDSP/11/0048
WDSP/11/0049
WDSP/11/0050
WDSP/11/0051
WDSP/11/0052
WDSP/11/0053
WDSP/11/0054
WDSP/11/0055
WDSP/11/0056
WDSP/11/0057
WDSP/11/0058
WDSP/11/0059
WDSP/11/0060
WDSP/11/0061
WDSP/11/0062
WDSP/11/0063
WDSP/11/0064
WDSP/11/0065
WDSP/11/0066
WDSP/11/0067
WDSP/11/0068
WDSP/11/0069
WDSP/11/0070
WDSP/11/0071
WDSP/11/0072
WDSP/11/0073
WDSP/11/0074
Sg. Kecil, Seberang Perai, Penang
Sg. Kecil, Seberang Perai, Penang
Kuala Juru, Seberang Perai, Penang
Kuala Juru, Seberang Perai, Penang
Permatang Kriang, Seberang Perai, Penang
Permatang Kriang, Seberang Perai, Penang
Ladang Byram, Seberang Perai, Penang
Ladang Byram, Seberang Perai, Penang
Ladang Byram, Seberang Perai, Penang
Ladang Byram, Seberang Perai, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Jerejak Rainforest Resort, Jerejak Island, Penang
Sg. Baru, Teluk Kumbar, Penang Island
Sg. Baru, Teluk Kumbar, Penang Island
Surau MK 2, Tg. Bungah, Penang Island
The Reef Condo, Batu Feringghi, Penang Island
Permatang Pasir, Balik Pulau, Penang Island
Surau MK 2, Tg. Bungah, Penang Island
Surau MK 2, Tg. Bungah, Penang Island
Surau MK 2, Tg. Bungah, Penang Island
Surau MK 2, Tg. Bungah, Penang Island
Surau MK 2, Tg. Bungah, Penang Island
Bkt. Gambir, Gelugor, Penang Island
Bkt. Gambir, Gelugor, Penang Island
Bkt. Gambir, Gelugor, Penang Island
Permatang Pasir, Balik Pulau, Penang Island
Bkt. Gambir, Gelugor, Penang Island
Bkt. Gambir, Gelugor, Penang Island
Jln. Perak, Penang Island
L
L
J
J
I
I
K
K
K
K
H
H
H
H
H
H
H
H
H
H
H
G
G
B
A
E
B
B
B
B
B
F
F
F
E
F
F
D
F
F
M
M
M
M
F
F
F
F
M
M
M
M
M
M
M
M
M
F
M
M
F
M
M
M
M
M
M
F
F
M
M
M
M
F
M
M
1
1
1
1
1
1
1
1
1
1
2
3
3
4
4
5
6
7
7
7
7
7
8
9
10
10
10
10
11
12
12
12
13
14
15
16
17
17
JX113316
JX113317
JX113318
JX113319
JX113320
JX113321
JX113322
JX113323
JX113324
JX113325
JX113326
JX113327
JX113328
JX113329
JX113330
JX113331
JX113332
JX113333
JX113334
JX113335
JX113336
JX113337
JX113338
JX113339
JX113340
JX113341
JX113342
JX113343
JX113344
JX113345
JX113346
JX113347
JX113348
JX113349
JX113350
JX113351
JX113352
JX113353
177
No
Sample Label
Location/GPS of capture
Location
in Map
Sex
Hap. No.
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
GenBank
Acc. No.
39
40
41
42
43
44
45
46
ZMW486
ZMW487
ZMW488
ZMW489
ZMW490
ZMW491
ZMW492
ZMW493
Tanjung Tokong, Penang Island
Tanjung Tokong, Penang Island
Tanjung Tokong, Penang Island
Tanjung Tokong, Penang Island
Tanjung Tokong, Penang Island
Tanjung Tokong, Penang Island
Tanjung Tokong, Penang Island
Tanjung Tokong, Penang Island
C
C
C
C
C
C
C
C
F
M
F
F
M
M
F
M
18
19
20
20
20
20
20
20
JX113354
JX113355
JX113356
JX113357
JX113358
JX113359
JX113360
JX113361
2.4 Population Analyses
To analyse patterns of geographical subdivision, estimate of population subdivision (Fst) (Hudson et al.,
1992), were calculated using DnaSP v5 (Table 2) between each populations and later comparing between
the island populations (Populations A-H) to the mainland populations (Populations I-L). Finally, to estimate
genetic distances among the populations (Table 2), pair-wise distances were also calculated by using the
Kimura two-parameter model (Kimura, 1980) as performed using MEGA.
Mainland
Island
Table 2 Fst index between the populations (above the diagonal; estimated using Hudson et al., 1992) and
the genetic divergence between the populations (below the diagonal; estimated using Kimura-2 parameter
model, Kimura, 1980)
Pop.
A
B
C
D
E
F
G
H
I
J
K
L
A
B 0.020
0.34
0.08
0.64
0.96
0.41
0.47
0.00
0.96
0.96
C 0.019 0.006
-0.09 0.03
0.86
0.37
0.32
0.27
0.84
0.84
D 0.020 0.001 0.005
E 0.012 0.013 0.012 0.012
-0.05 0.00
-0.04 -0.23 0.07
0.15
0.15
F 0.019 0.004 0.003 0.004 0.010
0.97
0.52
0.48
0.46
0.97
0.97
G 0.004 0.018 0.018 0.018 0.010 0.017
0.75
0.22
0.90
1.00
1.00
H 0.017 0.009 0.011 0.009 0.014 0.010 0.017
0.27
0.36
0.75
0.75
I
0.014 0.021 0.018 0.020 0.017 0.020 0.013 0.019
0.44
0.22
0.22
J 0.021 0.004 0.006 0.003 0.013 0.004 0.019 0.010 0.021
0.89
0.89
K 0.016 0.022 0.021 0.022 0.018 0.021 0.014 0.023 0.019 0.023
0.00
L 0.016 0.022 0.021 0.022 0.018 0.021 0.014 0.023 0.019 0.023 0.000
2.5 Phylogenetic Relationship and Dating the Divergence of Penang of M. fascicularis Haplotypes
To infer phylogenetic relationships, the haplotype data were used to generate phyolgenetic trees. Tree
analyses was constructed by using the neighbour-joining (NJ), maximum parsimony (MP) and maximum
likelihood (ML) methods as implemented in MEGA, and also Bayesian method using MrBayes
(Huelsenbeck et al., 2001) as a plug-in in the Geneious program. The NJ clustering was performed by
using the Kimura 2-parameter distance model (Kimura, 1980) with pairwise deletion option while the MP
analysis was done by using the Close-Neighbour-Interchange (CNI) option. The best-fit substitution model
for ML was calculated and the tree was constructed using HKY+G model using the Nearest-NeighbourInterchange (NNI) option. The Bayesian analysis was performed by using the default settings with the
HKY85 model (Hasegawa et al., 1985). Four heated Markov chains were initiated from random trees and
sub-sampled at every 200 cycles. All trees were rooted with the sequences of the other Macaca species;
M. mulatta (AY612638), M. thibetana (NC_011519), and M. sylvanus (NC_002764), and with the outgroup
species of the Tribe Papionini, Papio hamdryas (NC_001992) as obtained from the GenBank database.
To assess the robustness of the trees, bootstrapping (Felsenstein, 1985) with 10,000 replicates were
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conducted on all the NJ, MP, and ML trees. Finally, all the sequences in this study were registered with
the NCBI and were given accession numbers from JX113316-JX113361.
By using the 1.6 million years ago (Mya) estimate for the divergence time between M. mulatta and M.
fascicularis as proposed by Hayasaka et al. (1996), we attempt to date the divergence time between the
haplotypes by calibrating the divergence time according to the branch length of the ML tree.
3. RESULTS AND DISCUSSION
3.1 mtDNA CR Variation
Between 1,031 and 1,032 base-pairs (bp) of homologous DNA sequence of the control region gene were
obtained from the 46 individual samples. Considering all the other Macaca and outgroup sequences (N=
51), alignment produces a sequence length of 1,042 bp. All sequences obtained were searched with the
GenBank database (BLAST, available at http:// www.ncbi.nlm.nih.gov/BLAST/) for confirmation and found
to match with M. fascicularis control region segment with at least 97% in sequence similarity. This
confirms that the sequences obtained are of true control region segment origin and not the widespread
nuclear mtDNA sequences (numts) which have evolved as pseudogenes in chromosomal DNA.
Considering all sequences (N= 51), 695 bp of conserved sites, 344 bp of variable sites and 158 bp of
parsimony informative sites were detected. Considering only the Penang samples, 975 bp of conserved
sites, 57 bp of variable sites and 47 bp of parsimony informative sites were observed. A single indel was
detected at nucleotide position (np) 220 of the alignment. DnaSP detected 20 haplotype (Table 1) with
haplotype diversity (Hd) at 0.921. Nucleotide diversity (Pi) for the 20 haplotypes was 0.01237. Although
the island populations consists of 36 individuals (Population A-H), the Pi= 0.00927 was observed to be
lower than the mainland population (Population I-L; Pi= 0.01339) which were represented by 10
individuals. This condition could be explained by the situation where the island populations were
populated by closely related individuals.
3.2 M. fascicularis Population Structure
The mainland were represented with 5 haplotypes (Hd= 0.667, data not shown) while the island with 15
haplotypes (Hd= 0.895, data not shown). A single haplotype sharing between the mainland (Population I)
and island population (Population C) was observed (Hap4; Figure 1) suggesting close relationships
between both populations. Population H (Jerejak Island) constitutes the most haplotypes (NHap= 6, Hd=
0.836) while Population E and I shows the highest Pi value of 0.02.
Figure 1 Haplotype designation
based on the sampling location.
Dotted
arrows
indicated
haplotype
sharing
between
locations.
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The fixation index Fst value generally means “little differentiation” at 0 to 0.05, “moderate differentiation” at
0.05 to 0.15, “great differentiation” at 0.15 to 0.25, and “very great differentiation” at values >0.25 (Wright,
1965). Fst index for Population A and D were not available due to only a single individual representing the
population. Comparison between the mainland populations (I-L) and the island populations (A-H) revealed
an Fst index at 0.36 which suggest very great differentiation. This can be explained by sedentary nature of
females which delineates the population’s structure. Interestingly though, low level of population
structuring between population J of the mainland and B (Fst= 0), and E (Fst= 0.07) from the island
populations were observed.
Genetic distances calculated between all populations using the Kimura 2-parameter model (Kimura, 1980)
ranges from 0-0.23. Mainland populations were genetically different from the island population with
distance of 0.018. Within the island populations, the mean genetic distance is 0.009 while within the
mainland populations, the mean genetic distance is 0.014.
3.3 Phylogenetic Relationship and Dispersal Pattern of M. fascicularis into Penang
Phylogenetic tree constructed using the NJ, MP, ML, and Bayesian methods produced similar topologies
and thus we summarized them using the ML tree (Figure 2). Overall, the tree showed the monophyletic
grouping of M. fascicularis. Within the 20 Penang haplotypes, the topology generally separates them into
three major groups; (1) Group I which consists of three subgroups, (2) Group II which was formed entirely
by the Jerejak Island populations, and (3) Group II which consist of a mixture of haplotypes from the
mainland and the island populations. In Group I, subgroup I-1 consist of haplotypes from the north-eastern
part of the island, subgroup I-2 consist of haplotypes from the mainland which is adjacent to the subgroup
I-1, while subgroup I-3 constitutes of a mixture of haplotypes from all over the island.
In an attempt to infer the dispersal pattern of M. fascicularis into Penang, we calibrated the divergence
time of 1.6 Mya into the ML tree (at node A) and use the length of branches (Figure 2) to estimate the
times at node B-D under the assumption of a constant rate of mutation. With node A being calibrated at
1.6 Mya, node B, C, and D become 0.87, 0.50, and 0.15 Mya, respectively. Therefore, we would
hypothesize that at about 0.5 Mya the first group of M. fascicularis invaded both the mainland and island
of Penang. Haplotypes within Group III would then represent the most ancient form of M. fascicularis in
Penang based on the basal positioning as shown in the phylogeny tree. At about 0.15 Mya, separation
between Group I and II occurred, and the Jerejak Island population diverged and formed the unique
haplotypes. Group I would then represent the most recent and last connection with the mainland as
haplotypes from the mainland (subgroup I-2) were shown clustering with the subgroup I-1 and I-3 of island
populations.
Figure 2 Phylogenetic relationships
of the M. fascicularis haplotypes
used in this study as represented
by the ML tree. Values above the
branches represent bootstraps
confidence
level
(10,000
replications) for NJ, MP and ML
repectively. Values below the
branches represents the Bayesian
posterior probability.
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4. CONCLUSION
In conclusion, the findings in this study revealed a great level of differentiation both within and between
the mainland and island of the M. fascicularis population. This condition can best be explained by the
sedentary nature of female long-tailed macaques. Genetic diversity was lower in the island as compared
to the mainland suggesting close relationships between the individuals that populate the island
populations. These findings would certainly benefit other studies on the co-evolution of primates with the
diseases associated with primates (e.g. evolution of the simian malaria parasite), biogeographical history
of the Penang Island, selecting homogenous breeding stocks for biomedical research, and management
and conservation of the M. fascicularis particularly from the Penang Island. We also discovered that the
control region of the mtDNA is a powerful segment to be used in addressing population structure of M.
fascicularis. Finally, to infer current gene flow, further study incorporating Y-chromosome are currently
being done.
Acknowledgement
We would like to thank the DWNP for providing the facilities, equipments, and personnel to conduct this
pilot study. Also thanks to the State Director of DWNP Penang, Jamalun Nasir Ibrahim and his staffs for
their assistance. This pilot project is funded by the DWNP, and partly by the Proboscis genome research
grant awarded to MT Abdullah. The sampling for ZMW coded samples were funded by the USAID
PREDICT programme. We would like to thank also these following individual for their dedication and help
during sampling: Charles Keliang, Azroie Daniel, Ahmad Faizal, and Emilia Joeneh.
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MOLECULAR CLONING AND EXPRESSION STUDIES OF GROWTH FACTOR RECEPTOR BOUND
PROTEIN 14 (GRB14) GENE IN VERTEBRATE DEVELOPMENT
1
Louis Chin Vui Ngian and Lee Kui Soon
1
Animal Biotechnology Laboratory, Faculty of Resource Science and Technology, Universiti Malaysia
Sarawak,94300 Kota Samarahan, Sarawak.
E-mail: louisxanvier.87@gmail.com
Abstract
Grb14 is the most recently identified members of Grb7 superfamily of adaptor protein, which participate in
the functionality of multiple signal transduction pathways under the control of a variety of activated
tyrosine kinase receptors (RTK). To date, the functional roles of Grb14 adaptors is not fully understood. At
the present time, a role for this Grb14-RTK interaction was only established in the regulatory effect on
RTK signaling especially in insulin receptor signaling inhibition as well as cell migration and tumor
progression. In respect that most of the current knowledge and understanding of Grb14 expression come
from murine studies, therefore, this study aims to investigate the expression pattern of Grb14 throughout
the development of zebrafish, whose cell migration process during embryonic development is wellunderstood. Semi-quantitative RT-PCR was employed to examine the temporal expression pattern of
Grb14 in ten stages of zebrafish development. The results denoted that the gene was expressed in all
stages studied, despite the levels of expression differed among the stages. Comparison with previous
studies revealed that the temporal expression pattern of Grb14 corresponded to that of some of its known
binding receptors, inclusing insulin receptor (IR), fibroblast growth factor recptor (FGFR) and epidermal
growth factor receptor (EGFR). This suggests the possible occurrence of interaction between Grb14 and
these molecules during zebrafish embryonic development.In progression, the expression studies of Grb14
will be determined through in situ hybridization to localize gene expression to specific cell types in specific
region in zebrafish. Last but not least, gene knockdown will be performed as Morpholinos will be
microinjected into zebrafish to target Grb14 gene for studying its functional roles in vertebrate
development.
Keywords: Grb14, Zebrafish (Danio rerio), RT-PCR, molecular cloning, gene expression, in-situ
hybridization, morpholino gene knockdown.
1. INTRODUCTION
1.1 Growth Factor Receptor Bound Protein 14 (Grb14)
The growth factor receptor-bound protein 14, Grb14 was the latest described member of the Grb7
superfamily of adaptors protein that comprises of Grb7, Grb10 and Grb14. It was cloned initially using the
CORT (cloning of receptor target) by interacting with EGFR. The study of Daly, Sanderson, and
Sytherland (1996) proposed that Grb14 was identified in a screen of a breast epithelial cDNA library.
Structurally, Grb14 contain a C-terminal Src homology 2 (SH2) domain, a central region Plecktrin
homology (PH) domain, a putative RA (Ras associating) domain, a BPS (between PH and SH2) domain
and lastly a N-terminal (Kasus- Jacobi et al., 2000).
The studies of Baker, Sutherland and Daly (1996) discovered that human Grb14 gene is localized on
chromosome 2q22-24 close to ErB4 family by fluorescent in situ hybridization. According to the newly
adopted nomenclature, there are three variants of Grb14 which differ from their species of origin including
hGrb14, rGrb14 and mGrb14 (Daly, 1998). Peptide sequence alignment showed that human and rat or
mouse partake 83% sequence identity as most conserved region is found in the C terminus of the protein.
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Currently, Grb14 has been described in several vertebrate species including human (Holt and Daly, 2005),
rat (Kasus-Jacobi et al., 1998) and mouse (Reilly et al., 2000). In rat, Northern and Western blot analysis
revealed that Grb14 is specifically expressed in insulin sensitive tissue such as skeletal muscle, liver,
pancreas and kidney (Kasus-Jacobi et al., 1998). Expression of Grb14 can be detected in human in the
same organ mentioned above, however with some degree of difference. It is noteworthy to notice that
Grb14 seems to be preferentially expressed in insulin target tissues such as liver, skeletal muscle, white
adipose tissue and insulin sensitive organs like pancrease and brain. Grb14 also personate its roles in
tumor progression as it is found to be expressed in human cell lines including those derived from normal
breast epithelial strains breast cancer and prostate cancer (Cariou et al., 2004; Daly et al., 1996).
Expression of Grb14 in these human cell lines might underscore its potential implication in cancer biology.
However, the functional role of this protein still remains to be elucidated.
Grb14 binds to various receptor tyrosine kinases (RTKs) under ligand induction, for instance insulin
receptor (IR) and fibroblast growth factor receptor (FGFR). Several studies validate that Grb14 play a role
in regulating insulin signaling. Conspicuous studies by Kasus-Jacobi et al., 2000 showed that binding of
Grb14 to the Insulin tyrosine kinase loop may alter the catalytic activity of the receptor thus resulting in
inhibition of insulin signaling. Inhibitory action on the IR kinase activity is mainly mediated by BPS domain
of Grb14. Numerous researches proved that Grb14 plays role in insulin signalling. Besides that, there
were research reported that Grb14 may plays role in cell migration as well as tumor progression but the
functional role of this gene remains to be clarified.
Since cell migration is an important event in embryonic development, expression study of Grb14 in the
development of an organism is likely to provide some hints to the biological functions of the protein.
However, there is currently a lack of information on the expression pattern of the gene throughout an
organism’s development. In the interest of expression study, zebrafish is a marvelous animal model. The
relative ease with which zebrafish embryos and lavae can be studied, the powerful genetics which can be
applied for the generation of mutants and transgenic animals, the vast genomic resources including the
availability of many molecular markers and the completed genomic sequence, make zebrafish an
extremely versatile model for developmental studies (Willett et al., 2001). Therefore, this study focuses on
analysing the expression pattern of Grb14 throughout the embryonic development of zebrafish, whose cell
migratory process during development is well-characterised. Analysis of gene expression was performed
at mRNA level on twelve stages of zebrafish development, namely the 4-cell (1.00 hpf), 1000-cell (3.00
hpf), 50%-epiboly (5.25 hpf, 1-somite (10.33 hpf), 18-somite (18.00 hpf), prim-5 (24.00 hpf), long-pec
(48.00 hpf), protruding-mouth (72.00 hpf), first-feeding early larval (96.00 hpf), and adult (120.00 hpf)
stages, by using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).
Overall, the main objectives of this study are:
(i) To extract total RNA of 10 different embryonic developmental stages from zebrafish for cDNA
synthesis to perform Reverse Transcription RT-PCR.
(ii) To establish, detect and study the semi-quantitative expression analysis of Grb14 at different
developmental stage using RT-PCR.
(iii) To clone the Grb14 gene for the used for in situ hybridization on whole mount zebrafish embryo.
[In Proceeding]
(iv) To establish the spatial expression pattern in ten different embryonic developmental stages in
zebrafish. [Proceeding]
(v) To understand the function of Grb14 gene by performing morpholino knockdown. [Proceeding]
2. METHODOLOGY
2.1 Primer design and sequences
All the primer in this project was designed according to Lee (2008) as dominating guideline by using two
different programmes. Firstly, suitable primer pairs for PCR amplification were suggested by Primer3
(http://waldo.wi.mit.edu/cgi-bin/primer3.cgi/primer3www.cgi). Then the primer pairs were construing for
hairpin,
palindromes,
dimmers
and
melting
temperature
(Tm)
using
NetPrimer
(http://www.premierbiosoft.com/netprimer/netprimer.html).
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2.2 Sample preparation
Zebrafish embryos were obtained from the fish facility in Animal Biotechnology Laboratory, Department of
Science and Technology, Univeristy Malaysia Sarawak. A total number of 10 disparate developmental
stages were selected for total RNA isolation. The developmental stages include 8-cells, 3hpf, 11hpf, 16hpf,
24hpf, 48hpf, 54hpf, 3dpf, 4dpf and 5dpf (Lee, 2008). The embryos were excavate and finely minced using
gauge and syringe. Promptly, the homogenate were placed separately into eppendorf tubes with each tube
containing Tri Reagent (Chomczynski, 1993; Chomczynski and Mackey, 1995) for extraction purposes.
2.3 Isolation of total RNA using Tri Reagent
Total RNA of the zebrafish embryo aged 8-cells, 3hpf, 11hpf, 16hpf, 24hpf, 48hpf, 54hpf, 3dpf, 4dpf and
TM
5dpf was isolated using TRIzol
Reagent at a concentration of not more than 100mg tissue/ml Tri Reagent
as decribed in the manufacturer’s protocol.
2.4 Agarose gel electrophoresis on RNA isolated and Quantification of RNA.
1µl of loading dye (6x) and 4µl of distilled water was added to 1µl of RNA sample to load the gel. The gel
was run at 120V for approximately 30 minutes. Subsequently, the RNA concentration and purity were
determined by OD260 measurements. The absorbance value were recorded.
2.5 cDNA sytnthesis
TM
cDNA synthesis for each respective stage RNA was carried out using SuperScript
III First Strand
Synthesis System (Invitrogen) according to manufacturer’s instruction. 15 µl of reverse transcription
reaction aliquots prepared was then mixed with 5 µl of primer mix for a final reaction volume of 20 µl per
tube. On the other hand, the 25 µl of negative control were done for each respective stage by not adding
SuperScript RT yet replaced by 1 µl of nuclease free water. The cDNA synthesis reaction were stored at o
20 C or used for PCR immediately.
2.6 Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Amplification of Grb14 cDNA was performed by using Taq DNA Polymerase (Invitrogen, USA). First, the
following components were added into a PCR tube (Table 1):
Table 1 RT-PCR reaction conditions and volume for 1X reaction and master mix
1X reaction (Volume
per 25 µl reaction)
Components
10X PCR Buffer minus Mg
10 mM dNTP mixture
50mM MgCl2
1 µM Forward Primer
1 µM Reverse Primer
Taq DNA Polymerase
MiliQ water
Template DNA (added separately for each tubes)
Final Volume
2.50 µl
0.50 µl
0.75 µl
1.25 µl
1.25 µl
0.50 µl
16.75 µl
1.50 µl
25.00 µl
Master Mix (X 24)
(including negative RT
control)
60.00 µl
12.00 µl
18.00 µl
30.00 µl
30.00 µl
12.00 µl
402.00 µl
36.00 µl
600.00 µl
The contents of the tube were mixed well and centrifuged briefly. The tubes were then placed in the
SuperCycler SC200 Thermal Cycler (Kyratec, Australia) programmed with the following PCR conditions:
initial denaturation (95°C for 2 minutes); 35 cycles of denaturation (94°C for 30 seconds), primer
annealing (60°C for 45 seconds), and elongation (72°C for 1 minute); final extension (72°C for 5 minutes).
The PCR products were visualised on 1% agarose gel. The following primer pairs were used for
amplification of Grb14 cDNA: 5'-GTC CTG AGA TTC ACG GCT TC -3' (forward) and 5'-CGT ATG GCA
TAG CGA CAG AA -3' (reverse). In addition, positive control reactions were conducted with β-actin
primersμ 5’-TGC CCA TCT ACG AGG GTT AC-3’ (forward) and 5’-CTC GTG GAT ACC GCA AGA TT-3’
(reverse).
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3.0 RESULTS and DISCUSSION
3.1 mRNA expression of Grb14 during zebrafish development
Semi-quantitative two-step RT-PCR was performed to detect the presence of Grb14 expression at
developmental stages mentioned above. The expected PCR product size was 725bp. Zebrafish
was used as the positive control during the amplification reaction, and the expected positive
product size was 341 bp. On the other hand, negative controls were also set up by using the
reaction from cDNA synthesis as the template.
the ten
β-actin
control
no RT
Figure 1 RT-PCR samples result on ten different embryonic developmental stages from zebrafish. Lane M
(100bp DNA marker); Lane 1(4 cells); Lane 3 (3hpf); Lane 5 (5.33hpf); Lane 7 (11hpf); Lane 9 (18hpf); Lane
11 (24hpf); Lane 13 (36hpf); Lane 15 (48hpf); Lane 17 (3dpf); and Lane 19 (5dpf). Lane
2,4,6,8,10,12,14,16,18,20 (Negative control).
Figure 2 Beta actin (positive control) RT-PCR result on ten different embryonic developmental stages from
zebrafish. Lane M (100bp DNA marker); `Lane 1(4 cells); Lane 2 (3hpf); Lane 3 (5.33hpf); Lane 4 (11hpf);
Lane 5 (18hpf); Lane 6 (24hpf); Lane 7 (36hpf); Lane 8 (48hpf); Lane 9 (3dpf); and Lane 10 (5dpf).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a very essential test in the field of gene
expression and expression diagnostics because it gives researchers a mechanism to test whether any
specific gene is turned on (active) or turned off (inactive). It is a rapid and sensitive method for analyzing
gene expression, for determining the presence or absence of transcripts and for producing cDNA for
cloning. If a gene is expressed, its mRNA product will be produced, and an associated band will appear in
the final agarose gel with the correct molecular weight for the gene.
In this research, RT-PCR were performed with the objective to provide a first and precedence to point out
when a gene was expressed during zebrafish development so that expression could then be able to
confirmed or consolidate in more detail by mRNA in situ hybridization subsequently. The zebrafish
embryonic developmental stages that had been chosen in this research embody and represent maternal
expression, cleavage, blastula, segmentation, pharyngula, hatching and early larval period respectively.
The RT-PCR results showed that the Grb14 gene is expressed in all ten isolated embryonic
developmental stages of zebrafish with the expected product size. Based on Lee,K.S., (2008), the product
size was 725 base pair, which suggests that Grb14 in zebrafish was expressed in a correct manner. Bright,
clear bands were observed in stages of 3hpf, 5hpf, 11hpf, 18hpf, 24hpf, 36hpf, 48hpf and 5dpf on the
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agarose gel of RT-PCR. This phenomena may indicates high level of gene expression in the respective
stages. Meanwhile, the faint bands at 4 cells and 3dpf may indicate low expression level of gene in
embryonic developmental stages of zebrafish.
In addition, the even number lane shown in the result were the negative controls (with no reverse
transcriptase were added when cDNA was synthesis) of each respective gene correspond to the 10
embryonic developmental stages. A negative RT sample control was essential to ensure the RNA used is
free of DNA contaminants such as genomic DNA. εoreover, housekeeping gene β actin primer was used
as a positive control on each developmental stage to assure the validity of the analysis. The gel photo
showed that β actin was expressed in all developmental stages and was equally consistent with the
expressions of the genes of interest (Grb14).
The RT-PCR outcome in this experiment displayed that Grb14 gene exhibited maternal expression at 4
cells stage and early zygotic expression at 3hpf. These results interpret that Grb14 expressed at an earlier
stage compared to the mammalian orthologues in both mice and human.
Comprehensively, RT-PCR would be able to provide initial information regarding temporal gene
expression that could serve as a corresponded reference for further analysis that purvey more accuracy ,
particularly for in situ hybridization studies in this research.
3.2 Sequencing of purified PCR product
The purified Grb14 PCR product was sent to First BASE Laboratories, Malaysia, for sequencing. Two
single pass sequencings were performed, one by using the forward Grb7 primer for PCR and another one
by using the reverse primer. The sequences obtained were used for homology searches using NCBI
BLAST. Both forward and reverse sequencing results showed 99% identity with zebrafish Grb14.
Accession
Description
Query Coverage
E value
Max identity
XM 002661948.2
PREDICTED: Danio rerio growth
factor receptor bound 14 protein
like
99%
0.0
99%
4. CONCLUSION
This current study has successfully established the expression profile of Grb14 gene during the embryonic
development of zebrafish. The result obtained showed that the gene was expressed in all the ten stages
studied, including the pre-MBT stage, which indicates that the gene was maternally expressed. . In this
research, RT-PCR were performed with the objective to provide a first and precedence to point out when
a gene was expressed during zebrafish development so that expression could then be able to confirmed
or consolidate in more detail by mRNA in situ hybridization subsequently.
There are several limitations in the present study. First, the method used for gene expression analysis
(RT-PCR) was semi-quantitative. For a more accurate analysis of gene expression, quantitative
approaches such as Northern blotting or real-time PCR could be performed. However, the data obtained
in this study can serve as a good source of reference for any future work that is to be done. Second, only
temporal, and not spatial, pattern of gene expression was investigated. Interpretation and comparison of
the result obtained with previous studies can therefore rely only on the temporal pattern of gene
expression. To examine the spatial pattern of gene expression, in situ hybridisation is recommended.
Identification of these limitations points to several interesting directions for future work, and the results
obtained in this study could serve as a reference for these future works. For future work, in situ
hybridisation is recommended as a more comprehensive method for detecting gene expression. By using
in situ hybridisation, it is possible to localize gene expression to specific cell types in specific regions.
Therefore, this technique allows the specific sites of expression of Grb14 gene to be detected in zebrafish.
Finally, it is anticipated that the functional study of the protein be performed by using gene knockout or
knockdown techniques.
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REFERENCES
Baker, E., Sutherland, G.R., Sutherland, R.L., and Daly, R.J. (1997). Assignment of the human GRB14
gene to chromosome 2q22-q24 by fluorescence in situ hybridization. Genomics 36 (1): 218–20.
Bereziat, V., Kasus-Jacobi, A., Perdereau, D., Cariou, B., Girard, J. and Burnol, A.F. (2002). Inhibition of
insulin receptor catalytic activity by the molecular adapter Grb14. J Biol Chem 277:4845-4852.
Bustin, S.A (2002). Absolute quantification of mRNA using real-time reverse transcription polymerase
chain reaction assays. Journal of Molecular Endocrinology pp. 169–193.
Cailliau,K., Perdereau, D., Lescuyer, A., Chen,H., Garbay, C., Vilain, J.P., Burnol,A.F., and BrowaeysPoly, E. (1995). FGF Receptor Phosphotyrosine 766 is a target gor Grb14 to inhibit MDA-MB-231
Human Breast Cancer Cell Signaling. Anticancer Research 25: 3877-3882.
Camelia, V.M-G., Wilson, S.W., Holder, N., and Pachnis, V. (1997). The zebrafish homologue of the ret
receptor and its pattern of expression during embryogenesis. 14, 879-889.
Cariou, B., Capitaine, N., Le Marcis, V., Vega, N., Béréziat, V., Kergoat, M., Laville, M., Girard, J., Vidal,
H., and Burnol, A.F. (2004). Increased adipose tissue expression of Grb14 in several models of
insulin resistance. FASEB J;18: 965–967.
Cariou, B., Perdereau, D., Cailliau, K., Browaeys-Poly E., Bereziat, V., Vasseur-Cognet, M., Girard, J.,
and Burnol, A.F. (2002).The adapter protein ZIP binds Grb14 and regulates its inhibitory action on
insulin signaling by recruiting protein kinase Cz. Mol Cell Biol 22: 6959–6970.
Cooney, G.J., Lyons, R.J., Crew, A.J., Jensen, T.E., Molero, J.C., Mitchell, C.J., Biden,T.J., Ormandy,
C.J., James,D.E., and Daly, R.J. (2004). Improved glucose homeostasis and enhanced insulin
signaling in Grb14-deficient mice. The EMBO Journal 23, 582-593.
Daly, R.J., Sanderson, G.M., Janes, P.W. and Sutherland, R.L. (1996). Cloning and characterization of
GRB14, a novel member of the GRB7 family. J. Biol. Chem. 271: 12502-12510.
Eisen, J.S and Weston, J.A. (1998). Development of the neural Crest in Zebrafish. Development Biology
159, 50-59.
Froese, R. and D. Pauly. “Danio rerio". FishBase. Retrived July 2, 2010, from
http://www.fishbase.org/Summary/speciesSummary.php?ID=4653&genusname=Danio&speciesna
me=rerio
Gregory, J.C., Ruth, J.L., Crew, A.J., Thomas, E.J., Carlos Molero, J., and Daly, R.J. (2004). Improved
glucose homeostasis and enhanced insulin signaling in Grb14 deficient mice. 23, 582-593.
Han, D.C., Shen, T.L., and Guan, J.-L. (2001). The Grb7 family proteins: structure, interactions with other
signaling molecules and potential cellular functions. Oncogene 20 : 6315-6321.
Holt,L.J. and Siddle, K. (2005). Grb10 and Grb14: enigmatic regulators of insulin action-and more?
Biochem. J. 388, 393-406.
Kasus, J.A., Perdereau, D., Auzan, C., Clauser, E., Van Obberghen, E., Mauvais-Jarvis, F., Girard, J. and
Burnol, A.-F. (1998). Identification of the rat adapter Grb14 as an inhibitor of insulin actions. J.
Biol. Chem. 273:26026- 26035.
Kasus-Jacobi, A., Bereziat, V., Perdereau, D., Girard, J. and Burnol, A.-.F. (2000). Evidence for an
interaction between the insulin receptor and Grb7. A role for two of its binding domains, PIR and
SH2. Oncogene 19:2052-2059.
Karine, M., Isabelle, B., Valery, L., Dominique, P., Katia, C., Edith, B., Anne-Francoise, B., and Arnaud, D.
(2003). The PIR domain of Grb14 is an intrinsically unstructyred protein: implication in insulin
signaling. 554: 240-246.
Lee, K.S. (2008). Characterization of Grb7, Prel and GYGYF family genes in zebrafish (PhD
Thesis). UK: University of Bath.
Liu, F. and Roth, R.A. (1995). Grb-IR: a SH2-domaincontaining protein that binds to the insulin receptor
and inhibits its function. Proc. Natl. Acad. Sci. USA 92:10287- 10291.
Moncoq, K., Broutin, I., Larue, V., Perdereau, D., Cailliau, K., Browaeys-Poly, E., Burnol, A. F. and
Ducruix, A. (2003). The PIR domain of Grb14 is an intrinsically unstructured protein: implication in
insulin signaling. FEBS Lett. 554, 240–246.
Reilley, J.F., Mickey, G., and Maher, P.A. (2000). Association of fibroblast growth factor receptor 1 with
the adaptor protein Grb14. Characterization of a new receptor binding partner. J Biol Chem 275,
7771-8.
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Roger, J.D., Georgia, M.S., Janes, P.W., and Sutherland, R.L. (1996). Cloning and characterization of
Grb14, a novel member of the Grb7 family. The journal of biological chemistry. 271(21), p.p.
12502-12510.
Thisse, B., and Thisse, C. Protocol for In situ Hybridization on whole mount zebrafish embryos. Retrieved
nd
2 August 2010, from http://zfin.org/ZFIN/Methods/ThisseProtocol.html
Veronique, B., Kasus-Jacobi, A., Perdereau, D., Bertrand, C., Girard, J., and Burnol, A. (2002). Inhibition
of insulin receptor catalytic activity by the molecular adaptor Grb14. The Journal of Biological
Chemistry. 277(7), pp. 4845-4852.
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FACILITATING COMMUNITY AWARENESS ON NATURAL RESOURCE ENVIRONMENT AND
ISSUES - TOWARDS SUSTAINING LIVELIHOOD OF MATANG COMMUNITY
1
2
Khamurudin M.N. *, Rosta Harun , and Abdullah Mohd
1
2
Faculty of Forestry, Universiti Putra Malaysi, 43400 UPM Serdang, Selangor, . Faculty of Environmental
Studies, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor
*E-mail: kamardin@upm.my
Abstract
One of the most important interacting components of mangrove forest system is local communities. The
influence of local communities on mangrove forest ranges from simple fishing activities to permanently
changed of mangrove scape into other land use such as fish and shrimp ponds. Such challenge has made
managing mangrove forest for environmental stability and society’s economic needs balance a delicate
task. The objective of this paper is to share a facilitation approach employed by the authors in identifying
the community awareness of mangrove management issues and its importance to other stakeholders. A
focus group discussion was conducted with 34 individuals representing different community structures of
Matang Mangrove Forest Reserve in Perak. Various tools were used in the focus group discussion to
deliberate with the community in respect to their perception of mangrove, opportunities and challenges,
and issues of mangrove environment. Some of the major findings from this workshop indicated that the
community realized that they depend more than 80% of their livelihood on mangrove environment. Some
of the issues raised were required research to expand the value of mangrove product and matters related
employment issues, and the importance of mangrove forest to the state government.
Please contact the corresponding author for full details of this paper.
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UTILISING TOURISM CARRYING CAPACITY ASSESSMENT TO DEPRECIATE ENVIRONMENTAL
ASSETS AT TOURISM SITES
1*
Khalizul Khalid , Rosmini Ismail
2
1
Department of Management & Leadership, Universiti Pendidikan Sultan Idris (MALAYSIA)
2
Department of Accounting & Finance, Universiti Pendidikan Sultan Idris (MALAYSIA)
*Email: khalizul@fpe.upsi.edu.my
Abstract
This paper proposes utilising the Tourism Carrying Capacity Assessment as a method to estimate
depreciation for the environment/natural assets. For this, a Linear Programming Model is used for the
computation of multidimensional Tourism Carrying Capacity Assessment. The depreciation expense is
determined through this model by estimating the costs of damages caused by the exceeding number of
visitors of that particular site. Ultimately, the objective of this paper is to incorporate the concept into
Environmental Accounting Framework.
Keywords: Tourism carrying capacity. Carrying capacity, environmental assets, tourism management.
1. INTRODUCTION
Tourism has become an important component in Malaysian economy. There are significant increasing
trend since year 2000 until 2011. In 2011 Tourism Malaysia reports that the amount receipts from tourist is
RM56.5 billion (Tourism Malaysia, 2011). This figure is expected to grow over the years due to Malaysian
government effort in promoting Malaysia as an attractive destination in the world. Tourism may act as a
catalyst for economic development for instance providing employments and investments to the tourism
site. However, tourism often has a significant impacts on environment, social and economic structures and
dynamics, as well as lifestyles (Coccossis, 2004).
However, tourism activities also produce negative impacts to the demographics structure, social structure
and relations, economics activity, environment and land use, natural and cultural heritage and
environmental resources (Coccossis, 2004). Tourism development and management should recognise
this issue and not only focusing on simulating tourism industries by means of providing essential condition
for tourism growth. Natural capital depletion may lead to quality deterioration of tourism products and the
consequence are costly to the tourism site (Bryan & Taylor, 1987; Dolnicar, Crouch, & Long, 2008;
Mohamed, Mat Som, Jusoh, & Kong, 2006). To avoid this, the planning of tourism development and
management should be based on the limits indicators (environment, social, economic) (Pazienza, 2004).
These indicators can be measured in three components; (i) physical-ecological component; (ii)
sociocultural components and (3) political-economic component (Coccossis & Mexa, 2004) .This paper
discussed on the possibility of utilising tourism carrying capacity assessment and it applicability at tourism
islands of Malaysia.
2. TOURISM CARRYING CAPACITY
The growing needs of modern societies for recreational and leisure have turned tourism activities into a
complex industry. Tourism has become a major economic activity worldwide and a priority field in policy
making at local, regional, national, supranational and international level (Coccossis, 2004). World Tourism
Organisation (UNWTO, 2012) defined tourism as a "movement of people, spatially and temporally, out of
their own communities for leisure and business purposes" where a tourist is defined as a "visitor staying
for more than 24 hours in a country visited for business or leisure purposes". In other words, tourism
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involves the movement of people to places for more than 24 hours out of their own communities either for
leisure or business purposes. These movement may cause a changes to the place visited and give
impacts on the environment, social and economic structure and dynamics, as well as culture and
lifestyles. The demand of tourists related to facilities, spaces and needs for recreational and leisure has
increased physical development at the tourism site (Dolnicar, et al., 2008; Lim & McAleer, 2005; Lundie,
Dwyer, & Forsyth, 2007). It may lead to environmental, social and economic pressure (impact) that may
degraded the beauty, joy and wealth of the tourism site. Thus, the impacts of tourism at the tourism site
have to be monitored and controlled in order to ensure the sustainable tourism practices. Tourism carrying
capacity assessment can be used as one of the tools in tourism planning and management to cope with
such impacts.
Carrying capacity concept was introduced in mid-1930 and the first attempt to apply the concept was
made in 1λ60’s. Conceptualize under the ecology discipline, it initially investigated the relationship
between visitors and environmental condition of a site (Manning, 2002) .Then, Wager, 1964 (in Manning,
2002) found that there are variables involves other than human to determine carrying capacity of a place.
The concept later becomes more complex, adopted by other fields and applied to different types of area
for example, recreational (Manning, 2002), tourism (Pazienza, 2004) and aquaculture (McKindsey,
Thetmeyer, Landry, & Silvert, 2006) to name a few. Mathematic equation(s) is used in carrying capacity
to identify upper level of saturation point and, once it has been reached, the species population is at its
maximum sustainable level (Pazienza, 2004). Thus, Tourism Carrying Capacity is referring to “maximum
number of people may visit a tourist destination at the same time without irreversible or unacceptable
deteriorating the physical, economic and sociocultural environment and an unacceptable decrease in the
equality of visitor satisfaction without considerably diminishing user satisfaction” (World Tourism
Organization, 1981).
However, according to O’Reilly (1986), there are two school of thoughts concerned with the nature and
interpretation of tourism carrying capacity. First, tourism capacity predicted the capacity of the destination
area to absorb tourism before negative impacts of tourism are felt by the host county. The carrying
capacity will determine maximum number of tourist is allowed to visit rather than by how many tourists can
be attracted. This concept paid more attention to the country, population and the tourism site compare to
what demand tourism might offer in term of economic value. Second school of thought proposes that
tourism capacity be considered as the levels beyond which tourists flows will decline due to certain
capacities (physical, social, economy) as perceived by the tourist themselves have been exceeded. This
area will become less attractive and no longer satisfy the needs of tourist, therefore will seek alternative
destination (O'Reilly, 1986).
The study of carrying capacity must take consideration of physical (environmental and experiential), social
and economic impact of tourism activities (Johnston & Tyrrell, 2005; Manning, LeungYu-Fai, & Budruk,
2005; O'Reilly, 1986). Capacity levels influenced by (1) the characteristics of tourists, (2) the
characteristics of the destination are and its populations, (3) natural environmental features and
processes, (4) economics structure and economic development, (5) social structure and organizations,
(6)political organization and (7) level of tourist development (O'Reilly, 1986).Carrying capacity can
contribute to managing tourism on the basis of sustainability principle(Johnston & Tyrrell, 2005; Mexa &
Coccossis, 2004). Thus, carrying capacity can become guiding framework that contributes to achieve
consensus over the need to pursue sustainable tourism development (Mexa & Coccossis, 2004).
3. METHODOLOGY OF TOURISM CARRYING CAPACITY ASSESSMENT
The study Tourism Carrying Capacity Assessment (TCCA) requires a multi-dimensional approach, which
inclusive of environmental, social, political, and economic aspects of tourist destination under
consideration (Coccossis & Mexa, 2004). This research proposed to use Parpairis (2004) framework
(Figure 1) along with a model by (Pazienza, 2004) to evaluate the environmental system’s carrying
capacity will be used as a guide to model the carrying capacity in islands studied.
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Environmental
Factors
Methodology Design and
development
Tourism Activity
Area
Neighbouring
Areas
Environmental Factors
Impact (Direct, indirect and imminent)
Ecological
Cultural
Social
Economical
Political
Design & Handling Tools Parameters. Instructions & Model
Carrying Capacity for Tourism Development
Figure 1 Evaluating the environmental system’s carrying capacity (Parpairis, 2004)
The model suggest by Pazienza (2004) and Collins (1999) is used for the purpose of the study. Collins
(1999) claimed that sustainable development at a given tourism destination j, over time T requires,
(1)
In ideal situation this condition is difficult to fulfil since tourism activities will cause natural degradation.
However, the destruction of natural capital can be compensated by reconstruction and manmade capital
such as reconstructing, transplanting and restoring the affected natural asset. This concept is in parallel
with environmental accounting framework proposed by Ismail, Forgie & Khalid (2012) where real tourism
income is estimated by deducting degradation elements from total revenue of a tourism site. Whereas,
tourism balance sheet within the framework takes account on costs incurred to beautify and conserve the
environment are categorized environmental assets under the sub heading of environmental regulation
generated and market based assets. However, for penalties imposed by authority as a result of noncompliant to environmental regulation or cost incurred to mitigate damages due to negligence are
categorized under environmental liabilities.
Whereas, Fisher and Krutilla (1972) defined carrying capacity in two different terms according to
ecological terms and economic terms. In ecology terms, carrying capacity is defined as “a maximum
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number of visitors that can accommodate by given destination under conditions of maximum stress”,
where in economic terms as “maximum number of visitor that can accommodate at a constant quality of
experience”. Hence, in order to maximise economic value at any given destination, this may cause
possible adverse impacts of the physical, economics and social environment as perceived by the resident
population, are among the limitations factors (Canestrelli & Costa, 1991). Therefore, carrying capacity
would come to represent the upper limit of the potential number of visitors in a resource-based tourism
destination. So, in order to determine the optimal use level of a resource-based tourism attraction is given
by maximization of (Canestrelli & Costa, 1991; Fisher & Krutilla, 1972):
max
With
(2)
(3)
Where:
= The amount of net benefits from tourism activity,
B = Gross benefits;
C = The amount of costs implied to run tourism activity;
q = Use level of recreation attraction;
= the cost of damage to ecological environment;
= the amount of current expenditures;
= capital expenditures (e.g. the relevant interest and depreciation charge).
Following the equation, the problem finds its solution by differentiating the equation with respect to q and
set equal to zero (Canestrelli & Costa, 1991; Fisher & Krutilla, 1972; Pazienza, 2004) .
(4)
The optimum use level of the tourism destination resources, q* of resource can be found at the
intersection between cost benefits graph where optimal benefits and optimal cost is can be determined
from the graph plotted in Figure 2 (at the point between the highest - benefits is maximum and the lowest
– cost is minimum).
Figure 2 Optimal use of a tourism recreation resources; (Pazienza, 2004)
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The works of Canestrelli & Costa (1991) have made building multi-dimensional model to empirically
determining a measure of carrying capacity for tourism destinations using linear programming approach is
possible (Pazienza, 2004). Pazienza (2004) combine the idea of Canestrelli & Costa (1991) and Chiang
(1984) to suggest that linear programing model can be used for computation of multidimensional Tourism
carrying capacity, where:
(5)
Such that
(i=1 to n)
(6)
(j=1 to n)
(7)
And
Where:
= a level of sector activity (e.g. hotel tourism, non-hotel tourism activities), with j = 1 to n denoting the
number of possible activities;
cj = forecasted gross margin of a unit of the activity, with i=1 to m denoting the number of resources;
th
ri = the amount of the i resources available. (which represent the constraint).
This model is known as primal linear programming problem that generates the highest level of total gross
margin Z without either breaking any of the fixed resources constraints or involving any negative activity
levels. To solve the linear programming problems, simplex method is used to reduce the feasible sector
plans that need to be considered to a finite number identified with the number of basic solutions(Pazienza,
2004). Inequality constraint considered by equation (6) can be transform into equality constrain to arrive
at:
(8)
th
Where Si = the amount of the i resource not used in the sector plan.
Computer software or computer programming application can be used to automate this transformation
and search for the optimal result. TCC can be determined as an optimal use level where benefits are
maximized and costs are minimized. For the case of islands in Malaysia, linear programming model is set
by identifying numbers of supporting facilities which are considered as relevant to fulfil the basic needs of
tourist. These aspects are related to accommodation needs and the impact caused by the facilities
provided to fulfil the needs. Examples of these facilities are number of beds in hotel and non-hotel sectors,
solid waste and sewage disposal, to name a few. Secondly, tourist are categorized into two groups which
are hotel tourist (HT) (those who use hotel services) and non-hotel tourists (NHT) (those who use nonhotel services) i.e. staying camping areas and home stay villages. The objective function of linear
programming model can be express in the following terms (Pazienza, 2004)
Max HT,NHT
(9)
In this equation:
= net private benefits from the whole tourism sector;
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HT = hotel tourism
HNT = non-hotel tourist
pHT = per night price averages in the hotel
pNHT = per night price averages in non-hotel sectors
cHT = daily cost borne by the hotel management per each group of tourist
cNHT = daily cost borne by the non- hotel management per each group of tourist
The number of constraints in the model will be determined during the study. This would require valid
information on function forms which can be significantly describe and explain the relationship existing
between tourists and social and biophysical of the host community (Pazienza, 2004). From the equation 9,
we can describe the constraints of the objective function that can be summarize as follows (Pazienza,
2004)
i.
ii.
iii.
iv.
v.
HB = number of beds available in the hotel sector;
NHB = number of beds available in the non-hotel sector;
GD = waste disposal capacity (kg/day)
3
SD = sewage disposal capacity (m /day)
PP = number of boat landing places
Using this model, TCCA can be made by gathering all the data needed in studied tourism island. An
empirical application can be carried out. Consequently, the Tourism Income and Balance Sheet of the
environmental accounting framework as proposed by Ismail, Forgie & Khalid (mentioned earlier),
suggested incorporating TCC component into the framework as means of estimating depreciation
expenses (for income statement of tourism site) and accumulated depreciation value (for balance sheet of
tourism site). As a result, the tourism income would represent the real income – taking account on the
usage/exhaustion of natural capital from tourism activities.
4. CONCLUSION
This study proposes incorporating TCC for estimating depreciation of natural capital used for tourism
activities. The TCC models by several authors especially Pazienza’ s presented in the paper will be used
as guidelines for the application in tourism islands of Malaysia. It is expected that the model/equations will
be modified to correspond with Malaysian environment. Therefore, through TCCA model, the ‘real’ return
of these tourism destinations can be estimated to indicate whether the environmental resources at the
sites are sustainable or over exploited (degradation & depletion exceed its yield).
REFERENCES
Bryan, H., & Taylor, N. (1987). A. Use a preservation: the outdoor recreation policy dilemma. Policy
Studies Review, 7(2), 349-358.
Canestrelli, E., & Costa, P. (1991). Tourist carrying capacity: A fuzzy approach. Annals of Tourism
Research, 18(2), 295-311.
Chiang, A. C. (1984). Fundamental Methods of Matamethical Economics (3 ed.). New York: MacGraw-Hill.
Coccossis, H. (2004). Sustainable Tourism and Carrying Capacity: A New Context. In H. Coccossis & A.
Mexa (Eds.), The Challenge of Tourism Carrying Capaicty Assessment: Theory and Practice (pp.
1-15). Surrey, England: Ashgate.
Coccossis, H., & Mexa, A. (2004). Tourism carrying Capacity: Methodological Considerations. In H.
Coccossis & A. Mexa (Eds.), The Challenge of Tourism Carrying Capacity Assessment: Theory
and Practice (pp. 55-90). Surrey, England: Ashgate.
Collins, A. (1999). Tourism Development and Natural Capital. Annals of Tourism Research, 26(1), 98-109.
Dolnicar, S., Crouch, G. I., & Long, P. (2008). Environment-friendly Tourists: What Do We Really Know
About Them? Journal of Sustainable Tourism, 16(2), 197-210.
Fisher, A. C., & Krutilla, J. V. (1972). Determination of Optimal Capacity of Resource-based Recreation
Facilities. Natural Resources Journal, 12417-444.
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Ismail, R., Forgie, V., & Khalid, K. (2012). Bridging the environmental accounting gap between the
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of Sustainable Tourism, 15(5), 503-519.
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Capacity of Boston Harbor Islands. [Article]. Northeastern Naturalist, 12, 201-220.
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(pp. 37-54). Surrey, England: Ashgate.
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THE ECONOMIC IMPACTS OF SUSTAINABLE FOREST MANAGEMENT POLICY ON TIMBER
MARKET IN SARAWAK, MALAYSIA
AS Abdul-Rahim* and HO Mohd-Shahwahid
Faculty of Economics & Management, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Email: abrahim@econ.upm.edu.my
Abstract
The main objective of this study is to analyse the impact of Sustainable Forest Management (SFM)
practices on the timber market in Sarawak, Malaysia. A partial equilibrium model was applied in this study
covering domestic and international timber market namely supply, domestic demand and export demand
of timber. It was analysed by using a system of equations approach. All of the data are annual time series
basis from 1970 to 2008. Based on the first three scenarios, the results show that changing from the
conventional logging (CL) practices to SFM practices, reduce the equilibrium quantity of timber and
increase the price level. The welfare economic impacts of SFM provide empirical evidence that there is a
loss in welfare economic impacts on the timber industry in Sarawak, Malaysia. However, an increase in
the domestic price of timber would help to compensate for the loss volume of timber. In addition, the
scenarios of market access shows that Sarawak timber industry could benefit from this advantage.
Keywords: Consumer surplus; Producer surplus; Equilibrium price; Equilibrium quantity
1. INTRODUCTION
In Malaysia, Kumari (1996) has conducted a study of total economics valuation (TEV) approach in the
context of Conventional Logging (CL) and SFM practices. The TEV involves use value, non-use value,
direct use value, indirect use value, option value, existence value and quasi-option value.
To make it clear, timber market in this study is also known as log market. Most of previous studies on
timber market, the analysis typically deals with the prices, supply and demand in domestic and
international market. However, to the best of authors knowledge, none of the study has analysed timber
market by internalising the value of price and quantity as the value which incorporated the externality
effects. For example, a current domestic timber prices in Sarawak is just determined by the market driven.
It is expected that by internalising the externalities, the price would potentially higher than the current
prices. Hence, government intervention is really needed to correct the distortion. In this context, this
present study will provide some evidences that could be used by the government for decision making
process. Therefore, the optimum level of output and price at externality level will be quantified because
the net benefit is maximised when it takes into account the negative externality effects as well (Tietenberg,
2003).
Most of the studies conducted either locally or abroad revealed that there is incremental cost in operating
SFM other than the reduction in timber production [Schwarzbauer and Rametsteiner (2001); Abdul Rahim
and Mohd Shahwahid (2009a)]. All of these possible changes are directly related on harvesting
regulations and additional guidelines on timber harvesting activities. This is because information about
productivity, cost and applications of timber harvesting activities are important component for
management plans (Parsakhoo et al., 2009). Hence, this will reduce the volume of timber which can be
extracted from forest as well as incurred higher cost. In other words, in the short run, it may reduce
potential harvesting volumes and producers may have to bear higher cost in implementing SFM. However,
in the long run, this may support a sustainable level of production that will exceed of what would be
possible in later years if environmentally harvesting systems were to be continued (Thang, 2007). Beside
the issues of operation cost and timber production, several other elements in SFM that potentially give
direct impact to timber market are also identified; such as price premium and market access. With regard
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to the economic reasons, timber producers must acknowledge that there are some economic advantages
to participate in SFM.
With the above issues raised relating to the Malaysian timber industry, it is paramount that the market to
be understood in term of the relationship of its major parameters. It has become essential to know the
various impacts of SFM practices on timber market in Sarawak, Malaysia. This is where we have to come
out with several scenarios of SFM practices and carry out simulations analysis for examining the market
and welfare economic impacts.
2. METHODOLOGY AND DATA COLLECTION
While there are different issues in forest sector policies analysis, the analytical framework is quite similar.
The common approach is to develop a forest sector model and to simulate the impacts of the policy on
timber and product markets for domestic or international markets. A typical model building involves the
estimation of output consumption, price and trade of the timber products. The impacts of the policies were
evaluated by comparing the simulated results for with and without policy scenarios.
Studies on forest related policies such as Schwarzbauer and Rametsteiner (2001), Mohd Shahwahid
(1995) and Ahmad Fauzi (2005) have used such framework. However, this paper differs as it takes into
account the SFM policy by incorporating with several scenarios of SFM practices namely (1) reduced by
24-percent in harvested area, (2) increased by 49-percent in external cost of timber harvesting, (3)
increased by 47-percent in the cost of internalisation the externalities, and (4) 20-percent gain in market
access. In other words, the current input cost of timber harvesting operations has to be incorporated
together with the cost related to the externalities. This is where most of the prior studies have ignored the
externality effects in their econometric modeling.
2.1 Timber Market Model
This study adopted and modified the model developed by Mohd Shahwahid (1995), Ahmad Fauzi (2005)
and Abdul Rahim and Mohd Shahwahid (2009a). Timber that comes from natural forests will be analysed
in this study.
2.2 Supply of timber from natural forest
0 + 1 lnP t + 2 lnAH t + 3 lnIC t + 4 lnTS t 1 + t
The timber supply from natural forest is given by the equation:
lnTS t =
where:
lnTS t
= Supply of natural forest timber
lnP t
= Price of timber
lnAH t
= Harvested area in natural forest
lnIC t
= Total salaries and wages paid in logging industry
lnTS t 1
= Lag supply of timber supply by one year
t
t
= Years
= Error term
ln
= natural logarithm
(1)
Equation (1) estimates the total supply of timber from natural forest, which should be positively related to
the natural forest timber prices and harvested area in natural forest. TSt is the supply of natural forest
timber as endogenous or dependent variable. Pt is the price of natural forest timber, which is an important
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variable in determining the quantity of natural forest timber supply. AHt is the natural forested area open
for harvesting. ICt is total salaries and wages paid in logging industry represents to the production cost.
TS t 1 is previous year natural forest timber supply, which have influenced the natural forest timber supply.
Incorporation the cost of internalisation the externalities and external cost
Input cost under the scenario of SFM = input cost + cost of internalisation the externalities
(2)
Input cost under the scenario of SFM = input cost + external cost of timberharvesting
(3)
Equations (2) and (3) explain the situation where the timber market model is incorporated with the cost of
internalisation the externalities and external cost. Incorporation those elements are crucial, otherwise it
can lead to the market failure. Market failure associated with the externality effects resulting from timber
harvesting activities in forest. Without taking into account the externality effects, the timber production
from natural forest could be considered as being managed without sustainably produced. In other words, it
cannot achieve the optimum level of quantity and price of timber. Most of previous studies especially
studies using econometric modeling had ignored the monetary value of externalities and external cost of
timber harvesting in their research. Therefore, this study tries to incorporate those elements so that the
research outcome could represent the optimal level estimation of quantity and price in timber market.
2.3 Demand of timber from timber processing mills
*
lnDD t =
0 + 1 lnP t + 2 lnIPI t + 3 lnWMP t + 4
lnDD t 1 +
t
(4)
where;
*
DD t
= Domestic demand for timber
Pt
= Domestic price for timber
IPI
t
= Industrial production index
WMP t
= World import price of timber
DD t 1
= Lag of domestic demand for timber for by one year
t
= Years
ln
= natural logarithm
t
= Error term
Equation (4) describes the estimated total domestic demand for timber from natural forests. It suggests
that the lower the price offer, the higher the volume of forest timber demanded domestically. On the other
hand, the higher world import price of timbers would encourage further consumption of domestic timbers.
Similarly, the higher industrial production index (IPI) would promote timber processing mills (i.e., sawmills,
plywood and veneer mills) to demand more domestic timbers. Instead of using Malaysian income, IPI will
be used in this study because timber demand is considered as intermediate goods. IPI is also used to
measure the economic growth of timber-based manufacturing industries and it should therefore be
positively related to the timber demand. When there is a growth in timber processing mills, demand for
timbers would rise but domestic demand would have to compete with other substitute such import of
timber. Hence, we used world import price of timber (WMP) which represents substitute good. It suggests
*
that the higher the WMP, the higher the volume of domestic demand of timbers. lnDD t is the dependent
variable for domestic demand for timber, which is influenced by the domestic price of timber (P t ),
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Industrial production index (IPI t ), world import price of timber (WMP t ) and the previous year’s domestic
demand of timber (DD t 1 ).
2.4 Export Demand of timber from less forest endowed countries
lnXD t =
0 + 1 lnXP t + 2 lnMKA t + 3 lnER t + 4 lnSWP t + 5 lnXD t 1
+
t
(5)
where;
XD t
= Export demand for timber
XP t
= Export price for timber
ER
t
= Exchange rate
MKA t
= Market access
SWP t
= World average price of softwood timber
XD t 1
= Lag of timber export for by one year
t
t
ln
= Years
= Error term
= natural logarithm
Equation (5) is expected to estimate the total export demand for timber from natural forests. It is expected
to show a negative relationship with the export price for timber export. Whereas, positive relationship with
the exchange rate, market access and its substitutes good which refers to world average price of softwood
timber. MKA measures the ratio of market access. For example the ratio total timber exports of Sarawak
with total timber import by the importing countries that imported timber from Sarawak. There is a positive
relationship between exported demands of timber with MKA. This is because the higher the ratio, this
shows timber from Sarawak is the main favorite from timber importing countries as a result of SFM
practices. XD t is an endogenous or dependent variable for export demand equation. Timber export
demand is influenced by the export price of timber (XP t ), exchange rate (ER t ), market access (MKA t ),
world average price of softwood timber (SWP t ) and the previous year’s timber export (XD t 1 ).
2.5 Closing identities (total supply of timber)
The above timber market model has three main equations. To close the system, an identity equating
timber availability with summation of domestic and export demands of forest timbers is postulated as
equation below:
TSt = DDt + XDt
(6)
To analyse the timber market model, this study estimates timber supply and demand for domestic and
export market. Then, re-estimate the supply and demand simultaneously followed by simulation analysis
of several scenarios under the SFM practices. The export demand equation will be estimated by Ordinary
Least Square (OLS) while the domestic supply and domestic demand equations will be estimated by
system of equations approach. From Equation (6), a partial equilibrium of quantity and price of timber can
be generated. In addition, the producer and consumer’s surplus that represents welfare economic impacts
are also being quantified.
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2.6 Data Description
Data used in this analysis is time series data. With regards to the time series data analysis, this study
intends to evaluate the empirical performance of SFM practices in Sarawak timber market using annual
data from 1970 to 2008. Published and unpublished data on all variables in this study were gathered from
the Forestry Department of Sarawak, Malaysia, Department of Statistics, Malaysia and Ministry of
Plantation Industries and Commodities, Malaysia.
3. RESULTS AND DISCUSSION
Econometric analysis is capable of providing a quantitative analysis of the actual economic phenomenon
based on the concurrent development of theory and observation, related by an appropriate method of
inference Gujarati (2003). Since this analysis uses time series data, it is necessary to find out whether the
data are stationary or otherwise. For this reason, unit root test has been conducted using the Augmented
Dickey-Fuller (ADF) and Philips-Perron (PP) unit root test.
3.1 Unit Root Test on Time Series Data
Since the unit root test results are sensitive to different values of the autoregressive lag lengths, the
selection rule of the truncation lag parameter is crucial in determining the order of integration of the data.
In this study, the optimal lag length of the ADF test is chosen based on automatic selection by Schwartz
Information Criterion (SIC), while Newey-West Bandwidth criterion is used for the optimal lag length
selection in the PP test to ensure the errors are white noise. All the unit root tests are carried out using Eviews 6.0 software. In order to conserve the space in this paper, we decided not to present the results of
unit root test.
All variables are non-stationary in levels. Thus, we cannot reject the null hypotheses of a unit root in both
the ADF and PP tests. On the other hand, all series appear to be stationary after first differencing, that is
I(1). This result is consistent for both ADF and PP tests used in this study. Therefore, higher order of
differencing is not required to make the data into stationary process. The results imply that there is I(1)
variables in the Sarawak data, and no existence of I(2) variable.
There is a concrete support for the existence of a unit root stationary at I(1) by ADF and PP unit root tests
in Sarawak. The result of I(2) is automatically do not need to carried out because all the variables are
integrated at I(1). If, there are not integrated at I(0) and I(1), then it is necessary to analyse the unit root
test at I(2) level.
3.2 Estimated coefficients of timber market
Sarawak timber market model comprised of supply function, domestic and export demand functions of
timber and as a whole appeared to fit the data well (Table 1). Sarawak is still exporting its timber, unlike
Peninsular Malaysia has banned its timber exports. Table 1 showed the empirical results of the estimated
supply, domestic and export demand equations in Sarawak.
Table 1 Results of Timber Market for Sarawak
Supply Function
lnTS = 1.8206 + 0.1532lnP - 0.089lnIC + 0.8047lnAH + 0.2663lnTSt-1
(0.00)***
(0.02)**
(0.00) ***
(0.00)***
2
2
R = 0.98; Adj. R = 0.98; DW = 1.96; Ramsey RESET Test = (0.41);
Heteroskedasticity Test = (0.38); Wald Test = (0.00)***
Domestic Demand Function
lnDD = 4.2196 - 0.2237lnP + 0.2709lnIPI + 0.0332lnWMP + 0.7273lnDD t-1
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(0.37)
(0.05)*
(0.86)
(0.00)***
2
2
R = 0.92; Adj. R = 0.90; DW = 1.80; Ramsey RESET Test = (0.19);
Heteroskedasticity Test = (0.46); Wald Test = (0.00)***
Export Demand Function
lnXD = 4.1937 – 0.0818lnXP + 0.5941lnMKA + 0.0143lnSWP + 0. 8837lnREER
(0.19)
(0.02)**
(0.89)
(0.69)
+ 0.7488lnXD t-1 - 0.3794AR(1)
(0.00)***
(0.00)***
2
2
R = 0.94; Adj. R = 0.93; DW = 1.82; Ramsey RESET Test = (0.22);
Heteroskedasticity Test = (0.43); Wald Test = (0.00)***
Notes: ***Significant at 1 percent, **Significant at 5 percent, *Significant at 10 percent.
The values in the parentheses ( ) contain the p-value.
The supply and domestic demand equations were estimated using the system of equations approach as
endogenous variable exists in each of the equation. The remaining equation was estimated using OLS. All
of the variables coefficients in the model produced an expected sign consistent with the theory and similar
with previous studies.
For timber supply function, the policy variables (i.e. IC and AH) estimated coefficients are statistically
significant at the level of 5-percent and 1-percent respectively. This means that, they are the significant
determinants of timber supply. For IC, the result suggests that for every 10-percent increase in average
IC, ceteris paribus, timber supply would decreased by 0.9-percent. The significant coefficient of IC verified
the priori assumption that cost is a burden for timber producers. In other words, larger value in IC, will in
turn reduced the volume of timber produced. On the other hand, AH has a positive coefficient. Based on
the estimation, an increase of 10-percent in AH, ceteris paribus, timber supply would increased by 8percent. AH appeared to be highly significant at the level of 1-percent and almost elastic. This was
believed to be due to the direct relationship between timber harvesting activities and timber supply.
Therefore, the State Government of Sarawak has adopted one of the forest-related policies related to AH
which is the rate of Annual Allowable Cutting (AAC). This is the reason of declining in AAC since early
1990s. This to a certain extent would increase pressure to timber producers in producing timber.
Furthermore, timber producers would experience diminishing timber supply aroused from the increasing
important demand for Non-timber Forest Products (NFTPs) and environmental protection (Kumari, 1996).
Results from the present study confirmed some of the general themes and conclusions in previous studies
(see, Abdul Rahim et al., 2009b).
For the case of Sarawak, there is an export market for timber since early 1960s until today. Like Sabah,
Sarawak supplied timber to Peninsular Malaysia as well. In fact, the export of timber in Sarawak rose 50percent in year 2010 compared to the same period in 2009 (Anon, 2010). This is a reasonable explanation
for the exclusion of import demand function for timber in the Sarawak timber market model.
In the domestic demand equation, the coefficient estimates signs of P, IPI and WMP were as expected.
The insignificant result of P and WMP were similar to the study conducted by (Mohd Shahwahid, 1995).
Unlike timber supply function, P is not an important determinant of the domestic demand for timber in the
domestic market. Perhaps, this could be due to local timber processing mills having higher willingness to
pay in getting their raw material. Timber market is a supplier’s market. IPI is an important variable
influencing the demand for timber in Sarawak. IPI is significant at the level of 10-percent. An increase of
10-percent in the IPI was associated with a 2.7-percent increase in the demand for timber. This implied
that the growth in economic activities in Sarawak would influence the domestic demand for timber. In
contrary, Mohd Shahwahid (1995) found that IPI is not a significant variable influencing timber demand in
Peninsular Malaysia.
The estimates obtained for the export demand equation was inconsistent with the prior theory. The result
indicated that the own prices and exchange rate were not important determinants of the export demand
for timber in the international market. However, the export demand function was significant dependent on
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the MKA and XDt-1. An increase of 10-percent in MKA increases the quantity export demanded by 5.9percent.
The positive cross-price elasticity with respect to the export price of timber is 0.014, indicating that
softwood timber is a substitute for timber. However, the result obtained is not statistically significant. This
result is similar with the findings by Mohd Shahwahid (1995).
3.3 Validation of Timber Market Model in Sarawak
The overall fit of the equation between the explanatory variables and dependent variable can be explained
by the value of R-square. This is an important criterion for evaluating the quality of regression. For
example, the value of R-square obtained from the estimated supply equation is 0.92. This implied that 92
percent of the variation in timber supply could be explained by the explanatory variables in the model.
Other diagnostic tests that have been carried out for timber supply, domestic and export demand
equations were serial correlation, heteroscedasticity, Ramsey RESET test and Wald test (see, Table 1).
The results of Durbin-Watson (DW) and Heteroscedasticity tests have shown no evidence of serial
correlation and heteroscedasticity problems. The Ramsey RESET test has proven that the equation is
stable and has no functional misspecification. The Wald test revealed that there is a significant long run
cointegration at 1-percent level.
The root mean square error and Theil inequality test demonstrated that the deviation of simulated
variables is quite close to the average size of the variable in the equation. A historical simulation has been
carried out throughout the sample period of study. This is where the adequacy of the model in forecasting
and policy analysis. The detailed tests and results were depicted in Table 2. The Root Mean Square Error
(RεSE) and Theil’s inequality coefficient were found to be relatively small for the timber supply (TS),
domestic demand (DD) and export demand (XD). This suggests that the forecasting and policy analyses
can be considered accurate.
Table 2 Historical Simulation of Timber model for Sarawak
TS
DD
XD
Root Mean Square Error
0.06
0.18
0.25
Theil’s inequality coefficient
0.001
0.006
0.008
Bias proportion
Variance proportion
Covariance proportion
0.000
0.003
0.99
0.000
0.021
0.97
0.005
0.014
0.79
The value of bias proportion is equal to zero, indicating the non-existence of a systematic bias for TS, DD
and XD. The results of actual, fitted and residual provide strong evidence that the equation is stable
between the dependent and all independent variables.
3.4 Results of price and quantity equilibrium
Table 3 presents the empirical results of the average simulated value calculated from the timber partial
market equilibrium model for the period of 1995 to 2008. The impact analysis comprises of four scenarios;
(1) reduced by 24-percent in harvested area, (2) increased by 49-percent in external cost of timber
harvesting, (3) increased by 47-percent in the cost of internalisation the externalities, and (4) 20-percent
gain in market access. As mentioned before, the percentage of the reduction in harvested area and
percentage gain in market access was adopted from the study conducted by Ahmad Fauzi et al. (2002)
and Ahmad Fauzi et al. (2007) respectively. Whereas, the remaining two scenarios (i.e. incremental cost
of internalisation the externalities and incremental external cost of timber harvesting activities) were
borrowed from the study conducted by Abdul Rahim et al (2009) and Abdul Rahim and Mohd Shahwahid
(2011) respectively.
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In this impact analysis, the equilibrium price has been calculated from the estimation of the timber market
model at the market equilibrium was set-up. (see, Eq. 6). After substituting the equilibrium price into the
supply or demand model, the equilibrium quantity was obtained. In other words, the equilibrium price and
quantity of timber could be further quantified from the estimated coefficients. Therefore, the average
3
3
timber market equilibrium point for price and quantity was RM470/m and 2.60 millions m respectively.
This point corresponds with the baseline scenario.
After incorporating the SFM practices scenarios through simulation analysis, the equilibrium quantity
showed a negative effect. This is due to several changes such as reduction in harvested area, incremental
external cost of timber harvesting activities and incremental cost of internalisation the externalities from
timber harvesting activities. However, the price of timber has shown a positive effect as it increases under
the SFM practices scenarios. As shown in Table 3, the price of timber has increased by 123-percent, 5053
3
3
3
percent, 185-percent and 8-percent to RM1,050/m , RM2,846/m , RM1,344/m and RM507/m
respectively under the four scenarios of SFM practices. This result reflected the domestic and export
timber market in Sarawak and could be considered as price premium averaging from 8- to 507-percent.
This is because the incremental price of timber is due to the four scenarios under SFM practices. In this
context, the government could use this finding to formulate the price premium mechanisms such as
conducting programme to educate consumers and to promote the timber produced from forest that
practiced SFM.
Table 3: Average Simulated Values due to SFM Practices for Sarawak
Equilibrium
Quantity
Variable
m
Unit
RM/m
2,596,681
470
Reduced by 24% in
harvested area
2,152,674
1050
Rise by 49% in external
cost of timber harvesting
1,697,884
Baseline scenario
Scenarios % changes due to
SFM practices
3
Equilibrium Price
Rise by 47% in cost of
internalisation the
externalities
Rise 20% in market
access
1,992,579
2,616,273
3
3,546
1344
507
This may suggest that the price increased reflects the value of price premium for timber produced from
SFM practices or certified forests in Sarawak. Similar with the results of previous studies [see, i.e. Kollert
and Lagan (2007)], the price for timber produced from SFM practices or certified forest would potentially
fetch price premium ranging from 2- to 56-percent.
As seen in Table 3, the simulation results of the first three scenarios revealed that the equilibrium quantity
3
3
3
of timber has decreased to 2.15 million m , 1.70 million m and 1.99 million m which is 17-percent, 343
percent and 23-percent decrease from 2.60 million m . This finding is consistent with the study conducted
by Eriksson et al. (2007) who found that the reduction of timber supply in the long run was due to SFM
practices. This implied that the domestic timber processing mills might cut down their consumption of
timber as raw material from the natural forest in the long run. In addition, Woon (2001) revealed that the
total number of timber processing mills (i.e. sawmills, plywood and veneer mills) were expected to be
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drastically reduced due to SFM practices. On the other hand, when the market access scenario under
3
SFM was simulated, the equilibrium quantity of timber increased by 0.8-percent to 2.62 million m .
Based on these scenarios, the reduction in harvested area provides tremendous impact on equilibrium
quantity and price of timber. This is due to the elasticity of this variable is almost elastic (0.8). The result of
this study provides an empirical evidence of the implication of SFM practices on the timber market in
Sarawak. As price, harvested area and input cost were policy variables and revealed significant
determinant on the quantity of supply for timber, Sarawak’s State Government and Forestry Department
could use some mechanisms related to these variables to enhance the SFM practices. Furthermore, effort
should be made to convince tropical timber consumers that timber produced from SFM practices should
be accorded premium prices in view of high cost of timber harvesting operations due to internalisation of
externalities. In other words, the extra value of timber prices could reflect the price for environmental
resources.
The percentage of decreases in equilibrium quantity of timber under SFM scenarios would give an
explanation for timber to be extracted to meet the needs of the present generation without compromising
the ability of the future generations to fulfill their needs. However, with the use of appropriate technology in
the timber harvesting operations, the recovery rate for volume of timber to be extracted could be
enhanced.
3.5 Results of welfare economic impacts
Based on the simulated value calculated earlier as given in Table 3, the average annual estimated values
of welfare economic impacts were further calculated (Table 4). The similar scenarios as what in the
market impact analysis were adopted and simulated in this welfare economic impacts analysis. The
simulation results showed that the calculated value of producer surplus and consumer surplus changes
when incorporating the four scenarios under SFM practices.
The simulation result showed that the calculated value of producer surplus changed when the four
scenarios under SFM practices were incorporated. Under scenario one where HA was reduced by 24percent, the producer surplus reduced from RM27.01 million under the baseline scenario to RM22.10
million. Similarly, the consumer surplus also decreases from RM637.93 million under the baseline
scenario to RM637.75 million. Under scenario two where the external cost of timber harvesting rose by
49-percent, the producer surplus reduced from RM27.01 million under the baseline scenario to RM16.97
million. Similarly, the consumer surplus also decreased from RM637.93 million under the baseline
scenario to RM636.81 million. Under scenario three where the cost of internalisation the externalities went
up by 47-percent, the producer surplus reduced from RM27.01 million under the baseline scenario to
RM18.55 million. Similarly, the consumer surplus also decreased from RM637.93 million under the
baseline scenario to RM637.49 million. On the other hand, under scenario four where the export market
gained 20-percent of market access, the producer surplus increased from RM27.01 million under the
baseline scenario to RM28.43 million. Similarly, the consumer surplus also increased from RM637.93
million under the baseline scenario to RM762.03 million.
This result indicates that the variations in HA and IC were the causes of reduction in the calculated value
of the producer and consumer surplus. This situation would bring towards a loss in economic welfare on
the timber market in Sarawak. The economic welfare in this study referred to the calculated value of total
social benefit. Hence, this finding implied that when timber industry complied with SFM practices, it will
decline stakeholders’ economic welfare in the timber sector.
As noted by Wells and Wall (2005), there is an element of trade-offs between environmental protection
and timber production from the forest. SFM practices could ensure the source of timber supply from
natural forest is sustainable and to minimise the externality effects from timber harvesting activities.
Otherwise, the regeneration of timber from the natural forests would be affected and the nation would lose
the valuable NFTPs and environmental services. Kotwal et al. (2008) claimed that SFM practices could
enhance growing stock of timber and forest productivity of timber and non-timber forest produce.
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Table 4 Average Welfare Impacts due to SFM Practices for Sarawak
Item
Scenarios % changes under SFM
practices
Baseline scenario
Reduced by
24% in
harvested area
Rise by 49% in
external cost of
timber
harvesting
Rise by 47% in
cost of
internalisation
the externalities
Rise 20% in
market access
Producer surplus
Consumer surplus
Total Social
benefits
27,014,313
637,932,059
664,964,372
22,100,885
637,752,042
659,852,927
636,814,728
653,788,113
18,553,630
637,498,806
656,052,436
28,433,145
762,030,132
790,463,277
16,973,385
The simulation result showed that the calculated value of consumer and producer surplus increases when
the scenario of market access (20-percent gained in market access) was incorporated. The consumer
surplus increased to RM762.03 million which is 20-percent increase from RM637.93. The producer
surplus increased by 5-percent to RM28.43 million. This would bring economic welfare gain to the
stakeholders in the timber industry. This is because the timber produced from sustainably managed forest
could give consumers a credible guarantee that the timber is coming from environmentally responsible
and social beneficial forest (Perera et al., 2006). According to Rabiul et al. (2010) Malaysia will continue to
support the international efforts to ensure sustainability in forest management and market access of
Malaysian timber products, especially for the environmentally sensitive markets. Hence, the loss in value
of economic welfare under the first three scenarios of SFM practices could be offset by the market access
that could potentially be realised in the Sarawak timber market.
4. CONCLUSIONS
The results of timber market in Sarawak have shown that harvested area, market access and input cost
were the variables that gave significant impacts on the equilibrium price and quantity process, which in
turn affects the producer’s profits under SFε practices. εoreover, SFε practices would certainly affect
the stakeholders in timber-based industry as their interests normally require trade-offs from the
environmental and natural resource concerns. The interests of different stakeholders were rarely fully
mutually reinforcing (Rametsteiner and Simula, 2003). SFM deals with various stakeholders that were
related to the natural forests. In this study, stakeholders in the timber sector were evaluated.
For a partial equilibrium timber market analysis, the result showed that compliance with SFM practices
reduced the supply of timber. However, in the equilibrium process, compliance with SFM practices pushed
up the level of price. Hence, the equilibrium price and quantity of timber has increased and decreased
respectively as a result of internalising the externalities from timber harvesting activities. For the case of
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Sarawak, the scenario analysis showed that the market and welfare economic impacts due to SFM
practices were modest. Based on those scenarios, decreased harvested levels in connection with SFM
practices were likely to affect the market and economic welfare more than increased input costs. Similarly,
Schwarzbauer and Rametsteiner (2001) found that decreased in harvested levels gave an immense
impact on forest products market than increased in the operational costs due to SFM practices.
On the other hand, the existence of market access from consumers that consume only timber produced
from SFM practices, would give timber producers some advantages on their economic welfare. For
example, export of Sarawak timber has rose up to 50-percent in the first 4 months in 2010 compared with
the same period in 2009 (Anon, 2010). In addition, Sarawak timber industry has received recognition from
the Japan δumber Importers’ Association (JδIA) for the sustainably managed forests in Sarawak (ITTO,
2010). This would give a good indicator to the Sarawak timber industry. At the same time, “green” or price
premium and several incentives from the Sarawak State Government would offset the reduction in
economic welfare and could promote the implementation of SFM in Sarawak. Therefore, stakeholders
from the timber and other sectors which represent the society as a whole could at least have some mutual
benefits.
REFERENCES
Abdul Rahim, A.S. and Mohd Shahwahid, H.O. 2011. A panel data analysis of timber harvesting
operations and its impact on the cost of water treatment. Australian Journal of Basic and Applied
Science, 5: 598-601.
Abdul Rahim, A.S. and Mohd Shahwahid, H.O., 2009a. Short run and long run effects of sustainable
forest management practices on West Malaysian log supply: An ARDL approach. Journal of
Tropical Forest Science, 22: 369-376.
Abdul Rahim, A.S., Mohd Shahwahid, H.O., Zariyawati, M.A., 2009b. A comparison analysis of logging
cost between conventional and reduce impact logging practices. Int. Journal of Economics and
Management, 3: 354-366.
Ahmad Fauzi, P., 2005. The economic impact of sawntimber export levy removal on the Peninsular
εalaysia’s economy. Unpublished PhD Thesis, Universiti Putra εalaysia.
Ahmad Fauzi, P., Rohana, A.R., Nirini, H. and Nor Suryani, A.G. 2007. Chain-of-Custody certification:
Does it effect our export market? In Proceedings of the Conference on Forestry and Forest Products
Research, pp: 334–349.
Ahmad Fauzi, P., Salleh, M., Mohd Shahwahid, H.O., Abdul Rahim, N. Awang Noor, A.G. and Muhamad
Farid, A.R. 2002. Cost of harvesting operations in compliance with ITTO guidelines for SFM. In A
Model Project for Cost Analysis to Achieve Sustainable Forest Management. Volume II: Main
Report, pp: 63-84.
Anon. 2010. Sarawak timber exports up 33pc in first 4 months. Retrieve 28 July 2010 from
www.btimes.com.my.
Eriksson, L.O., Sallnass, O. and Stahl, G. 2007. Forest Certification and Swedish wood supply. Forest
Policy and Economics, 9: 452–463.
th
Gujarati, D.N., 2003. Basic econometrics, 4 ed. McGraw-Hill Book Company, New York.
ITTO. 2010. Tropical timber market reports. 15(15), 15 August 2010, 10 p.
Kollert, W. and Lagan, P. 2007. Do certified tropical forest logs fetch a market premium? A comparative
price analysis from Sabah, Malaysia. Forest Policy and Economics, 9, 862–868.
Kotwal, P.C., Omprakash, M.D., Gairola, S. and Dugaya, D. 2008. Ecological indicator: imperative to
sustainable forest management. Ecological Indicators, 8: 104-107.
Kumari, K., 1996. Sustainable forest management: myth or reality? Exploring the prospects for Malaysia.
Ambio, 25: 459-467.
Mohd Shahwahid, H.O., 1995. Forest conservation and its effects on Peninsular Malaysia log supply.
ASEAN Economic Bulletin, 11(3): 320-334.
Parsakhoo, A., Hosseini, S.A., Lotfalian, M. and Jalilvand, H. 2009. Efficiency and cost analysis of forestry
machinery usage in Hyrcanian forest of Iran. World Applied Science Journal, 6: 227-233.
Perera, P., Vlosky, R.P., Amarasekera, H.S. and De Silva, N. 2006. Forest certification in Sri Lanka.
Forest Products Journal, 56: 4-11.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Rabiul, I., Chamhuri, S., Shaharuddin, M.I. and Nurul Hidayah, C. 2010. Criteria and indicators for
sustainable forest management in Malaysia. American Journal of Environmental Science, 6: 212218.
Rametsteiner, E. and Simula, M. 2003. Forest certification - an instrument to promote sustainable forest
management? Journal of Environmental Management, 67: 87-98.
Schwarzbauer, P. and Rametsteiner, E., 2001. The impact of SFM-certification on forest product markets
in Western Europe – an analysis using a forest sector simulation model. Forest Policy and
Economics, 2: 241-256.
Thang, H.C., 2007. An outlook of the Malaysian forestry sector in 2020. Unpublished Report, Forestry
Department of Peninsular Malaysia (FDPM). 89 p.
Tietenberg, T., 2003. Environmental and natural resource economics. Sixth Edition. Addison Wesley,
USA.
Wells, J. and Wall, D. (2005). Sustainability of sawn timber supply in Tanzania. International Forestry
Review, 7(4), 332-341.
Woon, W.C. 2001. The impact of sustainable forest management (SFM) on wood-based industries in
Peninsular Malaysia. In Proceedings of the International Conference on Forestry and Forest
Products Research, pp: 286–292.
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PLANNING MATRIX TO INCREASE PADDY FARM INCOME IN EAST KALIMANTAN, INDONESIA
1,2*
Karmini
and Abu Hassan Md Isa
2
1)
2)
Faculty of Agriculture, University of Mulawarman, East Kalimantan, Indonesia
Faculty of Economics and Business, Universiti Malaysia Sarawak, Sarawak, Malaysia
*E-mail: karminiunmul@yahoo.com
Abstract
Agricultural development in East Kalimantan faces challenges in raising paddy farmer income. Paddy farm
income is lower than its average in Indonesia. Consequently, effort is needed to increase paddy farm
income and to develop agricultural sector simultaneously. The objective of this study was to develop the
planning matrix to increase paddy farm income in East Kalimantan, Indonesia. A two stage cluster
sampling was used to determine the 3 districts and the 9 subdistricts as the study areas and the 380
paddy households as respondents. A regression function was applied to determine factors influencing
paddy farm income. Descriptive analysis was used to make a planning matrix to increase paddy farm
income. The planning matrix consists of the integrated planning of agricultural development in the study
areas. It is designed to help and to give direction for all stakeholders to answer some issues such as
programs that should be carried out, the reasons why the programs should be done, locations, timing,
people involved and the methods to conduct the program. The matrix is made to ensure all activities
focus to increase paddy farm income.
Keywords: paddy farming, paddy farm income, planning matrix, East Kalimantan.
1. INTRODUCTION
Paddy farming is becoming the potential source of income for most of farm households in East
Kalimantan, Indonesia. It is concluded from the fact that the total paddy households in East Kalimantan in
2009 were 119,555 (Statistics East Kalimantan, 2009). Kustiari et al. (2008) found that agricultural sector
contributes to household income between 58 and 94%. Gross income of paddy farm in East Kalimantan
in 2008 was lower than its average in Indonesia (Statistics Indonesia, 2008). Consequently, effort is
needed to increase agricultural income and to develop this sector simultaneously.
Agricultural development faces challenges in raising paddy farmer income. Agricultural development is
needed not only to increase farmer’s income but also to achieve food security, improve competitiveness
and increase value added of agricultural products. It is needed the development of ideas and strategies in
order to help farmer getting better income. According to Hadwiger (1992), a government can provide a
framework to facilitate the development a productive and efficient agriculture.
The objective of this study was to develop the planning matrix to increase paddy farm income in East
Kalimantan, Indonesia. The planning matrix is important as a foundation and a guideline in agricultural
development planning. It provides information on what policies and decisions should be implemented to
manage natural, physical and human resources for agricultural and other sectors development.
2. METHODOLOGY
This study was conducted in East Kalimantan Province, Indonesia. A two stage cluster sampling was
used to determine the 3 districts (Kutai Kartanegara, Penajam Paser Utara and Bontang) and the 9
subdistricts as the study areas, also number of respondents. A total of 380 households of paddy farmers
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were selected in the sampling frame. Respondents reside in Tenggarong Seberang (128 households),
Loa Janan (17 households), Muara Muntai (4 households), Babulu (128 households), Penajam (84
households), Waru (16 households), South Bontang (2 households), North Bontang (1 household) and
West Bontang (0 household).
A regression function was applied to determine factors influencing paddy farm income. The regression
model consists of the relationship between paddy farm income and 8 socio-economic variables such as
age of household head, depreciation of tools, experience of household head in paddy farming, labor cost,
land cultivation cost, paddy farm size, raw materials cost and rice requirement of household. Descriptive
analysis was used in the making of the planning matrix to increase paddy farm income in East
Kalimantan, Indonesia.
3. RESULTS AND DISCUSSION
This research proposes a planning matrix to increase paddy farm income in East Kalimantan, Indonesia,
as shown in Table 1. In general, the planning matrix consists of the integrated planning of agricultural
development in the study areas. The planning matrix is designed to help stakeholders to answer some
issues such as programs that should be carried out, the reasons why the programs should be done,
locations, timing, people involved and the methods to conduct the program.
The programs that will be offered in the matrix are selected based on the reasons they are to be done.
This research discovers that, collectivity, age of household head, depreciation of tools, experience of
household head in paddy farming, labor cost, land cultivation cost, paddy farm size, raw materials cost
and rice requirement of household are significantly influencing paddy farm income in East Kalimantan,
Indonesia, ceteris paribus. Individually, the 3 independent variables have impact on the dependent
variable. Land cultivation cost and raw materials cost are very significantly and negatively influencing
paddy farm income in East Kalimantan, Indonesia, ceteris paribus. Then, paddy farm size is very
significantly and positively influencing paddy farm income in East Kalimantan, Indonesia, ceteris paribus.
Other variables, individually, are not significantly influencing paddy farm income in the study areas. This
research offers the 3 programs that have the potential ability to increase paddy farm income. It includes
increasing tractor number in agricultural area, extensification, intensification and diversification, and
developing of efficient and effective marketing system for agricultural inputs.
Economic development in rural areas must be integrated with other sectors. In the other words, every
sector has ability to influence other sectors for example a labor in agricultural sector could be a labor in
other sectors. This research proposes the 22 activities related to implementation the 3 programs to
increase paddy farm income. The stakeholders in agricultural development include farmers, private
entrepreneurs, government agencies, non government institutions and researchers. According to Henson
and Sekula (1994), in the longer term, the planning of food production was not be driven by any rational
assessment of market demand or requirements for efficiency in production. The authors argued that by
the perceptions of regional and/or national requirements for food products held by the central planners
and the wider social role of industry within the community such as ensuring local full employment or the
provision of social services should be the basis.
Therefore, stakeholders also have the role as environmental managers because environment is included
in the scope of agricultural development. According to Barrow (2005), they should take the initiative and
steer the development and implementation of programs and ensure co-ordination and regular review
among stakeholders. It could be happened difference the policy and the interpretation of policy among
stakeholders (Scobie et al., 1991). The integrated planning covers many levels of government such as
provincial, district, sub-district and village levels. Implementation of all proposed activities could be done
during a year depend on financial ability.
.
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Table 1 Planning matrix to increase paddy farm income in East Kalimantan, Indonesia.
No.
1.
2.
Background
Land
cultivation cost
is negatively
and very
significantly
influencing
paddy farm
income in East
Kalimantan,
Indonesia,
ceteris
paribus.
Paddy farm
size is
positively and
very
significantly
influencing
paddy farm
income in East
Kalimantan,
Indonesia,
ceteris
paribus.
Program
Increasing
tractor
number.
Extensification,
intensefication and
diversification.
Activity
Saving part of
income minimum as
much as
depreciation cost of
tractor.
Set up business in
selling, renting and
maintaining of
tractor.
Providing tractor to
agricultural
institution in village
level.
Facilitation access
to give credit for
small farmers to
buying tractor.
Researching
related to efficiency
of tractor utilization
to know optimal
number of tractor
needed in
agricultural area.
Increasing planted
& harvested area
through buying,
renting or using
marginal land.
Intensification of
inputs.
Diversification of
crop to optimize
land use.
Renting land to
farmer.
Expanding
agricultural area.
Stakeholder
Farmer
Place
Financial
institution
Time
Periodically.
Private
Rural and urban
areas.
During a
year.
Government &
non
government
institution.
Government, &
financial
institution.
Rural areas.
Depend on
target.
Rural areas,
financial
Institution.
During a
year.
Researcher,
government &
non
government
institution.
Research
institution, rural &
urban areas.
Depend on
target.
Farmer
Village
Short and
long term.
Farmer
Village
Farmer
Village
Short and
long term.
Short and
long term.
Private
Village
Government
Province, district,
village.
Developing
infrastructure in
rural and urban
areas (such as
road, irrigation,
market, social
facilities & etc) &
regular maintaining.
Researching
related to the
optimal farm size
for paddy farming.
The regular
research hearings
to know community
and farmer needed.
Government
Province, district
& village.
Short and long
term.
Researcher,
government &
non government
institution.
Researcher,
government &
non government
institution.
Research
institution, rural
areas.
Depend on
target.
Research
institution, rural
areas.
Periodi-cally.
212
During a
year.
Long term.
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Table 1. (continued).
No Background
Program
.
3.
Raw materials
cost
is
negatively and
significantly
influencing
paddy
farm
income
in
East
Kalimantan,
Indonesia,
ceteris
paribus.
Developing
of efficient
and effective
marketing
system for
agricultural
inputs.
Activity
Stakeholder
Place
Time
Using raw materials in
optimal number.
Buying
inputs
collectively
through
farmer group.
Facilitation
access
credit for farmer.
Supplying & selling
inputs.
Taking
action
to
stabilize market price.
Farmer
Village
Farmer
Village
Financial institution.
Village
Private
Government
Village,
district.
Province
Procuring purchase,
management
of
stocks
and
trade
measures.
Supplying/distributing
the subsidized raw
materials.
Providing extension &
empowerment farmer
groups, cooperatives,
agricultural institution
& financial institution
in rural areas.
Researching related
to the development of
input
& marketing
system for input &
output.
Government
Province
Planting
season.
Before
planting
season.
During a
year.
During a
year.
Short
and long
term.
Short
and long
term.
Government
&
non
government institution.
Province,
district,
village
Province,
district,
village
Depend
on target.
Village,
district,
province.
Depend
on target.
Government
&
non
government institution.
Researcher, government
&
non
government
institution.
Depend
on target.
According to Janseen (1993), the direct contribution of agricultural development can be viewed from the
aspect of creating more employment within agricultural production itself. The indirect effect is by
enhancing the forward and backward linkages with agro-industrial activities. Goodland and Day (1993)
mentioned that the priorities for sustainable economic development are population stability, renewable
energy, human capital formation (education and training, employment creation, technological transfer), job
creation, increasing society income, conservation and prudent management of natural resources.
4. CONCLUSION
The planning matrix is made to integrate and to ensure all activities and stakeholders focus their attention
to give the direct and indirect contributions to agricultural development. It consists of the 3 programs and
the 22 activities that have ability in raising paddy farm income in East Kalimantan, Indonesia. The
planning matrix offers many opportunities to develop business in agricultural and other sectors. This
research contributes to the practical level by providing information how to increase paddy farm income by
implementing resource management. It is hoped this research leads the improvement of existing
production systems in agricultural and other sectors to make them more productive and more sustainable.
REFERENCES
Barrow, C.J. (2005). Environmental management and development. New York: Routledge. p. 231.
Goodland, R. and Daly, H. (1993). Why northern income growth is not the solution to southern poverty.
Ecological Economics, 8:85-101.
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Hadwiger, D.F. (1992). Who creates food abundance? Agricultural policy decision structures and
productivity in developing countries. Food Policy, October 1992:337-348.
Henson, S. and Sekula, W. (1994). Market reform in the polish food sector: Impact upon on food
consumption and nutrition. Food Policy, 19(5):419-442.
Janssen, W.G. (1993). Economic and agricultural development in West Asia and North Africa. The need
for agricultural research. Food Policy, December 1993: 507-522.
Kustiari, R., Sugiarto, Supadi, Sinuraya, J.F., Ariani, M., Bastuti, T., Sunarsih, Hadi, P.U., Maulana, M.,
Purwoto, A., Winarso, B., Waluyo and Hidayat, D. (2008). National Farmer’s Panel/PATANASμ
Analysis of agricultural and rural development indicators. Jakarta: Ministry of Agriculture of
Indonesia. pp. 1-13.
Scobie, G.M., Jardine, V. and Greene, D.D. (1991). The importance of trade and exchange rate policies
for agriculture in Ecuador. Food Policy: 35.
Statistics East Kalimantan. (2009). Pendataan usahatani 2009 Provinsi Kalimantan Timur. Samarinda:
Statistics East Kalimantan. pp. 15, 33.
Statistics Indonesia. (2008). Trends of the selected socio-economic indicators of Indonesia. Jakarta:
Statistics Indonesia. pp.3-69.
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DEVELOPMENT OF POTENTIAL BIODIESEL FROM AGRICULTURAL WASTES
1*
1
Zainab Ngaini , Farra Diana Shahrom , Mohd Hasnain Hussain
2
1
Department of Chemistry, Faculty of Resource Science and Technology
Department of Biology Molecule, Faculty of Resource Science and Technology
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
2
*Email :nzainab@frst.unimas.my
Abstract
Biodiesel has gained an attention as an alternative energy source due to its non toxic, biodegradable and
non flammable properties. The production cost, however is not only quite high but also competing with
the edible sources as feedstock for biodiesel production. Therefore, production of biofuel from waste
source could be a better solution thus reduces its price. In this study, biomass from agricultural waste
was used in the production of biodiesel. Palm oil mill effluent, which was obtained from Bau Palm Oil Mild,
Sarawak underwent transesterification with alcohol in the presence of heterogeneous catalysts to afford
23% biodiesel. The biodiesel were characterized using Fourier Transform Infrared (FTIR) and Gas
Chromatography Mass Spectrometer (GC-MS). The properties of POME biodiesel such as odour,
viscosity and colour were also analysed. The biodiesel obtained from POME has low acidity, viscosity and
water content. It has potential to become one of the alternative sources for producing biodiesel.
Keywords: Agrowastes, palm oil mill effluent, transesterification, catalyst, biodiesel
1. INTRODUCTION
Biodiesel is an alternative fuel in substituting standard diesel fuel due to their similar properties. It is made
up from various biological ingredients other than petroleum such as plants and animals. It can also be
produced from new and used vegetable oils and animal fats (Bozbas, 2008), soybeans, peanuts,
rapeseeds as well as sunflowers (Ramesh, et al., 2002). It is non-toxic, biodegradable, sulphurless and
renewable (Cardoso et al., 2008).
Biodiesel can be used in the diesel engines without any alteration and is promisingly safe. It has similar
physical properties to those of diesel fuel in terms of cetane number, energy content, viscosity and phase
changes of fatty acid methyl ester (FAME) (Yee et al., 2008). The use of biodiesel may reduce the
hazardous gases emission such as carbon monoxide (CO) and sulphur monoxide (SO). Production of
biodiesel in Asian countries are based on crude palm kernel oil and crude coconut oil while USA and
Europe are using soybean and rapeseed as the feedstock (Yee et al., 2008)
Malaysia is one of the largest exporter countries in oil palm product. The total areas of plantation are
increasing every year. For example, in year 2000, plantation areas are increases to 3338 hectares from
320 hectares in 1970. Palm oil is extracted from palm fruit. One hectare of oil palm may produce
approximately 10 to 35 tonnes of fresh fruit bunches per year (Rupani et al., 2010). The increase in palm
oil production throughout the year also contributes to the rising of its by-product of the milling process.
Palm oil mill effluent (POME) is generated from oil extraction process which contain.50% of water (Okwute
et al., 2007). For every tonne fresh fruit bunch, 0.5- 0.75 tonnes of POME will be discharged.
POME is physically thick and brownish semi-liquid and contains high solids, oil and grease, COD and
BOD values (Rupani et al., 2010). Without proper treatment, POME is among the most dangerous waste
for the environment. It contains highly oxygenated compound which may deplete the dissolved oxygen
content in the water and cause the death of aquatic life due to insufficient oxygen (Rupani et al., 2010). In
this paper, we report on the development of biodiesel from POME in the presence of heterogeneous base
catalysts.
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2. MATERIALS AND METHODS
Preparation of Raw Materials: POME was obtained from cooling pond of Bau Palm Oil Mill (BAPOM),
Sarawak, Malaysia. It was heated to constant weight prior to transesterification process.
Transesterification of POME
Heterogeneous catalyst (20 g, 5%) was added into methanol (400ml) in the three-necked round bottom
flask and heated under reflux for 1h. POME (50ml) was then added onto the flask and continued heating
for overnight. The mixture was filtered and subsequent filtrate was dried in vacuo to afford brown liquid.
Characterization of Bio-oil
Raw and esterified oil were characterized for their physical and chemical properties such as odour,
viscosity, colour, FTIR analysis as well as GC-MS analysis.
3. RESULTS AND DISCUSSION
Preparation of biodiesel from POME was successfully carried in the presence of heterogeneous catalyst.
The biodiesel produced comprise of ester and glycerol, which was separated via centrifugation, to afford
23% by weight of biodiesel. The infrared spectra of POME after transesterification are shown in Figure 1.
-1
-1
IR showed the disappearance of peaks at 1708 cm and the presence of peaks at 1743.14 cm , which
attributed to the formation of ester. The IR peaks of POME biodiesel is corresponded to the commercial
biodiesel from rape seed oil (Dayong, et al., 2011)
Figure 1 FTIR Spectra of POME and POME biodiesel
Table 1 showed the GCMS analysis of the raw POME composition which is dominated by several types of
carboxylic acids. High palmitic acid and oleic acid were shown in the raw POME. The presence of various
types of carboxylic acid compounds in raw POME explained the characteristics of low pH, acidic
compound which also gave out strong, unpleasant smell. The carboxylic acid contents were decreased
after undergoing transesterification and ester was formed. The unpleasant odour was resulted from the
presence of high molecular compounds such as carboxylic acid compound such as palmitic and oleic
acids which released stronger odour (Bridgwater, 2003).
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Table 1 GC-MS of Raw POME
Peak
Retention Time
Area (%)
Molecular Weight
Compound
1
2
3
4
55.111
60.233
60.482
61.078
19.84
1.79
17.47
1.29
256
280
282
284
Palmitic acid (C16 H32 O2)
Linoleic acid (C18 H32 O2)
Oleic acid (C18 H34 O2)
Stearic acid (C18 H36 O2)
5
87.158
52.29
240
Palmitaldehyde (C16 H32 O)
After transesterification process, it was observed that the unpleasant odour of POME was converted into
pleasant smell product. This is due to the conversion of ester from carboxylic acid compound in the raw
POME. The colour of raw POME was changed from dark brown into clear brown after transesterification
process. The viscosity of raw and esterified POME wastes have also undergone changes from viscous
liquid to a less viscous liquid. This was due to the breaking of large, branched triglyceride into smaller and
straight chain molecule of alkyl esters (Lee et al., 2009). Table 2 showed the several types of ester found
after transesterification process.
Table 2 GC-MS of the Esterified POME Oil
Peak
Retention Time
Area (%)
Molecular Weight
1
2
3
4
5
53.688
58.872
59.135
59.845
60.222
33.16
10.17
31.06
6.36
1.04
270
294
296
298
280
Compound
Methyl Palmitate (C17 H34 O2)
Methyl Linoleate (C19 H34 O2)
Methyl Oleate (C19 H36 O2)
Methyl Stearate (C19 H38 O2)
Linoleic acid (C18 H32 O2)
POME biodiesel obtained was less viscous than the raw POME. The viscosities of biodiesels were much
less than those of pure oils (Demirbas, 2008). The presence of water in the biodiesel may reduce the
viscosity and enhance the stability however, water may decrease the calorific value of bio-oil even though
water is vital in many application (Sukiran et al., 2009).
The pH value for raw POME waste was 4.8 while the pH for POME biodiesel was 8.5. The increased in
pH value was supported by GC-MS result showed that the percentage area of carboxylic acid was
reduced after transesterification from 40.39% to 1.04%. The POME biodiesel became less acidic
compared to the raw wastes. The use of solid base catalyst was more effective in decreasing the
corrosiveness of the biodiesel and increasing its quality (Zhang et al., 2006).
4. CONCLUSION
POME from cooling pond of BAPOM Sarawak has been successfully converted into biodiesel via
transesterification process in the presence of methanol and heterogeneous catalyst. This green project
may help the BAPOM Company in solving the environmental problems due to POME waste. By the
production of biodiesel from agricultural waste, the country may increase the income and perhaps become
the biggest biodiesel exporter in this industry. POME biodiesel may become one of the commercial
biodiesel in the future.
ACKNOWLEDGEMENTS
The authors would like to thank Universiti Malaysia Sarawak and Kementerian Tenaga, Teknologi Hijau
dan Air (KTTHA) for the financial support to conduct this research.
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REFERENCES
Bozbas, K. (2008). Biodiesel as an alternative motor fuel: Production and policies in the European Union.
Renewable and Sustainable Energy Reviews, 12(2), 542-552.
Bridgwater, A. (2003). Renewable fuels and chemicals by thermal processing of biomass. Chemical
Engineering Journal, 91(2), 87-102.
Cardoso, A. L., Neves, S. C., & da Silva, M. J. (2008). Esterification of Oleic Acid for Biodiesel Production
Catalyzed by SnCl2: A Kinetic Investigation. Energies, 1(2), 79-92.
Dayong, J., Xuanjun, W., Shuguang, L, & HejunXi'an, G. (2011). Rapeseed Oil Monoester of Ethylene
Glycol Monomethyl Ether as a New Biodiesel. Journal of Biomedicine and Biotechnology, 2011
Demirbas, A. (2008). Relationships derived from physical properties of vegetable oil and biodiesel fuels.
Fuel, 87(8), 1743-1748.
Lee, D. W., Park, Y. M., & Lee, K. Y. (2009). Heterogeneous Base Catalysts for Transesterification in
Biodiesel Synthesis. Catalysis surveys from Asia, 13(2), 63-77.
Okwute, L. O., & Isu, N. R. (2007). The environmental impact of palm oil mill effluent (pome) on some
physico-chemical parameters and total aerobic bioload of soil at a dump site in Anyigba, Kogi
State, Nigeria. African Journal of Agricultural Research, 2(12), 656-662.
Ramesh, D., Samapathrajan, A., & Venkatachalam, P. (2002). Production of Biodiesel from Jatropha
curcas Oil by Using Pilot Biodiesel Plant. Agricultural Engineering College & Research Institute,
Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.
Rupani, P. F., Singh, R. P., Ibrahim, M. H., & Esa, N. (2010). Review of Current Palm Oil Mill Effluent
(POME) Treatment Methods: Vermicomposting as a Sustainable Practice. World Applied
Sciences Journal, 10(10), 1190-1201.
Sukiran, M. A., Chin, C. M., & Bakar, N. K. A. (2009). Bio-oils from pyrolysis of oil palm empty fruit
bunches. American Journal of Applied Sciences, 6(5), 869-875.
Yee, K. F., & Lee, K. T. (2008). Palm Oil As Feed Stocks For Biodiesel Production Via Heterogeneous
Transesterification: Optimization Study.
Zhang, Q., Chang, J., Wang, T. J., & Xu, Y. (2006). Upgrading bio-oil over different solid catalysts. Energy
& fuels, 20(6), 2717-2720.
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PHYLOGENY OF SELECTED MALAYSIAN PRIMATES INFERRED FROM MITOCHONDRIAL DNA
CYTOCHROME C OXIDASE I (COI) GENE.
Millawati Gani*, Licia Ho, Sarina Mat Yasin, Yuzine Esa and M. T. Abdullah
Department of Zoology, Faculty Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
*Email: gmillawati@hotmail.com
Abstract
In Malaysia, there are 19 species of primate comprises of nine genera. IUCN had listed six species as
endangered and seven species as vulnerable. Existing primate habitats are increasingly threatened by
human activities which are illegal trade, hunting, logging, forest clearing, and land conversion to
plantations or human settlement. These resulted in local extinction and remain populations trapped in
small forest fragments which caused declines in the number of primate species. The study of phylogenetic
on primates is important to determine their genetic relationship among the primate especially the
endangered species. Hence, this study aims to elucidate the species boundary and genetic diversity of
Malaysian primates by using mitochondrial DNA Cytochrome c Oxidase I (COI) gene. COI evolves more
rapidly than nuclear DNA which useful to distinguish closely related genera and able to show the genetic
differences between two individuals even though from the same species. Blood, tissue and fecal samples
were collected and extracted. A total of 580bp of the mtDNA COI gene from seven species of primates
were sequenced. Phylogenetic analyses of mtDNA COI gene were using neighbour-joining (NJ),
maximum parsimony (MP), maximum-likelihood (ML) and Bayesian method.
Keywords: Phylogeny, primates, mtDNA COI, Malaysia
1. INTRODUCTION
There are currently 19 described species in the Order Primate in Malaysia. Primates are mostly arboreal
animal and their rates of development are slower than other mammals. According to Bernard et al. (2010),
primates spend about half of their life on sleeping sites. The number of primates could decrease if there
was disturbance occur at their habitat or more precisely at their sleeping sites. Some of them move to
another area to survive and this may lead changes in genetic. The limited space of habitat would make
the population compete, split and then exchange gene with one another and share in the process of
genetic drift and adaptations ( Won and Hey, 2005). Primates have suffered critical declines and faces
possible extinction. They are facing critical habitat loss and their distribution was highly fragmented
because of land conversion to agriculture and human settlement as well as hunting, illegal trade, and
logging (Tisdell and Nantha, 2007).
This study addresses the ecology, morphology, biogeography and conservation of primate in Malaysia.
According to Abdullah (2011), in Malaysia there is none research on primates in detailed on ecology,
population including genetic study. The study of genetics in a species would be useful knowledge of
factors that might influence or control the divergence in species (Abdullah, 2011). Furthermore, there are
major gaps in the knowledge and lack of study done on the DNA ecology of the primate in Malaysia.
In this study, mitochondrial DNA (mtDNA) genes was chosen because mtDNA is common and widely
used for phylogenetic analyses of closely related species. Tissue, fecal and blood samples will be taken
from primates and phylogenetic analysis of mtDNA will be performed by using multiple mtDNA genes such
the COI gene. COI sequences were widely used in phylogenetic studies. According to Hebert et al. (2004)
COI might serve as a DNA barcode for identification of animal species.
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The objectives of this study were to investigate phylogenetic relationships of Malaysian primates based on
mtDNA genes and to construct phylogenetic trees of Malaysian primates using multiple mtDNA genes.
Other than that, this study is aimed to determine the species boundary and genetic diversity of Malaysian
primates.
2. MATERIALS AND METHODS
Sampling sites were chosen based on the distribution and the availability of primate species in Malaysia.
The study sites for this study include Bako National Park, Kuching Wetland National Park, Matang Wildlife
Centre, Zoo Melaka, Selangor and Maludam National Park.
A total of 47 samples which obtained from blood, tissue and fecal samples were collected from several
study sites. The targeted primates were tracked for the wild primate and was tranquilized using zoletil
(5mg/kg; 100mg/ml). Blood samples were taken from the blood veins using capillary tubes and then
stored in vials containing blood lysis buffer (Sibley and Ahlquist, 1990). Tissue samples were collected
from muscle and liver tissue only for targeted species. Fecal samples were collected and stored in -80°C.
To collect fecal samples in the wild in mangrove area, they were tracked by finding their feeding area
using a boat and waiting for dropping from the primates.
Genomic DNA from the blood samples was extracted using the Qiamp DNA Blood Mini Kit (Qiagen, Cat
No:51104) while from the fecal samples was extracted using Qiamp DNA Stool Mini Kit (Qiagen, Cat
No:51504). Total genomic DNA was extracted from tissue using C-TAB protocol (cetyl-tri-methyl
ammonium bromide).
Cytochrome Oxidase I (COI) of the mtDNA gene was amplified by PCR. Amplification was performed
using 25 µl total volume per reaction with 5 µl of 5X Colorless GoTaq Flexi Buffer, 1.5 µl of MgCl2 (25
mM), 0.5 µl dNTP mix, 1.0 µl of each primer forward and reverse, 14.8 µl of ddH 2O, 1.0 µl of DNA
template and 0.2 µl Taq polymerase. The oligonucleotide COI primers COIf (5’–
CCTGCAGGAGGAGGAGAYCC-3’)
and
COIe
(5’-CCAGAGATTAGAGGGAATCAGTG-3’).
The
temperature profile for 35 cycles was pre-denaturation (95°C for 5 minutes), denaturation (95°C for 1
minute), annealing (45-55°C for 1 minute), extension (72°C for 1 minute), post-extension (72°C for 5
minutes) and soak (4°C). The purified PCR products were sent to the private laboratory (Universiti
Science Malaysia and First Base Company Sdn. Bhd.) for DNA sequencing.
The result of DNA sequencing will be used to constructa phylogenetic analysis by using Chromas Pro
program version 1.5 (MacCarthy, 1998), CLUSTAL X program version 1.81 (Thompson et al., 1997),
Molecular Evolutionary Genetic Analysis (MEGA) version 5.05 (Kumar et al., 2007) and Phylogenetic
Analysis Using Parsimony (PAUP) version 4.0b10 (Swofford, 2001) and MrBayes version 3.1.2
(Huelsenbeck and Ronquist, 2003). Chromas Pro displayed the fluorescent-based DNA sequence result
which shows the four nucleotides. ClustalX can be used to perform multiple alignments, view the result of
alignment process and improve the alignment. MEGA used for detecting stop codon and utilized the
pairwise distance matrix which presents the estimation of genetic distance among primate species. PAUP
is designated for phylogenetic analysis using the neighbor-Joining (NJ), Maximum Parsimony (MP),
Maximum Likelihood (ML) and Bayesian analysis. The ML analysis was determined by using Modeltest
3.7 whereas modeltest was the most appropriate substitution model for ML analysis (Posada and
Crandall, 1998). Mrbayes v3.1.2 program was used to construct the Bayesian tree which selects the tree
that has the greatest probability that the tree is correct given under a specific model of substitution.
3. RESULTS
Genomic DNA from 47 samples consist of 11 species of Malaysian primate was successfully extracted. All
samples were amplified with COIe (reverse) and COIf (forward) primer. The estimated DNA fragment size
of the amplified COI gene was around 580 base pairs (bp). However, only 30 samples of them were
successfully optimized but not all of the samples resulted in bright bands. The annealing temperature for
this study ranged from 45°C to 55°C. From a total 30 samples, 25 samples were sent for sequencing and
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16 samples showed good sequences obtained. However, only 12 samples were used for analysis in this
study. In terms of compiling the data for the construction of the tree, the peak of the nucleotide was
observed based on the chromatograph (fluorescence-based) by using Chromas Pro. Multiple alignments
of the nucleotide sequence were done using the CLUSTAL X program. Pan troglodytes was used to
indicated the outgroup of the phylogenetic tree generated. The outgroup sequence was downloaded from
National Center Biotechnology Information (NCBI). From 511 bp of DNA sequences obtained, only 491 bp
were analyzed after the alignment. Based on the 491 bp analyzed, only 149 characters resulted in the
parsimony informative site while 348 sites were variables. Another 141 characters were on the conserved
sites.
Pairwise genetic distance was used to show variation between individual within the specific species which
calculated using the Kimura-2-parameter of COI for each individual. The pairwise genetic distance of 13
samples including outgroup was analyzed using the PAUP program. The highest divergence occurs
between ZM006 Hylobates lar and PRP009 Nasalis larvatus, indicated by 34.44%. However, the lowest
divergence between the primate species was modeled by the two individuals of Symphalangus
syndactylus (ZM003 and ZM004) with the genetic distance 0.21%. It was concluded that the divergence of
the selected primates ranged from 0.21% to 34.44%. Table 1 showed the average genetic distance
between Malaysia primate used in this study analysis. According to Md-Tamrin et al., (2011), percentage
of genetic distance >2% indicate the variation occur within species, 2%-11% indicate high probability
conspecific population and >11% indicate the large degree of genetic differentiation has been attained
between the species.
Table 1 Average genetic distance between Malaysian primate species using the Kimura-2-parameter
1
2
3
4
5
6
7
1
N. larvatus
-
2
T. cristatus
0.1787
-
3
M. arctoides
0.2800
0.1986
-
4
S. Syndactylus
0.2656
0.1902
0.2501
-
5
M. fascicularis
0.2724
0.1792
0.1136
0.2178
-
6
H. agilis
0.2803
0.2158
0.2522
0.0989
0.2208
-
7
H. lar
0.2841
0.2187
0.2542
0.1060
0.2322
0.0169
-
Nucleotide frequencies also were analyzed based on PAUP. The highest percentage of nucleotide
frequencies were indicated by Thymine (T) with the record of 30.77% and the lowest nucleotide
frequencies was shown by the Guanine (G) with the average of 17.86%. Adenine (A) and Cytosine (C)
were recorded to have 25.05% and 26.32% nucleotide frequencies respectively.
Four major trees were constructed using the neighbour-joining (NJ) with Kimura two-parameter, Maximum
parsimony (MP) with heuristic search option, Maximum likelihood (ML) with TrN+G (Tamura and Nei,
1993) model and Bayesian tree with the posterior probability distribution. One outgroup was used in the
phylogenetic tree, namely, Pan troglodytes to root the trees. All the phylogenetic topologies showed
slightly different topologies and groupings. The MP tree gave the most resolved topologies based on
observed by the higher bootstrap values (>70%) on each branch.
221
Group 2b
Group 2
(Hylobatidae)
Group 2a
Group 1b
Group 1
(Cercopithecidae)
Group 1a
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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st
Figure 1 Scores on the branches refer to bootstrap values from neighbour-joining (1 score), maximum
nd
rd
th
parsimony (2 score), maximum-likelihood (3 score), and Bayesian posterior probabilities (4 score); -indicates to no support value. Samples localities labelled by PRP= Bako National Park and Matang
Wildlife Center), PSA= Selangor and ZM= Zoo Melaka.
4. DISCUSSION
A comprehensive phylogenetic relationship of Malaysian primate based on 491 bp (after alignment the
original ~580 bp COI gene) ampilified from mtDNA COI gene in 12 individuals consist of seven species
with Pan troglodytes as an outgroup for this study. Based on Figure 1, the tree showed mtDNA COI
strongly support that the two main groups was formed. Group 1 formed a group of Family Cercopithecidae
which a group of leaf monkey and macaques while in Group 2 consist of individuals from family
Hylobatidae. The trees showed all individuals in this study are monophyletic.
In ML and Bayesian tree, the distinction of two genera of Malaysian primate in Group 1b which are N.
larvatus and T. cristatus is not clear as the clade resolved by COI gene in NJ and MP tree. They are
unresolved relationship shown between N. larvatus and T. cristatus. This might due to the partial primer
sequence used in this study which ~580 bp and after alignment, there are only used 491 bp to run the
data analysis. It is only 33% of the complete 1500 bp COI gene used. Besides, this occurrence might due
to not enough representatives of the species to analyze their relationship within or among species.
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However, in NJ and MP tree showed a strong relationship between both N. larvatusand T. cristatus with
supported by high bootstrap value which are >74%. Based on previous study done by Md-Zain et al.
(2011) on phylogenetic relationships of Malaysian monkey, Cercopithecidae, found that NJ and MP tree
showed that N. larvatus and T. cristatus was a sister taxa as well as result that showed in this study with
high bootstrap value. Other than that, study by Sterner et al. (2006) on mitochondrial data support an oddnosed colobine clade was also showed they was a sister taxa.
Group 1b consist a group of the macaques which they are in subfamily Cercopithecinae. They were sister
clade with with Group 1a with high bootstrap values which indicate 98% showed they had a strong
relationship within the macaque group. Group 1 showed the macaques as a basal is a sister clade with a
clade of N. larvatus and T. cristatus. This result was supported the previous study done by Perelman et al.
(2011), which their study tree also showed macaques is a sister clade with N. larvatus and T. cristatus.N.
larvatus,T. cristatus, M. fascicularis and M. arctoides was formed their own highly supported monophyletic
clade which are they also group as old world monkeys. The separation of the two subfamilies of Malaysian
Cercopithecidae which are Cercopithecinae and Colobinae is supported by NJ and MP analysis as well as
with the quite high average percentage of genetic distance between them based on the Kimura-2parameter distance matrix method which are 17.87%-28.0%. The value of genetic distance >11% is the
value was suggested that a large degree of genetic differentiation has been attained between species
(Md-Tamrin and Abdullah, 2011).
As shown in Figure 1, group 2 which consists of Family Hylobatidae which are S. syndactylus, H. lar and
H. agilis showed they are sister clade with Group 1 which this result also support the previous study done
by Perelman et al. (2011) showed Hylobatidae is a sister clade with Cercopithecidae in their ML tree. All
trees in this study showed S. syndactylus is a sister taxa with genus Hylobates. H. lar was clade together
with one representative of H. agilis with >70% of bootstrap value. The relationship of H. lar and H. agilis
was not showeda clear relationship might due to a limited number of representatives obtained per species
in this study. However, according to their genetic distance between H. agilis ZM001 and ZM005 showed
small variation which is 1.25% compared to genetic distance showed between H. agilis ZM005 and H. lar
is a little bit higher which is 1.46%. This showed H. agilis ZM001 and ZM005 more closely related
compared to H. agilis ZM005 and H. lareven though they are not in the same branch. Other than that,
based on average percentage of genetic distance between H. agilis and H. lar showed 1.69% which
suggested that they are intraspecific variation.
5. CONCLUSION
The phylogenetic analysis inferred from mtDNA COI gene successfully shows the relationship among
selected Malaysian primates. The tree topology formed a monophyletic clade in all four trees analysis
however N. larvatus having a problematic relationship with other species for both ML and Bayesian tree
might due to a limited number of individuals used in this study. However, in NJ and MP tree, the position
of N. larvatus are congruent with previous study with high bootstrap value in both trees. The unresolved
interspecies relationship might due to only partial COI gene used. COI is a conserved gene so it site more
to conserved gene than a variable gene. Thus, longer sequences should be used in order to determine
and elucidate the genetic relationship of Malaysian primates. For further studies, more samples should be
include to generate more reliable result in phylogenetic of Malaysian primates.
ACKNOWLEDGEMENT
The authors thank to other members of Primate Genome Project Mohd Hanif Ridzuan Mat Daud, Nur Aida
Md Tamrin, Jeffrine Rovie Ryan Japning, Mohamad bin Kombi and Madinah Adrus for their helps, hard
work and assistance throughout this study. We also thank to Faculty Resource Science and Technology,
Universti Malaysia Sarawak for providing facilities, logistic and administrative support. Our gratitude also
goes to the Science Officer of Faculty Resource Science and Technology, Wahap bin Marni for always
accompanied to do work sampling. This study was fully supported by Primate Genome Project Grant
awarded to Prof. Dr. Mohd Tajuddin Abdullah.
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Hebert, P. D. N., Stoeckle, M. Y., Zemlak, T. S. & Francis, C. M., (2004). Identification of Birds Through
DNA Barcodes. PLoS Biology | www.plosbiology.org: Volume 2: Issue 10/e312.
Kumar, S, Tamura, K, Dudley, J., & Nei, M.,(2007). MEGA4: Molecular Evolutionary Genetic Analysis
(MEGA) Software Version 4.0. Molecular Biology and Evolution 24: 1596-1599.
McCarthy, C. (1997). CHROMAS version 1.45 program. Schools of Health Science, Griffith University,
Gold Coast Campus, Queensland, Australia.
Md-Zain, B. M., Mohamad, M., Ernie-Muneerah, M. A., Ampeng, A., Jasmi, A., Lakim, M. & Mahani
M.C. (2010). Phylogenetic relationship of Malaysian Monkeys, Cercopithecidae, based on
Cytochrome c sequence. Genetics and Molecular Research 9 (4): 1987-1996 (2010).
Nur Aida Md Tamrin & M. T. Abdullah. (2011). Molecular phylogenetics and systematics of five genera
of Malaysian murine rodents (Maxomys, Sundamys, Leopoldamys, Niviventer and Rattus) inferred
from partial mitochondrial cytochrome c oxidase subunit I (COI) gene. Journal of Science and
Technology in the Tropics (2011). 7:75-86.
Perelman, P., Johnson, W. E., Roos C., Seuanez, H. N., Horvath, J. E., Moreira, M. A., Kessing, B.,
Pontius, J., Roelke, ε., Rumpler, Y., Schneider, ε. P. C., Silva, A., O’Brien, J., & Pecon-Slattery,
J. (2011). A Molecular Phylogeny of Living Primates. PloS Genetics. Volume 7: Issue 3/e1001342.
Posada, D. & Crandall, K.A. (1998). Modeltest: testing the model of DNA substitution. Bioinformatics,
14 (9): 817-818.
Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes v.3.1.2. Retrieved March 18, 2011, from
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Sterner, K. N., Raaum, R. L., Zhang, Y. P., Stewart, C. B. & Disotell, T. R. (2006). Mitochondrial Data
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Tamura, K., & Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of
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INSECT SPECIES COMPOSITION OF SUNGAI CHUKAI MANGROVE FOREST
Raja Nurul Nadia Raja Alang*, Nur Azura Adam, Wan Faridah Akmal Wan Jusoh and Nor Rasidah
Hashim
Universiti Putra Malaysia (MALAYSIA)
*Email: rajanurulnadia88@gmail.com
Abstract
Mangrove forests provide a unique ecosystem for its flora and fauna with special adaptations to harsh and
tidal conditions. The presence of insects in the mangrove forests is very common; however there have
been few studies conducted and not documented properly. In this study, we investigate the insect species
composition and their relationship of abiotic factors with the insect abundance in four different zones of
Sungai Chukai. Insect were sampled by using Malaise traps and Yellow Pan traps which were installed in
3 plots area for each zone. All the samples were collected after 3 days and preserved in 75% of ethanol.
In overall there are 1299 number of individuals comprises of 14 orders and 62 families of insects were
collected. The order of Hymenoptera, Coleoptera and Lepidoptera are the most common insect’s orders
found in each zones. While, Dermaptera, Neuroptera and Microcoryphia were the least order found and
only identified in zone 3 and 4. This study has shown that the insect species compositions in Sungai Cukai
Mangrove Forest are diverse and the assemblages of the insect in four zones have been influence by the
water salinity, distribution of the vegetation and the weather condition.
Keywords: Insect, zonation, diversity, abiotic.
1. INTRODUCTION
The existence of tropical trees and some shrubs that grow between land and sea (McKee 2002 and
Nagelkerken et al. 2008), variously known as tidal forests, mangrove forests, mangrove swamps or
mangal (Schaeffer-Novelli et al. 2000) has been classified as the major producer for human and animals
(Vanucci 2002). Thus, according to Hogarth (1999), Kathiresan and Bingham (2001), mangrove is one of
the most biologically diverse ecosystem in the world. Special adaptation of the mangrove trees with the
tidal inundation, high level of salt and wave action (Mastaller 1997) has support a wide range of fauna
including birds, mammals, crustaceans, reptiles and insects (Nagelkerken et al. 2008, Wan Jusoh et al.,
2010a; 2010b.).
Insects are one of the most essential components of the earth’s biota associated with every conceivable
type of environment (Ananthakrishnan and Sivaramakrishnan 2008). Insects are very diverse and
abundant in mangroves forest (Martin 1994). Insect frequently can be seen in the canopy and also under
the foliages of the trees which providing a significant habitat to meet their ecological need in different
trophic levels (Nagelkerken et al. 2007, Martin 1994 and Balasubramanyam et al 2000). However, the
assemblages of insects in natural habitat fragments and their long term persistence in a area may be
facilitate by sufficiently protection and adaptation for such possible habitat fragments (Hanski 2008). In
Indo-West Pacific mangroves forests, the insect diversity is consider to be higher than in the Atlantic-East
Pacific due to the higher plant diversity (Macintosh and Ashton 2002).
Generally, ant has been recorded as the most abundant insects in mangrove habitat (Clay and Andersen
1996). Ants are very significant in their role with plant interactions (Canicci et al. 2008). Preliminary study
on ant diversity has been conducted in Rembau-Linggi Estuary, Peninsular Malaysia which resulted 9
species of ants from four subfamilies found on the Berembang trees (Sonneratia caseolaris) (Alang et al
2010). In another study (Hashim et al. 2010), it was found that certain ants that were found in the
mangrove were absent in the adjacent oil palm plantations. In Thai mangrove ecosystem, a common
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predator ant, Oecophylla smaragdina has shown their positive interactions between mangrove trees,
Rhizophora mucronata in reducing number of herbivory from damaging the tree’s leaves (Offernberg et al.
2004). Whilst, in a mangrove forest at the mouth of Bangpakong river, the shaded area of the trees there
has provide the diversity of Culicidae (mosquitoes) and Ceratopogonodae (punkies) with 14 and 11
species were recorded (Prayoonrat 2004).
Besides, mangroves also provide important ecological and socio-economic values to humans (Bennett
and Reynolds 1993). The mangrove ecosystem provides income for locals such as by harvesting
mangrove trees for fuelwood, charcoal, timber and wood chips and also served as nurseries for important
fisheries activities, especially shrimp (Mcleod and Salm 2006). Unfortunately, the interference of man in
coastal areas has become an environmental problem to the world (Ndenecho 2007). This interference has
leading to depletion of the natural habitats of flora and fauna in mangrove forest where maybe some of the
species are not being identified yet. Indirectly it is also has disturb the natural habitat of insects in the
mangroves forest as it is adversely affects the species richness of these arthropods (Alongi 2002). This
alert has shown that the potential loss of insect diversity in any type of habitat through human interference
in the coming years is remarkable (Lewis and Basset 2007). Without further action and attention given to
this issue, we might lost the biodiversity of flora or fauna in this muddy ecosystem before we could
document them. Thus, this study on insect species composition of Sungai Chukai was conducted to
identify the species composition and the abundance of insect in different zonation along the riverine.
2. MATERIALS AND METHODS
2.1 Study area
This study was conducted in Kemaman Mangrove Forest Reserve, Terengganu, Peninsular Malaysia
(415'0.000"N, 10325'1.200"E) which comprises of 938 hectares. Sungai Chukai is located in the mangrove
forest reserve where there are several villages and Chukai town center located nor far from the forest
reserve. Total population at the area is about 82 434 where most of the local people there work as a
fisherman, factory worker and shipman. For this study purpose, Sungai Cukai has been divided into
zonation which total length of the river is 22KM.
2.2 Sampling methods
Sungai Cukai are divided into 4 zones based on two factors, namely type of vegetations and the water
salinity. Three plot areas are selected from each zone based on the type of focal trees and the
accessibility to the forest floor. Malaise trap and Yellow Pan trap were used as the main method in
capturing the insect. All the traps were installed in the forest up to three days. After three days, all the
samples were collected and preserved in the plastic bottle containing 70% of ethanol.
2.3 Sorting and identification
All the samples then were brought to the laboratory for the identification process. The samples were
sorted according to orders and family levels for each individuals. The key identification of insects were
based on Borror deLongs keys.
3. RESULTS AND DISCUSSIONS
In overall there are 1299 number of individuals comprises of 14 orders and 62 families of insects were
collected from all the 4 zones along Sungai Cukai. According to Figure 1, 5% of insects were collected in
zone 1. While in zone 2, the numbers of individual collected are 19%. In zone 3 and 4, the number of
insects were collected are 44% and 32% respectively.
The order of Hymenoptera, Coleoptera, Lepidoptera, Hemiptera and Blattodea are the most common
insect’s orders found in each zone. While, Dermaptera, Neuroptera, Microcoryphia, Odonata, Isoptera and
Mantodea were the least order found and only identified in zone 3 and 4. As in Table 1, for each zone, the
numbers of species composition identified were quite similar. However, in zone 1, the number of insect’
family recorded were low compare to the other zones which are 17 families from 8 orders of insect were
identified.
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Figure 1 The percentage of insects according to four zones in Sungai Chukai mangrove forest.
Table 1 Composition of insect in 4 zones of Sungai Chukai mangrove forest.
Order
Species (Family)
Zone 1
Zone 2
Zone 3
Zone 4
Blattodea
Blattellidae
1
1
1
1
Coleoptera
Anthribidae
0
0
1
0
Bostricidae
0
1
0
0
Brostidae
0
0
1
0
Carabidae
0
0
1
1
Cerambycidae
0
0
1
1
Chrysomellidae
0
1
1
1
Coccinelidae
0
1
1
1
Cucurlionidae
0
1
1
1
Elateridae
1
1
1
1
Lampyridae
0
0
0
1
Melyridae
0
0
0
1
Mordellidae
0
1
1
1
Nitidullidae
0
1
1
0
Omethidae
0
0
1
0
Scarabaeidae
1
1
1
1
Staphylinidae
0
1
0
1
Tenebrionidae
0
1
0
1
Collembola
Entomobryidae
1
0
1
0
Dermaptera
Forficulidae
0
0
0
1
Hemiptera
Cercopidae
0
0
1
0
Cicadellidae
1
1
1
1
Cixiidae
0
1
1
1
Delphacidae
1
1
1
0
Membracidae
1
0
0
0
Apidae
0
1
0
0
Bethylidae
0
1
1
1
Hymenoptera
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Braconidae
1
1
0
1
Ceraphonidae
0
0
1
1
Diapridae
0
1
0
1
Encyrtidae
0
1
0
0
Evaniidae
0
0
1
0
Formicidae
1
1
1
1
Gasteruptidae
0
0
1
0
Ichneumonidae
1
1
1
1
Mutillidae
0
0
0
1
Mymaridae
0
1
1
0
Perilampidae
0
1
0
0
Platygastridae
0
1
1
0
Pompilidae
0
1
0
1
Pteromalidae
0
0
0
1
Scelionidae
0
1
1
1
Sphecidae
0
1
1
1
Tetrigonidae
0
0
1
0
Tiphidae
1
1
1
1
Trigonolydae
0
0
0
1
Vespidae
1
1
1
0
Isoptera
Termitidae
0
0
1
0
Lepidoptera
Arctiidae
0
0
1
1
Cosmopterigidae
1
0
0
0
Eriocraniidae
1
0
0
0
Nymphalidae
0
1
1
0
Pyralidae
1
1
1
1
Pyraustidae
0
1
0
0
Tineidae
0
1
0
0
Mantodea
Mantidae
0
0
1
0
Microcoryphia
Meinertellidae
0
0
1
0
Neuroptera
Hemerobiidae
0
0
0
1
Odonata
Coenagrionidae
0
0
0
1
Orthoptera
Gryllidae
1
1
1
1
Tettigonidae
0
0
0
1
Thriphidae
1
0
0
0
38
35
Thysanoptera
Number of family(s)
17
34
*1 indicates that the data is present while 0 indicates the data is absent.
The highest number of insect species composition is recorded from zone 3 with 10 orders and 38 families.
Followed by zone 4, where there are 9 orders and 35 families were found. In zone 2, although the number
of insect’s order recorded is low, the number of family identified is considered high as there are 34 families
from 6 orders. Insect from the order of Hymenoptera has been recorded as the highest numbers of insects
identified both in zone 2 and 3. However, in zone 4 there are more beetles (order:Coleoptera) were found
compare to with the other insect orders.
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4.0 CONCLUSION
This study has shown that the insect species compositions in Sungai Chukai Mangrove Forest are
diverse. The diversity of insect in different zones has been influenced by the type of vegetation along the
river, salinity of the river water and also other abiotic factors namely weather condition, relative humidity
and temperature.
REFERENCES
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caseolaris tres in Rembau-Linggi mangrove forest, Peninsular Malaysia. Transylvanian Review of
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environment. 1st ed., Scientific Publishers, Jodhpur.
Balasubramanyam, K., Srinivasan, M., Kathiresan, K. 2000. Biology of mangroves and mangrove
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Bennett, E.L., Reynolds C.J., 1993. The value of a mangrove area in Sarawak. Biodiversity
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Canicci, S., Burrows, D., Fratini, S., Smith III, T.J., Offenberg, J. Dahdouh-Guebas, F., 2008. Faunal
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Clay, R.E., Andersen, A.N., 1996. Ant fauna of a mangrove community in the Australian seasonal tropics,
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Daniel ε. Alongi. 2002. Present state and future of the world’s mangrove forests. Environmental
conservation, 29 (3): 331-349. Australia.
Hanski, I. 2008. Insect conservation in boreal forest. Journal of Insect Conservation, 12:451–454.
Hashim, N.R., Wan Jusoh, W.F.A., Mohd Nasir, M.N.S., 2010. Ant diversity in a Peninsular Malaysia
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Hogarth, P.J., 1999. The Biology of Mangroves, Oxford University Press, New York.
Khatiresan, K. and B.L. Bingham. 2001. Biology of Mangroves and Mangrove Ecosystems. Advanced in
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Lewis, O.T., Basset, Y.,2007. Insect conservation in tropical forests. In: Insect Conservation Biology (eds
A.J.A. Stewart, T.R. New, O.T. Lewis). CABI Publishing, Wallingford.
Macintosh, D. J. and Ashton, E. C. (2002). A Review of Mangrove Biodiversity Conservation and
Management. Centre for Tropical Ecosystems Research, University of Aarhus, Denmark.
Martin, S. (1994). Mangroves II- the animals. Tropical Topics, Environmental Protection Agency,
Queensland Park and Wildlife Service. Australia.
Mastaller M., 1997. Mangroves: The Forgotten Forest between Land and Sea. Howes J. and Matthew J.
(Eds), Tropical Press, Malaysia. 5, 71-74.
McKee, K.L. 2002. Mangrove ecosystems: Definitions, distribution, zonation, forest structure, trophic
structure, and ecological significance, In: Mangrove ecology worskhop manual. Ed(s): Feller, I.C.,
Sitnik, M., Smithsonian Institution, Washington, 1-6 pp.
Mcleod, E. and R.V. Salm. 2006. Managing mangroves for resilience to climate change. IUCN, Gland,
Switzerland. 64pp.
Nagelkerken I., Blaber S.J.M., Bouillon S., Green P., Haywood M, Kirton L.G., Meynecke J.-O., Pawlik J,
Penrose H.M, Sasekumar A., Somerfield P.J. 2008. The habitat function of mangroves for terrestrial
and marine fauna: A review. Aquatic Botany 89:155–185.
Ndenecho, E.N.2007. Economic value and management of mangrove forests in Cameroon. International
Journal of Sustainable Development & World Ecology 14: 618–625. Cameroon.
Offernberg, J., Havanon, S., Arksonkoe, S., Macintosh, D.J., Nielsen, M.G., 2004. Observations on the
ecology of weaver Ants (Oecophy//a smaragdina Fabricius) in a Thai mangrove ecosystem and their
effect on herbivory of Rhizophora mucronata Lam. Biotropica, 36(3): 344-351.
Prayoonrat, P. 2004. A survey of insects in mangrove forest at the mouth of Bangpakong river in Thailand.
Asian Journal of Biology Education, 2: 81-85.
Schaeffer-Novelli, Y., Cintrón-Molero, G., Soares, M. L. G. and De-Rosa, T. 2000. 'Brazilian mangroves',
Aquatic Ecosystem Health & Management, 3(4): 561-570.
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Vanucci M., 2002. IndoWest Pacific Mangroves, Drude de Lacerda L.(Ed.). In: Mangrove Ecosystems;
Function and Management, Springer-Verlag, Berlin. Chapter 3, 123-173 pp.
Wan Jusoh, W.F.A., Hashim,, N.R., Ibrahim, Z.Z., 2010. Distribution and abundance of Pterotyx fireflies in
rembau-Linggi estuary Peninsular Malaysia. Environment Asia, 3: 56-60.
Wan Jusoh, W.F.A., Hashim,, N.R., Ibrahim, Z.Z., 2010.Firefly distribution and abundance on mangrove
vegetation assemblages in Sepetang estuary, Peninsular Malaysia. Wetlands Ecology and
Management, 18:367-373.
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TOURIST’S PERCEPTION ON CONSERVATION OF NATURAL RESOURCES AT BAKO NATIONAL
PARK, SARAWAK, MALAYSIA
1*
Hartini Binti Mahidin , Nurzawani Binti Md Sofwan
2*
1
Faculty of Applied Sciences, University Technology MARA (UiTM) Sarawak, MALAYSIA
Faculty of Health Sciences, University Technology MARA (UiTM) Sarawak, MALAYSIA
2
*Email: tiniewinnie@sarawak.uitm.edu.my, nurzawani@sarawak.uitm.edu.my
Abstract
The unique nature heritage of Bako National Park (BNP) that holds diverse vegetation types and animal
species with spectacular features of geomorphology has inspired the State government to gazette it as the
first national park in Sarawak. BNP is a green asset of Sarawak as it has long been drawn as one of the
main ecotourism destinations that attracted wide range of domestic and foreign tourists. BNP as a
conservation area carries conservation-themed interpretation that aims to increase tourist’s awareness on
conservation of natural resources, encourage them to engage with the conservation activities, and
develop environmentally responsible behaviour. The study aimed to explore tourist’s perception on
conservation of natural resources in terms of their awareness and engagement in conservation issues.
The study also assessed the tourist’s behaviours to ensure they understood the environmental impacts
that may cause from tourism activities. The outcomes from the visit were examined to indicate the benefits
that the tourists gained from the visit. Tourist’s satisfaction of their visit experience, comments, and
suggestions were also examined to assess their needs and expectations. The self-administered
questionnaire were employed to the one-site tourists and and key informants interview were conducted
with the Regional Manager of Bako National Park and a few park guides. Understanding and assessing
tourist’s perception on conservation of natural resources can be a useful and effective measure for BNP
improvement in the conservation efforts and tourism services for sustaining BNP in terms of economic,
environmental, and social interests.
Keywords: Perception; Awareness; Conservation; Tourists; Bako National Park
1. INTRODUCTION
The island of Borneo is one of three major world biogeographical regions of perhumid evergreen
rainforests which is known with its richness in biodiversity (Cranbrook, 2008). Sarawak which lies in the
north-west of the Borneo Island is well endowed with rich and diverse natural resources. Sarawak has
become one of the most extensive protected area networks in Malaysia with 18 national parks, four
wildlife sanctuaries and five nature reserves which cover a total area of 512, 387.47 hectares (Sarawak
National Park, n.d.). The idea for the establishment of modern parks and nature reserves in Sarawak first
arose in the late 1800’s after the realization of opening large areas of forests for economic development
has threatened the natural resources emerged. National park serves as a conservation area for natural
resources protection and environmental enhancement. Besides, it is undeniable that national park is a
green asset for the State as it offers economic benefits. The Sarawak Government through the State
Tourism Task Force has earmarked national parks for successful development of tourism. This is in line
with the objective of the State to promote Sarawak as a destination for adventure tourism.
BNP was declared as Sarawak's first national park in 1957. It is one of the smallest parks in Sarawak, yet
one of the most interesting as its offers a wonderful experience of Sarawak’s nature at its best (Hazebroek
& Morshidi, 2006). The BNP have long been drawn as an ecotourism attraction for local and international
tourists to Sarawak. BNP provides opportunities for the tourists to observe and explore the spectacular
views, a unique range of vegetation types and wildlife species. With more than 14,484 tourists visiting
BNP in 1990 and a tremendous increase with a total of 42,183 tourists in 2011, BNP has, without doubt
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becomes one of the main ecotourism spots in Sarawak which contribute to the State revenue. The
tremendous increase in the number of tourists however, poses a great challenge to the park management
in catering for the needs of the tourists without compromising the conservation of the natural resources.
Ecotourism or nature tourism involves experiencing natural ecosystems or wildlife for recreation or
education (Haysmith & Hunt, 1995).
Conservation area like BNP is a good platform to induce the conservation awareness, disseminate
knowledge about conservation, and promote environmentally responsible behaviour among the tourists
(Ballantyne & Parker, 2005; Ballantyne, Dierking, Hughes, & Parker, 2007). The act of visiting a national
park is an example of experiential learning where the tourists will learn through observation and interacts
with nature environment; hence encourage the process of learning more quickly. Past research has
shown that identifying tourist opinions, perceptions and experiences provide a crucial source of
information to park managers of protected areas such as national park and nature reserves (Boshoff,
Bradfield, Kerley, & Landman, 2006). The motivation for travel is a good indicator to identify tourist desires
and perceptions about the destination that they choose will serve their needs. Iso-Ahola (1999) stated that
motivation is understood as the underlying forces that arouse and direct those behaviours.
BNP as a conservation area carries conservation-themed interpretation that aims to increase tourist’s
awareness on conservation of natural resources, encourage them to engage with the conservation
activities, and develop environmentally responsible behaviour. BNP disseminates conservation messages
to the tourists by portraying itself as an outstanding conservation area. The awareness about conservation
issues are not centralized on the knowledge about the subject matter only but also the interest or
motivation to involve in the conservation practices. The willingness to pay for natural conservation, devote
time for ecotourism vacation, search information about conservation, and take part in nature volunteer
activities are examples of conservation engagement activities. A review from Ballantyne et al. (2007) on
visitor learning in captive and non-captive wildlife tourism settings suggested that tourists prefer to enjoy
the learning and discovery aspects of such experiences, and indeed, consider these to be an integral part
of the experience.
Behaviour can be viewed as a process of internal psychological factors (e.g., needs, wants, goals) which
can generate certain level of tension and leads to behaviours designed to release this tension in various
form (Beh & Bruyere, 2007) such as illustrated during the visit (Fodness, 1994). Tourist behaviour may
bring positive and negative impacts on natural resources depending on their level of awareness on
conservation. The positive impacts include providing income for the ongoing protection and sustainable
management of wildlife and wildlife habitats (Buckley, 2002; Fennell, 1999; Goodwin, Kent, Parker, &
Walpole, 1998; Wells, 1997; Wilson & Tisdell, 2001; Zeppel & Muloin, 2007); encouraging visitors to make
financial and non-financial contributions for environmental causes (Powell & Ham, 2008); and providing
socio-economic incentives for the conservation of natural resources (Higginbottom, Green, & Northrope,
2001; Schänzel & McIntosh, 2000). However, lack of awareness may influence irresponsible behaviour
that may be pursued by the tourists during the visit. Chin, Dowling, Moore and Wellington (2000) claimed
that ecotourism by its very commercial nature results in negative environmental impacts that include litter,
soil erosion, animal provocation and vegetation damage.
Outcome is the results or consequence of a situation. The assessment of tourist outcome will address the
benefits that they gained throughout the visit. If a national park positively raises conservation awareness
and influence environmentally responsible behaviour, it indicates a good achievement in conveying
conservation messages to the tourists. Understanding tourist’s level of satisfaction with and reaction to
their experience in the destination is a beneficial tool to give ideas for the management to improve the
services and products, thus effectively promote the destination itself (Yu & Goulden, 2006).
The aim of this research is to examine tourist’s perceptions in terms of awareness and engagement on
conservation of natural resources, to assess tourist’s behaviour during the visit to ensure compliance to
environmentally responsible behaviour, and to explore the tourists’ vast experiences, opinions, and
outcomes from the visit in BNP.
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2. METHODOLOGY
2.1 Study Area
The Bako National Park (BNP) is located at the northern tip of Muara Tebas peninsula with no direct road
access which contributed to an excellent buffer for the protection of the park resources (Figure 1). This
park land area of 2,727 hectares including the small Lakei Island (not including the marine waters) is
situated at latitude of between 1° 68’ and 1° 73’ south, and a longitude of 110° 42’ east.
Source : Sarawak Forestry Corporation
Figure 1 Geographical location of Bako National Park
BNP protects an extraordinary landscape with beautiful scenery. The park offers a lot of attractions
include unique range of vegetation types, diverse wildlife species, beautiful beaches, shore-life and birds,
plateau drained by small rivers, and fascinating geological formation. All animals, plants, and non-living
things in the park are protected under National Park and Nature Reserve Ordinance 1998.
BNP holds a considerable variety of vegetation types and animal species. Seven vegetation types are
recognized in the park which indicates good representative of Borneo’s forest. The vegetation types
include kerangas forest, open shrub land, mixed dipterocarp forest, riverine forest, mangrove forest,
beach forest, cliff vegetation, cultivated land and secondary vegetation (Hazebroek & Morshidi, 2006).
BNP has a record of about 600 species of flowering plants which include 49 species of orchids and 49
species of dipterocarps (BNP, 2011). Cliff vegetation in BNP is a unique feature with rising heights rising
up from 60 to 120 m. Most of the plant species found on the cliff are distinctive and are rarely found
elsewhere in Sarawak (Hazebroek & Morshidi, 2006).
Many tourists are attracted to BNP to get an excellent opportunity to see the wildlife there especially
Proboscis monkey, one of the world’s most wonderful primates. It is endemic in Borneo which means it
cannot be found elsewhere in the world. A significant diversity of animals lived in BNP with at least 37
species of mammals, 84 species of birds and 24 species of reptiles (Hazebroek & Morshidi, 2006).
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As an ecotourism park, BNP offers substantial accommodation and facilities to the tourists. BNP has three
unit hostels, a new unit of new hostel comprise of eight rooms, two semi-D lodges, six unit chalets and a
unit of VVIP lodge. Other facilities provided are Education Conservation Building (ECB), nature trails, park
shop, camping sites for 80 campers, canteen and a prayer room. The nature trails has a network of 18
trails including one at Lakei Island and it passes through most vegetation types (tall forests, grasslands,
shrub lands, bare rocks, waterfall and streams, scenic viewpoints and natural wilderness. The Community
Participant in park management includes the boat services by the Local Boat Society, canteen operator,
laundry services, employment, park guide, and maintenance as well as goods supplier. Activities like
jungle trekking, night walk, wildlife observation are the most popular activities pursued by the tourists in
the park. There are 17 trails in BNP which each of the trail encounters different experiences to be
explored by the tourists.
2.2 Questionnaire Design
A tourist perception survey was developed and distributed to 62 on-site tourists at Bako National Park.
The self-administered survey consists of five sections. The first section of the questionnaire aimed to
gather information on tourist’s demographic profile and visit characteristics. The second section was
designed to explore tourist’s awareness and engagement in the conservation issues. The third section
consisted of eight attributes aimed to assess the tourist’s behavior during the visit. This section was
developed to ensure the tourists recognized the environmental impacts that may arise from tourism
activities. While the fourth section was developed to determine the outcomes that tourists gained after
visiting the park. The last section of the questionnaire is for assessing the satisfaction of tourists on
conservation efforts and facilities provided in BNP. These attributes were measured using the likert scale
which scale point ranging from 1 (strongly disagree) to 5 (strongly agree). The findings of the survey or
responses would be the useful information for topic or issue under examination.
2.3 Key Informants Interview
Key informants interview were conducted with the Manager of Bako National Park and a few park guides.
The purpose of these interviews was to gather specialized information about the conservation efforts in
BNP, perceptions on the ecotourism aspect and shared their working experiences in BNP. The
information obtained from the interview was used to support the findings and discussion of the study.
2.4 Data Analysis
Statistical analysis was employed using Statistical Package for Social Sciences (SPSS) version 18.0. The
data analysis was summarized according to its respective sections consist of tourist’s demographic profile
and visit characteristics, tourist’s awareness and engagement in conservation, tourist’s behaviour during
the visit, outcomes from the visit and tourist’s satisfaction. Open ended responses regarding the
comments and suggestions for improvement of BNP were also analysed.
3. RESULTS AND DISCUSSIONS
3.1 Demographic Profile and Visit Characteristics
Respondent’s demographic profile and visit characteristics are summarized in Table 1. From the survey, it
was found that higher proportions of respondents were from European Country, 22.6% came from
Australasia (New Zealand and Australia), whilst the respondents from Asia accounted for 27.4%. Based
on the interview with the park guides, a frequent number of tourists from European Country such as
German and Holland prefer to visit BNP due to their interest in conservation of natural resources. Female
respondents outnumbered male respondents. Half of the respondents were between the ages of 25 to 30
years which are among young generation that favour adventurous trip like jungle trekking. Only one
respondent is under schooling age which is below than 18 years. It was interesting to note that 22.6% of
the respondents were between the ages of more than 41 years old. 74% of the respondents have good
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educational background which the highest number of respondents posed Master Degree, 30.6% have
Bachelor Degree, and 8.1% had obtained their doctorate.
The primary motivation for the respondents visit to BNP was for enjoyment of nature because the BNP
offers a wide range of natural resources such as vegetation, wildlife, geological formation, waterfall and
rain forest. Learning and discovery is also seen to be as an important factor for their visit which made up
24.2% of tourist’s choice. The motivation of the visit reflects the needs or interest of the tourist for their
visit. Besides, the understanding of tourist motivation can help the management to create an interpretive
program that will achieve the desired needs of tourist (Beh & Bruyere, 2007). In addition, the survey
revealed that 32.3% of the respondents received information about BNP from the travel agent. Interview
with the park guides addressed that the local travel agent has strong linkages with foreign travel agent;
hence helps in promoting BNP as a tourism attraction. The internet sources accounted for 24.2% which
are mainly derived from relevant websites like Sarawak Tourism Board that is used as one of the platform
for promotion strategy. The length of stay of the visit showed 61.4% of the respondents stayed for about
two to five days especially for those who took advantage of the package tour offered by the travel agent
whilst the remaining respondents made a day trip to BNP.
Table 1 Tourist’s Demographic Profile and Visit Characteristics
Characteristics
Country
Gender
Age
Frequency
Percentage
European Country
Australasia
Asia
31
14
17
50.0
22.6
27.4
Male
Female
26
36
41.9
58.1
1
9
31
5
2
14
1.6
14.5
50.0
8.1
3.2
22.6
5
22
19
6
9
8.1
35.5
30.6
9.7
14.5
15
11
29
7
24.2
17.7
46.8
11.3
15
20
12
12
3
24.2
32.3
19.4
19.4
4.8
24
38
38.7
61.3
Under 18 years old
18 - 24 years old
25 - 30 years old
31 - 35 years old
36 - 40 years old
More than 41 years old
Education
PhD
Master Degree
Bachelor Degree
Diploma
High School
Motivation
Learning and discovery
Leisure
Enjoyment of nature
Vacation
Information Source
Internet
Travel agent
Guide book
Friend
Media
Length of stay
1 day
2 - 5 days
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3.2 Tourist’s Awareness and Engagement
Respondents were asked about their awareness and engagement in conservation of natural resources.
The awareness and engagement of the respondents were arranged in descending order as shown in
Table 2.
Table 2 Awareness and Engagement of Tourists in Conservation of Natural Resources
Tourist's Perception
Mean
Standard Deviation
I realize that BNP is a conservation area
4.613
0.776
I am aware and understand the concept of conservation
4.452
0.694
I am interested to learn about conservation
4.306
0.781
I enjoy watching documentary about natural resources
4.226
1.015
I always visit national park or conservation areas
4.000
0.941
I am willing to pay for any conservation efforts
3.903
0.918
I always involve in conservation activities
3.468
0.970
I actively search information about conservation
3.242
1.082
From the survey, the awareness of the respondents about BNP as a conservation area gave the highest
mean score of 4.613 with standard deviation of 0.776. This suggests that BNP has been effective in
portraying its image as a conservation area to the tourists. A majority of the respondents were aware and
understood the concept of conservation and were interested to learn about it. From the analysis point of
view, the awareness and interest of the respondents in learning about conservation becomes the driven
factor to visit BNP.
The engagement or commitment of the respondents in conservation activities was assessed through four
questions. Generally, the entire respondents rated positive engagement in conservation activities with
mean ranging from 4.00 to 3.242. The study revealed that the willingness to pay for any conservation
efforts were nearly to agree with the mean score of 3.903. Previous research reported that income,
awareness, and educational level were the most significant predictors of the tourist’s willingness to pay
(Wang & Jia, 2012). The respondents were undecided about the regularity of involvement in the
conservation activities. The lowest engagement of the respondents was to search for information about
conservation which is probably due to lack of interest in the subject matter.
3.3 Tourist’s Behaviour during the Visit
The respondents were also asked about their behaviour during the visit. This was done to identify whether
the respondents were aware about their behaviour that may make an impact to the environment. Table 3
depicts the mean scores and standard deviation of tourist’s behaviour during the visit at BNP.
Table 3 Tourist’s Behavior during the Visit at Bako National Park
Tourist's Perception
Mean
Standard Deviation
I always walk within the walk trail to avoid environmental impacts
4.500
0.6954
I always keep my litter during the trekking
4.726
0.6317
I never pick the leaves or flowers during my visit
4.484
0.7839
I hardly provoke the wildlife
4.242
0.9698
I do not feed the wildlife
4.387
0.9296
I am aware not collect any nature specimens for personal collection
4.581
0.8006
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In this survey, an overwhelming respondent rated very positive behaviours during the visit with mean
score ranging from 4.242 to 4.726. Almost all respondents agreed that they always keep their litter during
the trekking to ensure the cleanliness of BNP. The respondents were also concerned that they must walk
within the walk trail to avoid any environmental disturbances and also for safety reason. Additionally,
majority of the respondents agreed that it was important to protect rare plant and animal species by not
picking any leaves or flowers, provoking and feeding the wildlife and not collecting any nature specimens
for personal collection. The positive behaviour practised by the respondents reflected that they understood
about the environmental impacts resulting from tourism activities. Jurowski (1994) stated that the
environmental attitudes of tourists may play a key role in determining the environmental impacts of
tourism. It was persuasive that experiential learning from visiting a conservation area like BNP can
encourage environmentally responsible behaviour among the respondents.
3.4 Outcomes from the Visit
The respondent’s outcomes from the visit to BNP were measured by six perception questions as tabulated
in Table 4. The mean score from the overall results were arranged from the highest outcomes to the
lowest outcomes gained by the respondents from the visit.
Table 4 Outcomes from the Visit at Bako National Park
Tourist's Perception
Mean
Standard Deviation
I have the responsibility to protect natural environment
4.290
0.7973
I understand the impacts of human activities on the environment
4.290
0.7973
I want to contribute to natural resources conservation
4.145
0.8067
I am motivated to learn more about natural resources conservation
4.048
0.9483
I will convey information about conservation to friends or family
3.952
0.9483
I gain a lot of knowledge about biodiversity
3.565
0.8800
The survey addressed that the respondents perceived that they have the responsibility to protect natural
environment and it is consistent with their perceptions that human activities related to tourism will bring
impacts on the environment. A number of respondents also emphasized on a few environmental impacts
that they observed in BNP such as litter along the forest floors and seashore, graffiti on the rocks, dead
mangrove and soil erosion. Besides, a majority of the respondents agreed that the visit increased their
motivation to learn more about conservation with mean score of 4.048. Their willingness to contribute and
convey information about conservation to family and friends will not only contributed to conservation
efforts, but also confer a good interpretation to BNP in return. It was interesting to note that the awareness
among the respondents was improved after the visit, hence influenced their motivation to learn more
about on conservation of natural resources. However, the knowledge about biodiversity gained by the
respondents from the visit was on the scale of undecided with mean score of 3.565 and standard
deviation of 0.8800. The reason for this probably caused by a few factors such as selection of trails,
activities pursued, lack of informative boards and lack of focus during the interpretation session by the
park guide due to large number of tourists in a group.
3.5 Tourist’s Satisfaction
The fourth section of the survey addressed tourist’s satisfaction in terms of conservation efforts and
facilities provided in BNP and their intention to return to BNP. The findings of the survey are shown in
Table 5 and Table 6.
From the analysis point of view, it was found that the overall satisfaction was quite positive with the mean
score ranging from 3.758 to 3.821. 25.8% of the respondents were strongly satisfied, 45.2% were
satisfied, 16.1% were undecided, 11.3 were dissatisfied and 1.6% were strongly dissatisfied with the
conservation efforts at BNP. The conservation efforts included the interpretation session by the park
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Table 5 Tourist’s Satisfaction at BNP
Tourist's Satisfaction
I am satisfied with the conservation efforts at BNP
Mean
3.823
Standard Deviation
1.0004
I am satisfied with the facilities provided at BNP
3.758
1.0509
guides, informative board displayed and cleanliness of the BNP. The mean rating of satisfaction with the
facilities provided is 3.758 with standard deviation of 1.0509. Only 3.2% respondents rated strongly
dissatisfied and 9.7% dissatisfied with the facilities whilst the rest gave positive perceptions. The fairly
satisfaction of the tourists can give an ample room for improvement in BNP.
Table 6 Tourist’s Intention to Return
Intention
Frequency
Percent
Yes
39
62.9
Maybe
22
35.5
No
1
1.6
It was apparent that 62.9% of the respondents wished to return to BNP in the future. While only one
respondent was not interested to return and 35.5% respondents were unsure. The intention to return was
influenced by their satisfaction with their visit experience in BNP.
3.6 Tourist’s Comments and Suggestions
The respondents were asked to provide comments about their visit and suggestions to improve the
conservation efforts at BNP by using an open-ended question. Although the respondents were positive
about their experience in BNP, but their comments are mostly concerned on the accommodation facilities
such as lodging, need for drinking water refill, and food services at the cafeteria.
The respondents were very supportive with the conservation efforts as they were fascinated with the effort
made to safeguard of natural resources in BNP. On the other hand, they were concerned about a few
issues that may create negative impacts to the natural resources in BNP such as uncontrolled number of
visitors, inadequate number of rubbish bins provided, and lack of regulations enforcement to protect
natural resources by the park officers.
In addition, the respondents also suggested a number of approaches that can be implemented in the
future for the improvement of conservation efforts in BNP. One of the respondent said that cleanliness is
one of the key factor in preserving natural resources. It is because; rubbish may attract wildlife to scatter it
around and eat which may endanger the health of wildlife. Besides, there must be a limitation to the
number of tourists for each visit because it is almost impossible to control people misbehaviour and some
of the tourists could not concentrate on the explanation given by the park guide in a large group. The
respondents also recommended the management to provide more explicit signs about the relevant
regulations and warning signs as well as interpretation boards about flora and fauna species. Besides,
they recommended BNP to provide more conservation messages through signage for instance
conservation of water usage and energy. BNP must react to tourist’s perception and make greater works
to improve the conservation efforts as well as tourism services.
4. CONCLUSION
The study suggests that the tourists to BNP overwhelmingly support the conservation effort made, aware
about the conservation and positively engaged with the conservation activities. The awareness level
influenced the tourist’s behaviour and it is proved by a majority of the tourist’s practised environmentally
responsible behaviour during the visit. Generally, the tourists were fairly satisfied with the conservation
efforts and the accommodation facilities provided in BNP. However, they were critical about cleanliness,
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accommodation facilities and uncontrolled number of visitors. The achievement of the BNP in portraying
itself as a conservation area and convey the conservation messages can be interpreted from the findings
of the study. More room for improvement can be reviewed and implemented by the management in
ensuring BNP as a successful conservation model of natural resources in Borneo.
ACKNOWLEDGEMENT
The authors wish to thank the management of Universiti Teknologi MARA Sarawak for the support given
to this research. We gratefully acknowledge those who contributed to the data collection at Bako National
Park especially the Regional Manager of Bako National Park, park guides, and cooperative tourists.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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MANAGEMENT OF NATURAL RESOURCES IN LENGGONG VALLEY IN DEER FARMING FACET
Siti Hajar Abd Aziz*, Siti Shuhada Mustaffa, Zuraini Zakaria & Norsuhana Abdul Hamid
Biology Program, School of Distance Education
Universiti Sains Malaysia, 11800 Minden
Penang, Malaysia
*Emailr: sitihajar15789@gmail.com
Abstract
The immediate environment in Lenggong Valley, Hulu Perak, located between Banjaran Titiwangsa and
Banjaran Bintang, is a dense tropical forest. This vast area signifies for a wide variety of flora and fauna
densities suitable for deer rearing. Cervus timorensis is one of the imported deer species that is highly
bred in Lenggong. The six deer grazing farms are in Kampung Luat, Kampung Chuas, Kampung Chepor,
Kampung Temelong, Bukit Sapi (Sungai Soh A) and Bukit Sapi (Sungai Soh B) with a total acreage of 465
acres. These Lenggong farms are cultivated with various types of crops such as woody and non woody
plants, herbs, bunch or tufted, stoloniferous, rhizomatous and leguminous. There are also efforts by the
government to improve the rearing farm management by supplying the grass seed varieties namely
Brachiaria decumbens (Signal grass), Brachiaria humidicola (Koronivia grass), Panicum maximum (Horse
grass) and Pennisetum purpureum (Elephant or Napier grass) for forage. These farms have gone through
several processes of development and maintenance such as cleaning of the forests, site preparation,
grass seed fertilization, farm fencing, weeding and transferring livestock. The farms have in fact become
one of δenggong’s tourism sites and prime supplier of local fresh meat.
Keywords: management, natural resources, deer farms, Lenggong
1. INTRODUCTION
Multiple types of livestock rearing is developing in Malaysia especially from mammals which contribute to
greater benefit in terms of economy specifically in husbandry and tourism. Directly it provides diverse
specification of job opportunities whilst indirectly it conserves the natural resources. This industry has a
great attraction nationwide; even people from rural areas such as in Lenggong, Hulu Perak have become
involved in the livestock rearing specifically deer rearing. Deer might present as pests to certain places
and endangered in some other places (Zeng et al. 2005). One of the methods taken to overcome the
problems is by cultivating the deer grazing farm.
The deer farming in Lenggong has a great potential in the tourism facet particularly amongst internal
tourists because it is one of the rare mammals to heave in sight compared to other livestock farming such
as cattle and goat. Plus, the unique infrastructure in terms of plant biodiversity which makes it more
fascinating as a farm.
Lenggong is one of the districts in Peninsular Malaysia with a high acreage of deer grazing farm and in
fact, it is growing significantly. Most of the resources for the deer farmers are provided by Department of
Veterinary Services, Lenggong following a sharecropper system. The beginners especially, are provided
with knowledge of training programs for management purposes. They are also presented with the first
deer breed as a starter, and a grazing farm area with suggestions of plant species to cultivate and stock.
There are six grazing farms in the Lenggong District supervised under the management of the Department
of Veterinary Services. The six grazing farms are located in Kampung Luat, Kampung Chuas, Kampung
Chepor, Kampung Temerlong and Bukit Sapi (Sungai Soh A and Sungai Soh B).
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Efficiency in conservation awareness towards natural resources and its ecosystem are based on
understanding of the biological and historical of the deer (Rooney, 2001). The farm plays an important role
in producing the venison and preventing of illegal hunting of endangered wild deer species (Peter, 2005).
The farmers in δenggong’s grazing farms only breed Cervus timorensis commonly known as Rusa deer,
which is an indigenous species from Indonesia islands of Bali and Java. The species was initially imported
and introduced to the farmers. C. timorensis is well known for its viable commercial value in most deer
farms in the tropics region. It has the durability and flexibility when exposed to extreme environments and
prolific characteristics compared to other deer species (Dahlan, 2009).
Biodiversity of the plants cultivated around and within the farms are usually based upon the deer’s
behavior which include grazing, browsing, mallowing, shedding, breeding and for shelter (Fuller et al.
2001). Rusa deer is known to be adaptable in habitats with a wide range plant diversity namely grasses,
herbs, shrubs, saplings, young shoots of ferns and bracken. Several research have been done to study
the impact of deer on natural ecosystem. The damages on the natural ecosystem may sometimes due to
the deer’s habit on over browsing and grazing, trampling and shedding behavior (Steeve et al. 2004). The
damaging effects in the deer grazing farm could be controlled by cultivating various types of plants within
the farm which increase the deer’s preferable behavior and provide choices of plant species as their diet.
Groups of plants mostly cultivated are species of Graminae and Leguminosae.
2. METHODS
2.1 Study site
Lenggong is a semi-rural area surrounded with villages, oil palm estates and limestone hills. The
immediate environment in Lenggong is a dense tropical forest. Located in between two mountain ranges
in Peninsular Malaysia, that is Titiwangsa Range and Bintang Range, this location signifies for a wide
variety of flora and fauna densities (http://whc.unesco.org/en/tentativelists/5481/). The farms are located in
Kampung Luat, Kampung Chuas, Kampung Chepor, Kampung Temerlong and Bukit Sapi (Sungai Soh A
and Sungai Soh B).
2.2 Data collection
Data was obtained by interviewing the native farmers from the six deer grazing farms and also officers
from the Department of Veterinary Services, Lenggong.
3. RESULTS AND DISCUSSION
The six deer grazing farms differ from each other in terms of its geographical location (Table 1).
Meanwhile the natural biodiversity structure in the farms corresponds to their acreage.
Table 1 Farm’s depiction
Farm
Location
Kampung Luat
Near to the villages and surrounded with wild forest
Kampung Chuas
Naturally surrounded with wild forest, isolated by Perak River and hilly
Kampung Chepor
On the side of Gerik Highway, facing cattle grazing farm and hilly
Kampung Temerlong
In oil palm and rubber plantation
Sg Soh A
At Bukit Sapi, hilly and have to go through small tributary
Sg Soh B
At Bukit Sapi, hilly and have to go through small tributary
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Table 2 Pasture grazing pattern
Grazing range (days)
Grass species
17 – 21
Brachiara decumbens
Brachiara humidicola
Brachiara mutica
Setaria sphacelata cv. kazungula
Brachiara ruziziensis
Digitaria setivalva
Cynodon
Pennisetum purpireum
Panicum maximum
21 – 28
35 – 42
Table 3 Recommended plant diversity
Utility
Common names
Scientific names
Pasture/fodder
Star grass
Signal grass
Para grass
Humidicola grass
Congo grass
Guinea/Horse grass
Napier grass
Kazungula grass
Splendida grass
Crab grass
Blanket grass
Buffalo grass
Blady grass
Centro
Stylo
Puero/Kudzu tropica
Calapo
White Leadtree
Jackfruit
Soapbush
Fern
Ficus tree
Elephant’s ear
Kanyere
Shrubs
Peach-leaf poison bush
Tampines tree
Golden gardenia
Chonoo
Purple milletia
Malabar melastone
Sensitive plant
Cynodon plestostachys
Brachiara decumbens
Brachiara mutica
Brachiara humidicola
Brachiara ruziziensis
Panicum maximum
Pannisetum purpureum
Setaria sphecelata var. kazungula
Setaria sphecelata var. splendid
Digitaria setivalva
Axonopus compressus
Paspalum conugatum
Imperata cylindrica
Centrosema pubescence
Stylosanthes guianensis
Pueraria phaseoloides
Calapogonium mucunoides
Leucaena leucocephala
Artocarpus heterophyllus
Clidemia hirta
Stenochlaena palustris
Ficus spp.
Macaranga gigantea
Bridelia monoica
Greenia corymbosa
Trema spp
Streblus elongatus
Gardenia tubifera
Croton argyratus
Millettia atropurpurea
Melastoma malabathricum
Mimosa pudica
Browsing/shelter
Deer grazing farm management is based on the farm groundwork and internal management forces. The
first process of any site preparation is cleaning of the forest, followed by plowing and harrowing of the soil
to improve the soil texture, making it available for root growth of seeds and saplings (Lal, 1984). Site is
then ready after a period of two months. Lime is applied to the soil two weeks before planting with
suggested amount of 2,000 kg per hectare. This is to increase the pH value of the soil as much as 0.5
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(Soedamardi & Kismono, 1985). Generally, the acidity pH for soils in Malaysia is lower; consequently,
liming enhances the fertility of the soil which is at best within the pH range of 5.0-7.0.
Based on Department of Veterinary Services farm’s management plan, the planting course is separated
into seeds and vegetative plantings. The seeds are dispersed in the farm following the proportion of seed
weight (kg) per hectare. Non seeded plants are planted via roots or rhizomes. The forage is available for
grazing once the flowering period commences. The growing pasture and fodder is maintained according
to the frequency of cutting or grazing. The frequency of grazing and cutting of forage depends on the
hardiness and prolific characters of each type of the fodder and pasture planted. The types of pasture and
grazing pattern being practiced in the farms are as stated in Table 2.
Pasture feeding method is either by grazing or cutting. Deer grazing is suitable for spacious farms (Figure
1) whereas cutting off pasture is reasonable for small grazing farms which prevents the over consumption
of forage. Deer browsing is the feeding behavior on the plant parts preferably the leaves, shoots,
branches and cambium layer of woody plants, shrubs and trees.
Figure 1 Grazing farms at Kampung Chepor (left) and Kampung Chuas (right)
Figure 2 Grazed paddock (left) and spelled paddock (right)
The common types of grasses planted in the grazing farms are Guinea grass, Humidicola grass, Napier
grass and signal grass. Table 3 shows the recommended plant species which serve as pasture/fodder
and browsing/shelter. The Lenggong farms are high on the density of livestock forage, able to cater to the
population and sustain the carrying capacity of the farm ecosystem. The resources correspond with each
paddock’s planting times. The purpose of having paddocks in the farm is to recycle and spelling the
resources. Once the paddock has been used for certain times, the deer are then transferred to another
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paddock. This establishes the spelling paddocks via the regrowth and increased density of the fodder in
the paddock. The spelling process takes place twice a year. Figure 2 shows the effect of paddock rotation.
Stocking density suggested by the Department of Veterinary Services is 20-24 deer per hectare. The
value marked as the maximum size of capital that a particular farm can sustain its resources. Densely
populated area leads to over consumption and insufficient of food resources, and compaction of soil
condition due to trampling effect that degrades the soil quality making it impossible for plants to grow
(McDowell et al. 2004). This factor plays an important role for a good management practice in the farm.
The growth of the fodder is facilitated with the application of organic (animal manure and compost) and
chemical (commercial) fertilizers on the soil for a long term effect. Fertilizing the fodder reduces the
degradation of soil fertility and provides essential nutrients demanded for plant growth (Verma et al. 2007).
Mixed pasture is preferable to enhance the quality of the forage and increase the availability of nutrients
for plant growth. The yield of mixed pasture is usually greater than the pasture alone.
Presence of Rhizobium sp. on the legumes planted along with the pasture reduces the utilization of
fertilizers that eventually lead to the degradation of the ecosystem (Fuskhah et al. 2009). Legumes are
higher in protein compared to grasses. Hence, the efficacy of pasture is upgraded, and the uptake as a
dry matter product increases. The forage is consumed 6 to 8 percent of wet matter and 2 to 3 percent of
dry matter per day. There is also a need on variety of palatable plants to be cultivated in the farms. This
increases the availability of forage resources for the deer and decreases the browsing effect (Kamler et al.
2009). Common legumes used for this particular purpose are Acacia magnium, Acacia auriculiformis,
Glyricidia sepium, Leucaena Leucocephala and Sesbania grandiflora. These types of fodder are more
significantly useful during the drought season.
(a)
(b
Figure 3 Shelters at Kampung Temerlong (a) and Kampung Chepor (b)
Shelter and cover are important for deer rearing farms. Most of the shelters are big trees with a dense
concentration of foliar such as herbs and ferns. These apparently also come from woody plants (Bendfeldt
et al. 2001). The characteristics of shelters should be dense, high and wide enough to cover the body of
the deer population in the farm. Areas dense with trees and shrubs are also suitable for deer to wallow
endlessly, and protect the deer from hard weather such as excessive sunlight and heavy rain (Figure 3).
Density of the shelter increases the biodiversity and humidity of the ecosystem. The shelter must exist in
each of the paddocks (Figure 4). Since deer has a wide range of eating preference, shelter will diversify
the food plants for deer, reducing the effecs of browsing and grazing as some might prefer leaves and
twigs (Erik et al. 2006).
The measurement on diversification of plant varieties in the farms is crucial in the management process
(Sanderson et al. 2004). One of the natural habits of deer is shedding the antler, causing the peeling off
the bark. Therefore a better understanding is desirable for controlling the ‘bark stripping effect’ on the
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trees and over utilized caused by the deer (Josefina Zidar, 2011). Damaging the vascular structure of the
trees will ultimately lead to bacteria and fungus infections.
Figure 4 Salt licks at Kampung Chepor paddocks
Salt mineral or salt lick is an accumulation of natural mineral that provides other sources of essential
natural mineral for the deer in a lack mineral-ecosystem. The deer are fond of travelling afar just to lick on
the salt (Figure 4). The hard weather condition causes the deposition of the mineral naturally. It was
observed that almost each of the deer farm in Lenggong area has the mineral lick particularly in opened
farms. Weeding on the farms is also another practice which is necessary to prevent overgrowing of
unwanted plant species especially the hosts for disease transmissions and protecting the livestock’s
quality.
Figure 5 Fencing surrounds the whole grazing farm and also between paddocks
Finally, fencing is the end step of farm preparation. Fencing is compulsory as it prevents the escaping of
deer (Figure 5). An escaped deer might not survive long as it is threatened by the surrounding ecosystem
as some of the deer grazing farms are located near to the highway. Fencings are built according to the
recommended specification of the fence available for the deer farms (Kurt et al. 2009).
4. CONCLUSION
Deer farming is a great approach in conservation facet. The crucial parts of the approach are the
management forces. Good management practices contribute to better farm condition and less harmful
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effects on human and natural ecosystem (Hoffmann, 2010). Diversifying the plants species in the farm
increases the farm’s quality and quantity of its products.
REFERENCES
Dahlan. 2009. Characteristics and Cutability of Farmed Rusa Deer (Cervus timorensis) Carcasses for
Marketing of Venison. Asian-Aust. J. Anim. Sci. Vol. 22, No. 5 : 740-746
Eny Fuskhah, R.D. Soetrisno , S.P.S Budhi & A. Maas. 2010. Growth and Production of Forages as The
Result of Association with Rhizobium in Saline Media. Seminar Nasional Kebangkitan
Penternakan – Semarang
Erik E. Stange Ret. & Kathleen L. Shea. 2006. Effects of Deer Browsing, Fabric Mats, and Tree Shelters
on Quercus rubra Seedlings. Restoration Ecology Vol.6 No. 1: 29-34
E. S. Bendfeldt, C. M. Feldhake & J. A. Burger. 2001. Establishing trees in an Appalachian silvopasture :
Response to shelters, grass control, mulch, and fertilization. Agroforestry Systems 53: 291–295,
2001
Hoffmann. 2010. Livestock biodiversity. Rev. sci. tech. Off. int. Epiz., 29 (1), 73-86
Josefina Zidar. 2011. Factors affecting bark-stripping by red deer (Cervus elaphus) :the
importance of
landscape structure and forage
Jirˇı´ Kamler Æ εiloslav Homolka Æ εiroslava Barancˇekova´ Æ & Jarmila Krojerova´-Prokesˇova. 2010.
Reduction of herbivore density as a tool for reduction of herbivore browsing on palatable tree
species. Eur J Forest Res 129:155–162
Kurt C. V., Michael J. L. & Scott H. 2006. Fences and Deer-Damage Management: A Review of Designs
and Efficacy. Wildlife Society Bulletin 34(1):191-200
M. A. Sanderson, R. H. Skinner, D. J. Barker, G. R. Edwards, B. F. Tracy & D. A. Wedin. 2004. Plant
Species Diversity and Management of Temperate Forage and Grazing Land Ecosystems. Crop
Sci. 44:1132–1144
Peter Jesser. 2005. Deer pest status review : Land protection. The State of Queensland Department of
Natural Resources and Mines. ISBN 1 921062 31 2
R. LAL. 1984. A Soil Suitability Guide for Different Tillage System in the Tropics. Soil & Tillage Research,
(5)179-196
R.W. McDowell, J.J. Drewry & R.J. Paton. 2004. Effects of deer grazing and fence-line pacing on water
and soil quality. Soil Use and Management 20:302–307
Soedarmadi & Ign. Kismono. 1985. Pengaruh Pemberian kapur Terhadap Nodulasi dan Produksi Lima
Jenis Kacang Makanan Ternakan. Media Penternakan , 10(1) : 25-39
Steeve D.Cˆot´e, Thomas P.Rooney, Jean-Pierre Tremblay,Christian Dussault & Donald M.Waller. 2004.
Ecological Impacts of Deer Over abundance. Rev. Ecol. Evol. Syst. 35:113–47
S. Verma & Pradeep K. Sharma. 2008. Long-term effects of organics, fertilizers and cropping systems on
soil physical productivity evaluated using a single value index (NLWR).Soil & Tillage Research
98:1–10
T. P. Rooney. 2001. Deer impact on forest ecosystem : A North American perspective. Forestry, Vol. 74,
No. 3.
Z.G. Zeng, Y.L. Song, J.S. Li. W. Teng, Q. Zhang and F. Guo. 2005. Distribution, status and conservation
of Hainan Eld’s deer (Cervus eldi hainanus) in China. Folia Zool. 54(3): 249–25
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CONSERVATION VALUE OF SELECTED GOLF COURSES FOR BIRDS IN SARAWAK
1*
1
Mary Buloh Balang , Andrew Alek Tuen and Mustafa Abdul Rahman
2
1
Institute of Biodiversity and Environmental Conservation Universiti Malaysia Sarawak, 94300 Kota
Samarahan, Sarawak (MALAYSIA)
2
Faculty of Resource Science and Technology Universiti Malaysia Sarawak, 94300 Kota Samarahan,
Sarawak (MALAYSIA)
*E-mail: bmary88@gmail.com
Abstract
The conservation value of golf courses for birds was studied by comparing species sampled via mistnetting at four selected golf course in Kuching- Kota Samarahan division. Eighty two species of birds were
recorded and species with conservation value include 13 species that are protected under the Sarawak
Wild Life Protection Ordinance 1998 and 14 species that are recognized as Near Threatened (NT) species
by The IUCN Red List of Threatened Species 2012. These were captured in golf courses surrounded by
large tracts of natural forest. The most abundant and common species recorded at all sites are the Little
Spiderhunter (Arachnothera longirostra), Pied Fantail (Rhipidura javanica) and Olive-winged Bulbul
(Pycnonotus plumosus). Species that is endemic to Borneo are Dusky Munia (Lonchura fuscans) and
Yellow-rumped Flowerpecker (Prionochilus xanthopygius).
Keywords: Avifauna, Tropical region, Golf course, Surrounding Landscapes, Conservation value
1. INTRODUCTION
Golf tourism has been spreading globally as a recreational activity and sport which has made golf courses
an important form of land use. Malaysia and other South-east Asian countries are enthusiastically
investing in golf courses because of the potential contribution it makes to the country’s economy. At the
same time, the rainforest of South-east Asia is heavily threatened by deforestation with estimated 1% of
the forest being cleared annually (Sodhi et al., 2004) and this rate is among the highest in the world and it
is still increasing (Sodhi et al., 2010).Thus the development of golf courses in the island of Borneo,
specifically in Sarawak, is the major concern of this study. The reason is because the island of Borneo is a
part of ‘Sundaland’ which has been identified as ‘biodiversity hotspot’ for conservation priorities (Myers et
al., 2000).The island of Borneo has a variety of habitats ranging from peat swamp, mangroves, mixed
dipterocarp and montane forest that support local biodiversity and endemism. In the past few decades,
most of these habitats have been transformed into agricultural and urban landscapes, including golf
courses. The avian community in golf courses surrounded by remnants of these forest habitats is
expected to reflect that of the original habitat.
Birds are well known for their sensitivity to environmental change and habitat fragmentation (Furness &
Greenwood, 1993; Sorace & Vicentin, 2007) which make them a good biological indicator. Previous
studies in non-tropical regions such as the United States, United Kingdom and Australia have suggested
that golf courses could be a surrogate habitat that offers refuge and food sources to the wildlife (Terman,
2000; White & Main, 2004; Zwartjes & DeLong, 2005; Tanner & Gange, 2005; Sorace & Vicentin, 2007)
but this may be doubtful for the golf courses in the tropical region. Therefore, the aim of this study is to
determine the avifauna species that inhabit the forests immediately surrounding selected golf courses in
Kuching-Kota Samarahan division, Sarawak and to highlight the existence of species of conservation
importance, which are protected by the local as well as the international law too.
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2. METHODOLOGY
2.1 Study Site
Bird surveys were conducted in four golf courses in Kuching-Kota Samarahan division, Sarawak. These
were Sarawak Golf Club (KGS), Petra Jaya (N 01°35.824’ E 110°20.554’), Damai Golf & Country Club
(DGCC), Santubong (N 01°44.650’ E 110°18.225’), Borneo Highlands Hornbill Golf & Jungle Club (BHR),
Padawan (N 01°07.060’ E 110°12.562’) and Sarawak Club Golf Resort (SCGR), Kota Samarahan (N
01°28.645’ E 110°25.67λ’) (Figure 1).
Figure 5 Satellite image showing the study sites: Sarawak Golf Club (A), Damai Golf & Country Club (B),
Hornbill Golf & Jungle Club (C) and Sarawak Club Golf Resort (D) (Source: Google Earth, 2011).
The KGS is an urban golf course located in the suburb of Petra Jaya, approximately 5 km north of
Kuching city centre. It is surrounded by residential and commercial areas. The forest habitat that
surrounds this golf course is a small area of remnant swamp forest. The vegetation comprised mostly
ferns, saplings and palms, and some woody plants. The common woody plant is Ficus sp. and planted
Acacia mangium. The second site, DGCC is located at the coastline of Santubong approximately 30km
north of Kuching city. The forest habitat in DGCC is mixture of regenerated secondary forest and patches
of scrubs near the beach. The scrub patches are dominated by ferns, grass, saplings, A. mangium and
conifer trees while more woody plants and palms such as Daemonorops sp. were observed in the forest
habitat on Mount Santubong side of the golf course. The third site, BHR golf course is located
approximately 70 km away from the Kuching city in the Penrissen Mountain Range. The golf course was
constructed on logged-over forest approximately 1,000 meters above sea level near the SarawakKalimantan border. The forest patches within the golf complex are regenerated logged-over forest while
the forest further up the steep mountain slopes are largely undisturbed sub-montane forest. The
vegetation around the golf course comprised mostly large size trees such as Koompassia excelsa and K.
malaccensis. Other non-woody plants such as gingers, ferns and pitcher plant were also commonly seen
in the area. This golf course and its surroundings have been noted as an Important Bird Area by Malayan
Nature Society (MNS). Lastly, SGCR is located in Kota Samarahan, approximately 18 km from Kuching
city. It is mostly surrounded by patches of remnant peat swamp forest, mangrove forest and scrubs.
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Several rubber trees (Hevea brasiliensis), Nibong (Omcosperma tigillarium), Daemonorops sp. and forest
edge species such as Gleichenia sp. and Melastoma malabathricum were among the common plants
observed in the forest habitat surround SGCR.
2.2 Mist-netting
The survey was conducted for three consecutive days at each site respectively in March, June and
September 2011. The mist-nets were deployed in the forest habitats immediately surrounding the golf
courses. Twenty mist-nets of 2.5 m (W) x 9 m (L) x 36 mm mesh size with four pockets were set up for
three days as longer period of mist-netting will resulting in increasing of the bird’s recapture rate (Rahman
et al., 2002). All of the mist-net were left open from morning (0600) until evening (1800) and checked once
every two hours. Every bird caught in the nets was identified using field guides by Myers (2009) and
Phillipps (2009). The morphological features (length of tarsus, bill, wing, weight, moult and brood patch)
were recorded and the birds marked with uniquely numbered metal rings bearing the address of Universiti
Malaysia Sarawak (UNIMAS) before being released at the sampling sites.
3. RESULTS
Table 1 shows the species and conservation status of birds recorded at DGCC, KGS, SCGR and BHR.
Overall, 82 species from 27 families were recorded from the four golf courses. At KGS, a total of 101 birds
from 26 species were mist-netted while at DGCC, it was 104 birds from 29 species. Meanwhile, BHR has
the highest total birds mist-netted with 220 birds from 52 species and SCGR has the lowest total of
individuals mist-netted with 83 birds from 21 species.
This study had recorded a species from Pittidae family, Hooded Pitta (Pitta sordida) that are listed in Part I
(Totally Protected Animals) of the Sarawak Wild Life Protection Ordinance 1998. Additionally, all species
of Owls (Strigidae), Kingfishers (Alcedinidae), Swiftlets (Apodidae), Woodpeckers (Picidae), Asian
paradise fly-catcher (Terpsiphone paradisi) and White-rumped Sharma (Copyschus malabaricus) are
listed in Part II (Protected Animals) of the Sarawak Wild Life Protection Ordinance 1998. Rufous-chested
Flycatcher (Ficedula dumetoria), Reddish Scoop Owl (Otus rufescens), Green Broadbill (Calyptomena
viridis), Lesser green Leafbird (Chloropsis cyanopogon), Buff-necked Woodpecker (Meiglyptes tukki),
Crested Jay (Platylophus galericulatus), Chestnut-naped Forktail (Enicurus ruficapillus), Puff-backed
Bulbul (Pycnonotus eutilotus), Buff-vented Bulbul (Iole olivacea), Short-tailed Babbler (Trichastoma
malaccense), White-chested Babbler (Trichastoma rostratum), Sooty-capped Babbler (Malacopteron
affine), Rufous-crowned Babbler (Malacopteron magnum), Brown Fulvetta (Alcippe brunneicauda) are
categorized as Near Threatened (NT) species while the others are Least Concern (LC) in The IUCN Red
List of Threatened Species 2012. The Dusky Munia (Lonchura fuscans) and Yellow-rumped Flowerpecker
(Prionochilus xanthopygius) are the only two Bornean endemics recorded in this study.
On the other hand, the forest habitats in KGS and DGCC shared the same abundant species which is the
Olive-winged Bulbul. There are 14 individuals of Olive-winged Bulbul; corresponding to relative
abundance of 13.86% recorded in KGS forest habitats while 17 individuals, equal to relative abundance of
16.46% recorded in the DGCC forest habitats. Meanwhile, the most abundant species in BHR forest
habitats is Little Spiderhunter of 40 individuals (relative abundance of 18.18%) and 12 individuals (relative
abundance of 14.45%) of Bold-stripped Tit-babbler recorded in SCGR forest habitats.
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Table 1 List of bird species in the forest habitats surrounding KGS, DGCC, BHR and SCGR and their
conservation value (TP: Totally Protected, P: Protected, NT: Near Threatened, LC: Least Concern).
Sarawak
WLPO 1998
status
IUCN
status
SCGR
BHR
KGS
DGCC
Total
Treron vernans
LC
1
-
-
2
3
Chalcophaps indica
LC
-
1
-
-
1
Cuculus optatus
LC
-
1
-
-
1
Surniculus lugubris
LC
-
1
-
-
1
Centropus sinensis
LC
-
-
1
-
1
Species
Columbidae
Cuculidae
Strigidae
Otus rufescens
P
NT
2
-
1
3
6
Ninox scutulata
P
LC
-
-
1
-
1
LC
-
-
4
-
4
Caprimulgidae
Caprimulgus macrurus
Apodidae
Aerodramus salanganus
P
LC
-
1
-
-
1
Collocalia esculenta
P
LC
-
-
-
2
2
Todiramphus chloris
P
LC
-
-
1
1
2
Pelargopsis capensis
P
LC
-
-
3
-
3
Alcedo meninting
P
LC
2
-
-
-
2
Ceyx erithacus
P
LC
5
4
-
-
9
LC
-
-
-
5
5
Alcedinidae
Meropidae
Merops viridis
Picidae
Sasia abnormis
P
LC
2
4
-
-
6
Picus puniceus
P
LC
-
-
1
-
1
Meiglyptes tukki
P
NT
-
3
3
4
10
Calyptomena viridis
NT
-
3
-
-
3
Cymbirhynchus macrorhynchos
LC
-
-
1
-
1
LC
-
-
1
1
2
LC
1
-
-
-
1
Hemipus hirundinaceus
LC
-
-
-
2
2
Lalage nigra
LC
1
-
1
1
3
Pericrocotus flammeus
LC
-
1
-
-
1
Eurylaimidae
Pittidae
Pitta sordida muelleri
TP
Hirundinidae
Hirundo tahitica
Campephagidae
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Aegithinidae
Aegithina tiphia
LC
-
-
-
1
1
Chloropsis cyanopogon
NT
-
1
-
-
1
Irena puella
LC
-
2
-
-
2
Pycnonotus eutilotus
NT
-
6
-
-
6
Pycnonotus atriceps
LC
-
9
-
-
9
Pycnonotus goiavier
LC
6
-
8
14
28
Pycnonotus plumosus
LC
6
-
14
17
37
Pycnonotus brunneus
LC
-
1
-
-
1
Pycnonotus simplex
LC
-
5
-
1
6
Pycnonotus erythrophthalmos
LC
-
5
-
-
5
Criniger bres
LC
-
8
-
-
8
Criniger ochraceus
LC
-
6
-
-
6
Criniger phaeocephalus
LC
-
1
-
-
1
Iole olivacea
NT
-
1
-
-
1
Hemixos flavala
LC
-
2
-
-
2
LC
1
-
6
3
10
LC
-
3
-
1
4
LC
-
2
-
-
2
Pellorneum pyrrogenys
LC
-
6
-
-
6
Trichastoma rostratum
NT
-
-
-
2
2
Malacocincla malaccense
NT
2
1
-
-
3
Malacocincla sepiarium
LC
-
3
-
-
3
Malacopteron magnum
NT
-
2
-
-
2
Malacopteron magnirostre
LC
-
8
-
-
8
Malacopteron affine
NT
-
2
-
-
2
Pomatorhinus montanus
LC
-
1
-
-
1
Macronous gularis
LC
12
-
7
3
22
Stachyris nigriceps
LC
-
11
-
-
11
Stachyris poliocephala
LC
-
8
-
-
8
Stachyris erythroptera
LC
6
3
-
-
9
Alcippe brunneicauda
NT
-
21
-
-
21
Yuhina zantholeuca
LC
-
1
-
1
2
Prinia flaviventris
LC
-
-
3
-
3
Orthotomus sericeus
LC
11
4
6
4
25
Orthotomus ruficeps
LC
-
-
3
2
5
Pycnonotidae
Turdidae
Copsychus saularis musicus
Copsychus malabaricus
Enicurus ruficapillus
P
Timaliidae
Sylviidae
Muscicapidae
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Rhipidura perlata
LC
-
1
-
-
1
Rhipidura javanica
LC
10
-
12
12
34
Culicicapa ceylonensis
LC
-
2
-
-
2
Cyornis concretus
LC
-
1
-
-
1
Cyornis rufigastra
LC
4
-
3
-
7
Cyornis banyumas
LC
-
10
-
-
10
Ficedula dumetoria
NT
-
2
-
-
2
Hypothymis azurea
LC
-
1
-
-
1
Terpsiphone paradisi
LC
-
3
-
-
3
LC
-
-
-
1
1
Prionochilus xanthopygius
LC
-
3
-
-
3
Prionochilus maculatus
LC
2
-
-
-
2
Dicaeum trigonostigma
LC
-
1
3
6
10
Anthreptes simplex
LC
-
4
-
1
5
Anthreptes malacensis
LC
2
-
10
3
15
Hypogramma hypogrammicum
LC
-
2
1
-
3
Nectarinia jugularis
LC
-
2
2
1
5
Arachnothera longirostra
LC
3
40
-
5
48
Arachnothera modesta
LC
-
4
-
-
4
LC
3
-
1
3
7
LC
1
1
4
2
8
NT
-
1
-
-
1
Total Individual
83
220
101
104
508
Total Species
21
52
26
29
82
Total Families
15
17
17
19
24
Number of NT species (IUCN 2011)
2
11
2
3
Number of TP & P species (WLPO 1998)
4
5
7
6
Sittidae
Sitta frontalis
Dicaeidae
Nectariniidae
Sturnidae
Aplonis panayensis
Estrildidae
Lonchura fuscans
Corvidae
Platylophus galericulatus
4. DISCUSSION
The conservation value of a particular region or country has often been described in terms of species
richness, endemism and presence of rare and endangered species (Myers et al. 2000, Sodhi et al. 2004).
Guidelines for identifying forest with high conservation values (HCV) was first published by the Forest
Stewardship Council in 1999 (Jennings 2004) and criterion HCV1 (forest containing significant
concentration of biodiversity values – richness, endemism, rarity, etc.) is particularly relevant to the
discussion on conservation value of golf courses. The relatively small size of individual golf courses (<50
hectares) in a mega-diverse region such as Borneo makes it unlikely for any of the forest patches in and
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around golf courses to have the concentration of biodiversity values greater than the original forest from
which it was created. The presence of any endemic, threatened and protected species in relation to its
surrounding landscape is enough to identify the conservation value of each golf course.
In this study, the forest immediately surrounding the golf courses varies greatly in terms of degree of
disturbance; the most disturbed would be KGS while the least disturbed would be BHR. The total
individual and species in the forest habitat immediately surrounding the golf courses (Table 1) indicate
that different type of forest habitats support different species regardless of the golf courses existence.
No threatened species were recorded in this study but 11 near-threatened species (42 individuals) were
recorded in BHR compared to not more than three species in the other golf courses. When the number of
totally protected and protected species, near-threatened and endemic species is taken into account, BHR
still has a greater conservation value compared to the other golf courses. The remoteness of BHR, its
sub-montane location and the course design, in which relatively large tracts of logged-over hill forest were
left behind in between the fairways, probably all contributed to its relatively high conservation value. The
conservation value of this golf course for bird is testified by its declaration as Important Bird Area (IBA) by
Malaysian Nature Society (Orenstein et al., 2010).
In contrast, the other golf courses are more exposed to urban landscapes and human activities share
more similar common species especially Yellow-vented Bulbul and Asian Glossy Starling that are
adaptable to disturbed habitats. Additionally, the forest canopy cover in these three golf courses is more
exposed to the sunlight has enhance the growth of flowering plant such as Melastoma malabathricum has
benefited nectarivorous species.
The bird species in this current study were compared to the previous studies done in the nearby and
almost similar habitat to the study sites just to see if there is any evidence of differences and similarities in
species. A previous study by Tuen et al. (2000) compared the disturbed and undisturbed site together with
gradual changes in altitude of Mount Santubong. The forested habitats that surround DGCC in this current
study share the almost the same species (13 species) with the forest of Mount Santubong as reported by
Tuen et al. (2000).
On the other hand, forested area surrounding BHR were connected by range of mountains, which
eventually also covers the Padawan limestone area where previous study done by Mansor et.al (2011).
Almost half of the species (24 species) recorded in this study were similar to Mansor et al. (2011)
suggested that these species do occur in forest that has slightly higher elevation. The forest area that
surrounds BHR ranged from 700-1000 m above sea level and could be recognized as lower montane
forest.
Before the construction of the UNIMAS west campus and also the SCGR was completed back in 2007,
the area was peat swamp forest mixed with mangroves riparian forest along the Sebayor river. Tuen and
Darub (1999) has conducted preliminary investigation into the diversity and abundance of understorey
birds in this particular peat swamp forest in 1996 and 1997 before a large area of it was converted into a
golf course and university new campus. Out of 21 species reported in this study, four species were not
recorded in Tuen and Darub (1996); these are Pink-necked Pigeon, Pied Thriller, Asian Glossy Starling
and Oriental Magpie Robin. The possible reason why these species were not recorded previously was
because these species were observed to favor the open habitat such as the golf open ground and scrubs
for foraging but still taking refuge in the adjacent surrounding forest from their predators and human
disturbance activities. For example, the human disturbances activities that are identified include as
golfers going round the course to complete their games, noise of heavy machineries from the construction
of UNIMAS Campus facilities and also vehicle that pass through the adjacent forest and near the golf
course.
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5. CONCLUSION
The existence of species of conservation value in the surrounding forest habitats around the golf courses
indicate that the bird species still can tolerate that particular habitat regardless of the existence of the golf
courses. Golf courses with a large tract of surrounding forest habitat and with environmentally friendly turf
practices (e.g. BHR) can sustain a better bird community. Other golf courses though having smaller
surrounding forest tracts still could support some species of conservation values.
ACKNOWLEDMENT
We would like to thank the staff and students of the Institute of Biodiversity and Environmental
Conservation, UNIMAS especially Mohd Hasri Al-Hafiz, Rahah bt Mohd Yakup, Bong Hsien Huon, Nurul
Ashikeen, Cecilia Emang, Luisa Duya and Lyhmer Jack for full support in completing this study. We also
owe many thanks to Isa Sait, Nasrun Ahmad, Zahirunisa Abdul Rahim, Lim Yang Ping and Lim Kiau Cheh
from the Zoology Department for helping in the fieldwork. We are grateful to the management of Sarawak
Club Golf Resort, Damai Golf & Country Club, Borneo Highlands Hornbill Golf & Jungle Club and Sarawak
Golf Club for permission to conduct the research in their golf courses. The research was supported by
FRGS grant number FRGS/07(05)/788/2010(69).
References
Furness, R. W., & Greenwood, J. J. (1993). Birds as Monitor of Environmental Change. London:
Chapman and Hall.
Jennings, S. (2004). HCVF for conservation practitioners. Oxford, UK: Proforest.
IUCN 2012. The IUCN Red List of Threatened Species. Version 2012.1. <http://www.iucnredlist.org>.
Downloaded on 19 June 2012.
Mansor, M. S., Sah, S. A., Lim, C. K., & Rahman, M. A. (2011). Bird Species in the Padawan Limestone
Area, Sarawak. Tropical Life Sciences Research 22(2) , 65-80.
Myers, N., Mittermeier, R. A., Mittermeier, C. G., Fonseca, G. A., & Kent, J. (2000). Biodiversity hotspots
for conservation priorities. NATURE 403 , 853-858.
Myers, S. (2009). A field guide to the Birds of Borneo. London: New Holland Publishers.
Orenstein, R., Wong, A., Abghani, N., Bakewell, D., Eaton, J., Yeo, S. T., et al. (2010). Sarawak-a
neglected birding destination in Malaysia. BirdingASIA 13 , 30-41.
Phillips, Q., & Phillips, K. (2009). Field guide to the birds of Borneo Sabah, Sarawak, Brunei and
Kalimantan. United Kingdom: John Beaufoy Books.
Rahman, M. A., Salleh, M. A., & Tuen, A. A. (2002). Bird Diversity of the Crocker Range National Park,
Sabah, Malaysia. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) , 1-8.
Sodhi, N. S., Koh, L. P., Brook, B. W., & Ng, P. L. (2004). Southeast Asia biodiversity: an impending
disaster. Trends in ecology and evolution, 19(12) , 654-660.
Sodhi, N. S., Posa, M. R., Lee, T. M., Bickford, D., Koh, L. P., & Brook, B. W. (2010). The state and
conservation of Southeast Asian biodiversity. Biodiversity Conservation 19 , 317-328.
Sorace, A., & Vicentin, M. (2007). Avian diversity on golf courses and surrounding landscapes in Italy.
Landscape and Urban Planning 81 , 81-90.
Tanner, R. A., & Gange, A. C. (2005). Effects of golf courses on local biodiversity. Landscape and Urban
Planning , 137-146.
Terman, M. R. (2000). Ecology and golf: Saving wildlife on human landscapes. Golf Course Management
, 52-54.
Tuen, A. A., & Darub, A. W. (1999). The Diversity and Abundance of Understorey Birds in a Peatswamp
Forest of Sarawak. Malayan Nature Journal 53:4 , 287-294.
Tuen, A. A., Osman, A., & Putet, C. (2000). Didtribution and Abundance of Small Mammals and Birds at
Mt.Santubong, Sarawak. The Sarawak Museum Journal , 236-254.
White, C. L., & Main, M. B. (2004). Waterbird use of created wetlands in the gold course landscapes.
Wildlife Society Buletin 33(2) , 411-421.
Zwartjes, M. M., & DeLong, P. J. (2005). Southwestern Golf Courses provided needed riparian habitat for
birds. Turfgrass and Environmental Research Online 4(14) , 1-18.
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WILDLIFE UTILIZATION SURVEY IN SELECTED AREAS OF SARAWAK
S.B. Kirupaliny and Mohd-Azlan J*
Department of Zoology, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
*Email: azlan@frst.unimas.my
Abstract
A survey on wildlife utilization and regulation in Sarawak was carried out from November 2011 to April
2012. Interview and market survey was made at selected areas of South and Central region of Sarawak
while secondary information from Forest Department Sarawak was gathered. A minimum of twelve
species was recorded with some species receiving protection from Sarawak Wild Life Protection
Ordinance 1998. The pangolin (Manis javanica) is listed in the IUCN 2012 Endangered category. Bearded
pig (Sus barbatus) is the most frequently utilized wildlife and sold as preserved meat during the survey.
Increased resources to the enforcing agencies with evenly distributed enforcement staff according to
regional areas may help mitigate this matter. This is a complex issue that needs careful attention.
Additionally there is an urgent need to revise the existing legislation. Strong socio-political advocacy is
desperately needed which will assist this process.
Key words: conservation category, regulation, wildlife utilization, Wildlife Protection Ordinance 1998
1. INTRODUCTION
The forest in Sarawak can be classified into beach forest, mangrove forest, brackish swamp, peat swamp
forest, heath forest, limestone forest, lowland evergreen rainforest, lower and upper montane forest
(Kavanagh, 1985). This diverse habitat is host to a variety of large animals, which are frequently utilized in
local livelihood including for traditional medicine (Abdullah, 1996; Mohd-Azlan and Mohd. Faisal, 2006).
In the 1990s the wild meat trade was conservatively estimated to be more than 1000 tons per year, with
almost all of the meat coming out over logging roads where the annual catch by hunters in a single
logging camp resulted in 1149 animals or 29 metric tons of meat per year roads (Wildlife Conservation
Society and Sarawak Forestry Department, 1996).
The rate of deforestation in Sarawak is higher than in Sabah and Peninsular Malaysia (Mohd Azlan and
Lawes, 2011). The logging activity contributes significantly to hunting activity, especially through providing
easy access to hunters (Caldecott, 1986). The hunting activity is intensive once logging roads had been
built (Francis et al., 2001). This has created an extensive road network which created a vast commercial
trade in wild meat in Sarawak (Bennett et al., 2002).
Demand for wildlife meat becomes increasing in many parts of Southeast Asia partly due to the region’s
economic status and cultural needs (Abdullah, 1996; Caldecott, 1986; Bennett et al., 2002; Lee et al 2005;
Mohd-Azlan and Mohd. Faisal, 2006). Wildlife trade plays an important role in economic development and
affects many aspects of livings especially to the local community especially in rural areas (Corlett, 2004).
Because wild meat has a high value compared to other forest products, it is a valuable commodity and
has become the main target by poachers with special interest and skill (Robinson et al., 1999). The
commercial trade is extensive and supplies mainly urban market and for traditional medicine (Sumrall,
2009). Illegal wildlife trade can do maximum harm and possess serious threat to a number of endangered
and vulnerable species (Yiming, 2005). The effect can be amplified in areas with high endemicity such as
Borneo.
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Sarawak is the only state in Malaysia which has the legal authority on wildlife protection and conservation
under single unit, the Sarawak Forestry. Sarawak Forestry encompasses the Sarawak Forest Department
and Sarawak Forest Corporation and enforces Wild Life Protection Ordinance (1998) to protect and
manage plants and animals. However the effectiveness of any legislation is dependent entirely on the
level of implementation especially on how well the legislation is used as a weapon to deter or prosecute
wildlife criminals. Therefore understanding the wildlife trade is very important in formulating management
policy for wildlife conservation as the State Government is serious in controlling and mitigating this matter
(Megang, 2001). In view of this, the main objective of this preliminary study is to document the wildlife
trade in major markets around Sarawak.
2. METHODS
Effective methods for estimating occurrence and trade levels are limited and often unreliable due to bias in
reports and inconsistency in species identification (Mohd-Azlan et al, in Press). Interview about wildlife
species including carnivores is a common tool used in Borneo and throughout South East Asia to
investigate species distribution (Wilting et al., 2010), hunting (Bennett and Robinson, 2001) and
ethnozoological studies (Mohd-Azlan and Mohd. Faisal, 2006). Collection of data is done through
examining annual reports and survey on wildlife traded in selected local markets. These markets are
located in urban city such as Sibu and rural towns of Kapit, Kanowit, Julau and Selangau. These weekend
markets were visited periodically from November 2011 to April 2012. The local vendors were asked on the
local names of the animal sold, their selling prices and estimated quantity of wild meat or animal parts
being sold.
3. RESULTS
Twelve species of wild life were recorded including two species which were sold alive namely the softshelled turtle and Malayan box turtle (Table 1). This translates to a minimum of 427.2 kg of meat traded in
these local markets during the study period (Table 1).
Table 1 Summary of wildlife sold according to weight and price based on taxonomic categories obtained
from surveys of all five markets. Scientific names are given in Table 2.
Species
∑weight
sold
Price per kg (RM)
Julau
Sibu
Kanowit
Selangau
Kapit
Pangolin
Soft-shelled turtle
Tortoise
31 kg
44 kg
8 kg
20-25
35/head
-
30
38/head
15/head
25
-
35/head
-
20
-
7 kg
50 - 200 kg
Up to 1 kg
Wild boar
196 kg
20
20
18-20
20
20
57 -83 kg
Python
Python (Ripong)
35 kg
76 kg
10
16
-
-
16
12
-
59 -113 kg
Up to 16 kg
Porcupine
10.2 kg
28
-
-
-
-
3.5 -5.5 kg
Monitor lizard
Deer
4 kg
10 kg
18
25
-
-
-
20
Up to 25 kg
250 -300 kg
Barking deer
3 kg
-
-
-
-
18
20 -30 kg
Civet
3 kg
-
-
-
-
15
5.8 -6.6 kg
Sun bear
7 kg
-
-
30
-
20
48 -63 kg
Body mass
The most frequently encountered species in the market are the wild boar (19%) and pangolin (18%)
(Fig.1).The pangolin is classified as endangered in the IUCN 2012 red list (Table 2).
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Figure 1 Frequency of animal observed (%) in selected weekend markets in Sarawak from November
2011 to April 2012.
Table 2 Animal species sold at selected areas during weekend market in Sarawak from November 2011
to April 2012 and its protection and conservation status.
Class
Order/Family
Animal Species
SWLPO
(1998)
P
NL
NL
NL
P
P
CITIES
(2012)
II
NL
NL
NL
I
NL
IUCN
(2012)
EN
NL
NL
NL
VU
LC
Pangolin (Manis javanica)
Bearded pig (Sus barbatus)
Sambar deer (Cervus unicolor)
Red muntjac (Muntiacus muntjak)
Sun bear (Helarctos malayanus)
Ursidae
Hystricidae
Thick-spinned porcupine
(Thecurus crassispinis)
Viverridae
Civet
P
NL
N/A
Malayan water monitor (Varanus salvator)
Reptiles
Varanidae
P
II
LC
Reticulated python (Python reticulatus)
Pythonidae
P
II
NL
Borneo short-tailed python
P
II
NL
(Python curtus breitensteini)
Trionychidae
Malayan Soft-Shelled Turtle
P
NL
NL
(Dogania subplana)
Malayan box turtle (Cuora ambionensis)
Bataguridae
P
II
VU
*P- Protected under Sarawak Wildlife Protection Ordinance 1998 (SWLPO 1998); Convention on
International Trade in Endangered Species of Wild Flora and Fauna (CITES), I –Appendix I, II – Appendix
II; International Union for Conservation of Nature (IUCN), EN - Endangered, VU- Vulnerable, LC-Least
Concern, NL – Not Listed, N/A- Not Applicable.
Mammalia
Manidae
Actidactyla
Most of the animals sold in the market have been processed into smoked and preserved meat which
made direct identification difficult. Most of the market operated early morning daily and most of the traders
there are Ibans. While in Sibu most of the traders are Chinese. Julau is one of the busiest weekend
markets. Most of the item sold here comes from Kapit region. The meats were usually brought to Julau by
using express boat all over from Kapit by middlemen. The same vendors were observed during the
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surveys. Mostly animals were sold in small numbers and occasionally alive. In Kanowit the wildlife are
traded rapidly and only available for a short period of time during the day, owing to the high demand and
the rarity of the resources. In Kanowit only two vendors were observed during the survey with three
species of wild life meat recorded excluding the quills of thick-spinned porcupine. Less variety of wildlife
sold in Selangau compared to the other markets. Four vendors were recorded here and most of the wild
life sold was alive except for the wild boar and Borneo short-tailed python, “Ripong”. εost of the wild life
sold in Selangau was brought in from Mukah. Kapit is known for its notoriousness in wild life trade. Four
Iban vendors were recorded during the survey. All of the wild life meat sold was from this region mostly
obtained directly from the hunters. Most of the wildlife trade occurs during the weekend to take advantage
on the influx of people visiting the markets. Most vendors are selling the wildlife bought through a
middleman while most of the vendors are associated with underground mobsters.
At least 75% of the animals observed in the local markets are protected under SWLPO 1998 while
approximately 42% are listed in the IUCN 2012 red list.
Figure 2 Linear regression showing number of officers from Sarawak Forest Department for the year
2
2010 plotted against the size of nine regional areas in Sarawak (F 1,8 =0.04, R =0.00,P=0.84) showing no
significant relationship.
4. DISCUSSION
Twelve species consists of mammals and reptiles were frequently observed in weekend markets. The
intrinsic values of these animals possess in local livelihood (e.g. meat, skins, charms) receives continuous
demand from the community. This rapid survey shows substantial amount of wild meat is being utilized by
the local community in urban and sub-urban areas. This in turn may contribute to the rapid rate of
depletion of wildlife resources in its natural habitat. Small bodied-size animals are often observed in
market probably because of the depleting number of large bodied-size animals in the wild (Jerozolimski
and Peres, 2003). When large animals are difficult to hunt due to its low diversity, non-target animal such
as small carnivores (civets) could potentially harvest for sale. It is also feared that the hunting activity may
encroach into protected areas when wildlife resources are difficult to get in un-protected forest. In the
absence of habitat disturbances even minimum level of hunting can significantly depress wildlife
populations while heavy hunting can drive targeted species to local extinction (Robinson et al., 1999).
The most frequently sold animal is the Bearded pig (Sus barbatus) (19%) as the meat receives continuous
demand from local ethnic groups especially from the Iban community. Other animals such as civet and
barking deer are rare in the market because they are hard to hunt and mostly are for personal
consumption. Many of the protected animals are being traded in the local market owing partly to low
awareness of the legislation. Additionally the number of enforcement officers is not proportional to the
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district area (Figure 2). For example Kapit the largest district has fewer officers compared to other areas in
Sarawak. Kapit is where most of the wildlife trade was recorded in this study.
Hunting and trading activity is mostly driven by demand, socioeconomic status and cultural needs
involving more than one individual at any one time (Caldecott, 1986).This is not a straightforward but a
complex issue as it involves socio-political, cultural practices, believes and local livelihood. Therefore
there is an urgent need to address this issue from a short and long term perspective. In view of this we
recommend the following short term strategies based on this preliminary study. 1) Continuous dialogue
with local community to strengthen awareness practice the limits previously set for personal consumption.
2) rapid assessment on the intensity and regularity of the trade and address them promptly with the
available tools including the Sarawak Wildlife Protection Ordinance (SWLPO) 1998. Whilst the long term
strategies to address these issues are; 1) there is a need to review the existing SWLPO (1998) in order to
strengthen and to reduce loop holes. 2) Continuous monitoring and law implementation especially on the
trend and effect of hunting need to be monitored as it is crucial in addressing overhunting and wildlife
trade 3) review the protection status and the possibility of extending it to the non-protected species 4) gain
political support from the State authority to overcome the some of the more complex issues.
Given the current rapid decline in wildlife resources in many parts of Southeast Asia, conservationist,
stakeholders and government agencies need to sit together with local community to come up with a winwin solution. We understand that this is not easy to achieve but a road map is needed to get the ball
rolling. Therefore we conclude that in order to address this issue a multi-faceted approach is needed with
strong socio-political support and will.
ACKNOWLEDGEMENTS
We are grateful to Forest Department Sarawak and Sarawak Forestry Corporation for their help and
assistance in providing the research permit (NCCD.907.4.4(Jld.7)-131).
REFERENCES
Abdullah, M.T. 1996. Some Aspect of Wildlife Utilisation in Sarawak. Sarawak Museum Journal 50: 201207.
Bennett, E.L. and Robinson, J.G. 2001. Hunting of Wildlife in Tropical Forests. Implications for Biodiversity
and Forest Peoples. Biodiversity Series 76. The World Bank. Washington D.C.
Bennett, E.L., Milner-Gulland, E.J., Bakarr, M., Eves, H.E, Robinson, J.G. and Wilkie, D.S. 2002. Hunting
The World’s Wildlife To Extinction. Oryx, 36.
Caldecott, J. 1986. Wildlife Hunting and Management In Sarawak. IUCN. Cambridge University Press
1990.
Corlett, R.T. 2007. The impact of hunting on the mammalian fauna of tropical Asian forest. Biotropica
39(3): 292-303.
Francis, E.P., Geoffrey, M.B, Kent, H.R, Robert, F. and John, R. 2001. Tropical Forest Management and
Conservation of Biodiversity: An Overview. Conservation
Biology, 15: 7-20.
Jerozolimski, A. and Peres, C.A. 2003. Bringing Home the Biggest Bacon: A Cross-Site Analysis of the
Structure of Hunter-Kill Profiles in Neotropical Forests. Biological Conservation, 111: 415–425.
Kavanagh, M. 1985. Planning Considerations for a System of National Parks and Wildlife Sanctuaries in
Sarawak. Sarawak Gazette 112: (l491): 15-29.
Lee, R.J., Gorog. A.J., Dwiyahreni, A., Siwu, S., Riley, J., Alexander, H., Paoli, G.D. and Ramono, W.
2005. Wildlife Trade and Implications for Law Enforcement in Indonesia: A Case Study from North
Sulawesi. Biological Conservation, 123: 477-488.
Megang, M. 2001. International Trade In Wild Flora and Fauna: Case Studies From Tebedu, Sarawak.
Hornbill, 5: 156-159.
Mohd. Azlan, J. and Muhd. Faisal, F. 2006. Ethnozoological Survey in Selected Areas in Sarawak. The
Sarawak Museum Journal. Vol: LXII.
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Mohd-Azlan J. and Michael J. Lawes. 2011. The Efficacy of Protected Areas and Future Challenges for
Wildlife Conservation in Sarawak. In Mazlin Mokhtar & Sharina Abdul Halim (Eds.), RIMBA2:
Regional Sustainable Development in Malaysia and Australia, pg. 136-146. Bangi. LESTARI
Publisher. ISBN 978-967-5227-50-9
Mohd-Azlan, J., Belant, J.L. and Meijaard, E. in press. Using Secondary Information to Study Carnivores
in Borneo. Raffles Bulletin of Zoology.
Robinson, J.G, Redford, K.H. and Bennett, E.L. 1999. Wildlife Harvest in Logged Tropical Forests.
Science, 284: 595-596.
Sumrall, A.K. (2009). Confronting Illegal Wildlife Trade in Vietnam: The Experience of Education for
Nature-Vietnam. University of Michigan: 2-6.
Wildlife Conservation Society and Sarawak Forest Department. 1996. A Master Plan for Wildlife in
Sarawak. Wildlife Conservation Society and Sarawak Forest Department, Kuching.
Wilting, A., Cord, A., Hearn, A.J., Hesse, D., Mohamed, A., Traeholdt, C., Cheyne, S.M., Sunarto, S.,
Mohd. Azlan, J., Ross, J., Shapiro, A.C., Sebastian, A., Dech, S., Breitenmoser, C, Sanderson, J.
Duckworth, J.W. and Hofer, H. 2010. Modelling the Species Distribution of Flat-Headed Cats
(Prionailurus planiceps), an Endangered South-East Asian Small Felid. PLoS ONE 5:3.
Yiming, L. and Wilcove, S. 2005. Threats to Vertebrate Species in China and The United States.
Bioscience Vol.55: 147-150.
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FAMILY COMPOSITION OF SEEDLINGS AND SAPLINGS AT EARLY SECONDARY SUCCESSION
OF FALLOW LANDS IN SARAWAK
Karyati
1,2*
1
1
, Isa B. Ipor , Ismail Jusoh and Mohd. Effendi Wasli
1
1
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300,
Kota Samarahan, Sarawak, Malaysia.
2
Faculty of Forestry, University of Mulawarman, Kampus Gunung Kelua, Samarinda,
East Kalimantan, Indonesia, 75119.
*Email: karyati.hanapi@yahoo.com
Abstract
Information about the composition and distribution of seedlings and saplings are useful for predicting
future trends in population change, especially on secondary succession of fallow lands. However, there is
a scarcity of information on the distribution of seedlings and saplings on fallow lands at Sarawak. This
study was conducted to determine family composition of seedlings and saplings at early stages of
secondary succession. Samplings were carried out in two stages of fallows or period of abandonment
such as 3 years old fallows lands (hereafter called Temuda I) and 5 years old secondary forest (hereafter
called Temuda II) at Sabal, Sarawak. Twenty five plots of 20 × 20 m were established from every study
sites. All seedlings and saplings within the plot with diameter at breast height (DBH) of < 5 cm were
enumerated and identified. There were 45 families of seedlings and saplings were recorded from study
sites. Thirty nine and 38 families were observed in Temuda I and Temuda II respectively. According to
number of species and genera, Euphorbiaceae, Moraceae, and Rubiaceae were among the most families
in Temuda I and Temuda II.
Keywords: Distribution, seedlings, saplings, secondary succession, and fallow land.
1. INTRODUCTION
Typical swidden fallow secondary forest vegetation includes many forest patches at different stages of
succession. The structure of swidden fallow secondary forests changes rapidly at the young stages
(Perera, 2001). Denslow (1987) mentioned that species and succession processes characteristic of very
large clearings and repeatedly disturbed areas are different from those of large but shorter-lived gaps.
Abazari and Talebi (2008) described that the duration of development of a stand and transition from one
stage to another varies among the forest communities. The growth characteristics and dynamic of the
stands varies in different development stages. Inadequate fallow vegetation recovery is influenced by
competing resource use which affects not only the structural and functional attributes of the secondary
forest ecosystem, but also the social and economic values of current and future regenerating forest
(Ramakrishnan & Kushwaha, 2001). In terrestrial vegetation, seeds and seedlings are implicated in
various ecological phenomena. The contribution of seedling regeneration is important to the structure and
dynamics of the plant communities, ecosystems and larger units of the landscape (Grime & Hillier, 2000).
Many studies have been conducted on the floristic and structure of seedlings and saplings in the tropic.
However, there is insufficiency information available on the family composition of seedlings and saplings
at secondary succession in Sarawak. This study was conducted in order to determine the family
composition of seedlings and saplings at early stage of secondary succession in fallow lands.
2. MATERIALS AND METHODS
2.1 Study Sites
The study was carried out in Temuda I (01°04'35.6''N 110°58'49.7''E) and Temuda II (01°04'43.3''N
110°59'02.0''E) in Sabal, Sri Aman, Sarawak, Malaysia. Climatically, Sarawak is mostly influenced by the
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Indo-Australian Monsoon within the tropical monsoon belt (Whitmore, 1984). During the last 20 years
o
(1990-2009), the study sites received average annual 3,334 mm/yr of rainfall, 26.5 C of monthly
temperature, and 85.3% of relative humidity (Karyati et al., 2011).
2.2 Species Survey
Twenty five plots of 20 × 20 m were established from every study site. All seedlings and saplings within
the plot with diameter at breast height (DBH) of < 5 cm were enumerated and identified. Nomenclature
was checked in the flora records of the study area (Anderson, 1980; Jawa & Chai, 2007).
3. RESULTS AND DISCUSSION
3.1 Family Occurrence
A total of 45 families of seedlings and saplings were recorded from the study sites (see Table 1). Thirty
nine and 38 families were recorded in Temuda I and Temuda II respectively. There were 32 families
observed in both Temuda I and Temuda II. Six families such as Flacourtiaceae, Meliaceae,
Scrophulariaceae, Tiliaceae, Ulmaceae, and Urticaceae were only found in Temuda I. Anacardiaceae,
Anisophylleaceae, Rosaceae, Sapotaceae, Simaroubaceae, and Thymelaeaceae only appeared in
Temuda II. The occurrence of families of seedlings and saplings was similar in the study sites.
Table 1 Families occurrence of seedlings and saplings in the study sites.
No.
Family
Temuda I
1
Actinidiaceae
√
2
Ampelidaceae
√
3
Anacardiaceae
4
Anisophylleaceae
5
Annonaceae
√
6
Apocynaceae
√
7
Aquifoliaceae
√
8
Asteraceae
√
9
Burseraceae
√
10
Clusiaceae
√
11
Dilleniaceae
√
12
Elaeocarpaceae
√
13
Euphorbiaceae
√
14
Fabaceae
√
15
Fagaceae
√
16
Flacourtiaceae
√
17
Ixonanthaceae
√
18
Lauraceae
√
19
Lecythidaceae
√
20
Loganiaceae
√
21
Melastomataceae
√
22
Meliaceae
√
23
Moraceae
√
24
Myristicaceae
√
25
Myrsinaceae
√
26
Myrtaceae
√
27
Proteaceae
√
28
Rhamnaceae
√
29
Rhizophoraceae
√
30
Rosaceae
31
Rubiaceae
√
32
Rutaceae
√
33
Sapindaceae
√
34
Sapotaceae
263
Temuda II
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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35
36
37
38
39
40
41
42
43
44
45
Family
Scrophulariaceae
Simaroubaceae
Sterculiaceae
Symplocaceae
Theaceae
Thymelaeaceae
Tiliaceae
Ulmaceae
Unknown family
Urticaceae
Verbenaceae
Total
√ indicates the presence of family.
Temuda I
√
√
√
√
√
√
√
√
√
39
Temuda II
√
√
√
√
√
√
38
The successful survival of some seedlings of every species is critical because it is under less the
development and sustainability of plant communities (Leck et al., 2008). The process of succession is fast
in the initial stages and changes in community structure and composition could be marked from season to
season or from year to year. The young plant community develops with the passage of the time and
matures in due course (Misra, 1992).
3.2 Family Composition
The 3332 seedlings and saplings were recorded in Temuda I. These were belonging to 97 species, 74
genera, and 39 families. In Temuda II, 3149 seedlings and saplings were included to 93 species, 72
genera, and 38 families (see Table 2).
In terms of structural and life-form complexity, the second-growth forests were clearly simpler than the oldgrowth forests (Martin et al., 2004). Changes in stand structure were especially fast in the first 5 years of
succession, and decreased rapidly afterwards, which resulted from similar stand-level changes in relative
mortality, growth, and recruitment rates (Van Breugel et al., 2006).
Table 2 Total number of genera, species, and number of individuals for seedlings
different families recorded in the study sites.
Temuda I
Temuda II
No.
Family
F
G
S
N
F
G
S
1
Actinidiaceae
1
1
1
3
1
1
1
2
Ampelidaceae
1
1
1
52
1
1
1
3
Anacardiaceae
1
3
3
4
Anisophylleaceae
1
1
1
5
Annonaceae
1
4
4
35
1
3
3
6
Apocynaceae
1
1
2
29
1
1
2
7
Aquifoliaceae
1
1
1
33
1
1
1
8
Asteraceae
1
1
1
4
1
1
1
9
Burseraceae
1
2
2
2
1
1
2
10
Clusiaceae
1
2
4
283
1
2
4
11
Dilleniaceae
1
1
2
291
1
1
3
12
Elaeocarpaceae
1
1
1
9
1
1
1
13
Euphorbiaceae
1
11
18
682
1
10
14
14
Fabaceae
1
3
3
15
1
3
3
15
Fagaceae
1
2
2
7
1
1
1
16
Flacourtiaceae
1
1
1
2
17
Ixonanthaceae
1
1
1
1
1
1
1
18
Lauraceae
1
2
4
26
1
1
3
19
Lecythidaceae
1
1
1
4
1
1
1
20
Loganiaceae
1
2
3
62
1
1
1
21
Melastomataceae
1
2
2
336
1
2
2
264
and saplings from
N
3
85
4
9
20
137
44
17
2
185
326
5
404
20
3
11
12
5
22
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No.
Family
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
F
1
1
1
1
1
1
1
1
Temuda I
G
S
1
1
2
8
1
1
1
1
1
4
1
1
1
1
1
1
N
1
386
5
2
178
14
68
13
F
Meliaceae
Moraceae
1
Myristicaceae
1
Myrsinaceae
1
Myrtaceae
1
Proteaceae
1
Rhamnaceae
1
Rhizophoraceae
1
Rosaceae
1
Rubiaceae
1
7
7
111
1
Rutaceae
1
2
2
140
1
Sapindaceae
1
1
1
1
1
Sapotaceae
1
Scrophulariaceae
1
1
1
117
Simaroubaceae
1
Sterculiaceae
1
1
1
1
1
Symplocaceae
1
1
1
3
1
Theaceae
1
3
3
257
1
Thymelaeaceae
1
Tiliaceae
1
1
1
1
Ulmaceae
1
2
2
25
Unknown family
1
3
3
6
Urticaceae
1
1
1
67
Verbenaceae
1
2
2
60
1
Total
39
74
97
3332
38
F = number of family; G = number of genera; S = number of species,
hectare.
Temuda II
G
S
N
2
2
1
2
1
1
2
1
8
1
1
2
9
2
1
4
1
1
2
1
8
1
1
2
359
12
5
166
1
24
9
2
127
127
2
10
2
1
1
2
2
2
1
1
2
2
9
2
1
277
116
3
3
106
72
93
3149
and N = number of individuals per
Euphorbiaceae, Moraceae, and Rubiaceae were the most common families at Temuda I (18, 8, and 7)
and Temuda II (14, 9, and 8) according to number of species. Based on number of genera,
Euphorbiaceae was also the dominant family in Temuda I and Temuda II with 11 and 10 genera.
Meanwhile Rubiaceae was co-dominant family in Temuda I (7) and Temuda II (8) in terms of number of
genera.
The capacity to survive through alternating periods of fast and slow growth facilitates the persistence of
seedlings over time, allowing access to a greater number of small, temporary gaps, and so should be
selectively advantageous if these seedlings have a chance of reaching maturity (Delissio et al., 2002).
The study by Zhu et al. (2010) revealed that ecological species groups changed significantly in the
fragment with floristic shifts. The mature-forest (climax and shade-tolerant) species declined and early
succession space became more important. One of the distinct changes is an increase in ruderal species.
4. CONCLUSION
The survey at early stages of secondary succession on fallow lands showed similar variation with their
family composition. Moreover, the number of family, genera, and species decrease in succession stage
after 2 years abandonment. At the same time, number of individuals per hectare decreases as they are
compared with stand ages. Family composition of seedlings and saplings may explain differences in
floristic composition and structure, particularly on secondary succession of fallow lands. The composition
of seedlings and saplings at early stages of secondary succession on fallow lands is useful for soil and
water conservation, biodiversity conservation, and social and economic values for future forest.
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Acknowledgement
The authors are grateful to all support staffs at Faculty of Resource Science and Technology, En. Hidir
Marzuki, En. Sekudan Tedong, En. Salim Arip, and En. Muhd Najib Fardos for their excellent field
assistance.
References
Abazari, B.D. & Talebi, K.H.S. (2008). Diameter and Height Increment Process of Oriental Beech (Faglls
Orientalis) in Natural Caspian Forests; Kelardasht Region. Iranian Journal of Forest and Poplar
Research Winter, 15(4(30)): 320-328.
Anderson, J.A.R. (1980). A Check List of the Trees of Sarawak. Malaysia: Forest Department Sarawak.
364 pp.
Delissio, L.J., Primack, R.B., Hall, P. & Lee, H.S. (2002). A Decade of Canopy-Tree Seedling Survival
and Growth in Two Bornean Rain Forests: Persistence and Recovery from Suppression. Journal
of Tropical Ecology, 18: 645-658.
Denslow, J.S. (1987). Tropical Rainforest Gaps and Tree Species Diversity. Annual Review of Ecology
and Systematics, 18: 431-451.
Grime, J.P. & Hillier, S.H. (2000). The Contribution of Seedling Regeneration to the Structure and
Dynamics of Plant Communities and Larger Units of Landscape. In Seeds: The Ecology of
nd
Regeneration in Plant Communities, 2 Edition. (Fenner, M., ed.), United Kingdom: CABI
Publishing, pp. 349-364.
Jawa R. & P. Chai, P.K. (2007). A New Check List of the Trees of Sarawak. Kuching, Sarawak,
Malaysia : Lee Miing Press Sdn. Bhd. 340 p.
Karyati, Ipor, I.B., Jusoh, I. & Wasli, M.E. (2011). Suitability of Plant Species for Agroforestry Program at
Sri Aman, Sarawak. In Taxonomy and Ecology: Beyond Classical Approachs (Fasihuddin, B.A.,
Sepiah, M., Ipor, I.B., Zainudin, R., Wasli, M.E., Meekiong, K. & Assim, Z., eds.). pp. 184-194.
Malaysia: Universiti Malaysia Sarawak.
Leck, M.A., Simpson, R.L. && Parker, V.T. (2008) Why Seedlings?. In Seedling Ecology and Evolution
(Leck, M.A., Parker, V.T. & Simpson, R.L., eds.), pp. 3-13. New Yok: Cambridge University
Press.
Martin, P.H., Sherman, R.E. & Fahey, T.J. (2004). Forty Years of Tropical Forest Recovery from
Agriculture: Structure and Floristics of Secondary and Old-Growth Riparian Forests in the
Dominican Republic. Biotropica, 36(3): 297-317.
Misra, K.C. (1992). Manual of Plant Ecology. New Delhi : Raju Primlani. pp. 109-241.
Perera, G.A.D. (2001). The Secondary Forest Situation in Sri Lanka: A review. Journal of Tropical Forest
Science, 13(4): 768-785.
Ramakrishnan, P.S. & Kushwaha. S.P.S. (2001). Secondary Forests of the Himalaya with Emphasis on
the North-Eastern Hill Region of India. Journal of Tropical Forest Science, 13 (4): 727-747.
Van Breugel, M., Martinez-Ramos, M. & Bongers, F. (2006). Community Dynamics During Early
Secondary Succession in Mexican Tropical Rain Forests. Journal of Tropical Ecology, 22: 663674.
Whitmore, T.C. (1984). Tropical Rain Forests of the Far East. United Kingdom: Oxford University Press.
pp. 228-238.
Zhu, H., Wang, H. & Zhou, S.S. (2010). Species Diversity, Floristic Composition and Physiognomy
Changes in a Rainforest Remnant in Southern Yunnan, China after 48 Years. Journal of Tropical
Forest Science, 22(1): 49-66.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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EFFECT OF THINNING ON GROWTH PERFORMANCE OF Eucalyptus degulpta AND Eucalyptus
pellita PLANTATION IN KAPIT DIVISION, SARAWAK: A Preliminary Analysis
Arianti Atong* and John Sabang
Sarawak Forestry Corporation, Sarawak Forest Tree, Seed Bank Semenggoh,
KM20 Jalan Puncak Borneo, 93250 Kuching
*Email: arianti@sarawakforestry.com
Abstract
A preliminary study was carried out to evaluate tree growth performance of Eucalyptus deglupta
and Eucalyptus pellita based on two silviculture treatments within trial plots established in Licensed
Planted Forest (LPF), Ulu Mengiong, Kapit. The silviculture treatments include thinning and
fertilizing. Two sample plots each, measuring 20 x 20 m were used to assess the stand growth
performance between the silviculture treated and untreated areas. Individual trees within the
sample plots were marked and measured for diameter at breast height (dbh), bole height and
crown width. Tree stem diameter and crown width showed positive effect of thinning on growth
performance of both Eucalyptus species. In general, E.deglupta showed higher growth
performance compared to E. pellita.
Keywords: Eucalyptus plantation, silviculture treatment, tree density, diameter at breast height
(dbh), crown width, bole height,
1. INTRODUCTION
The genus Eucalyptus is one of the four main tree species planted in Sarawak (Sabang, 2010). This is to
meet the increasing demand from local and international market for end product such as sawn timber and
veneer. The management of Licensed Planted Forest (LPF)in Ulu Mengiong, Kapit had established one
trial plot each for Eucalyptus deglupta in Block 1 and Eucalyptus pellita in Block 37, Coupe 4.The trees
are approximately five years old (planted in 2007). The trial plots were established to determine the effect
of thinning and fertilizer application.
Eucalyptus plantation for high-value timber production requires integrating of thinning and pruning
treatments (Ronggui et. al., 2003). Basic principal of thinning in any plantation species is to minimize intraspecific competition in order to increase residual trees diameter (Beadle, 2006). Thinning is a silvicultural
treatment which aims to enhance tree growth and its vitality as well as stem and wood quality (Jactel et
al., 2011). Type of thinning, frequency, intensity, and timing alongside with selection objectives may
change tree density composition and influence the vertical and horizontal profile of the stand structure
(Jactel et al., 2011). Early thinning treatment will create room for more rapid diameter growth of residual
trees to a desirable sawlog size (Gerrand et al., 1997, Medhurst et al., 2001).
Fertilizing in planted forest was proposed to enhance tree growth performance and to improve tree vigor
(Sabang, 2009) which in some circumstances expected to reduce tree susceptibility to pests and
pathogen. However, fertilizer recommendations should be site –specific (Dickens et al., 2003). There are
several factors that need to be considered and evaluated before initiating fertilizing programs. For
example, soil type and land use history are some of the basic information needed prior to formulate
fertilizing plans. The economics of fertilization need to be considered so that it is cost effective in order to
maximize the fertilizing benefits. The study plots have been treated with fertilizers. The growth
performance information can be used to compare stand structure elsewhere.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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In view of this, the objectives of this preliminary study are1) to evaluate the growth performance of
Eucalyptus deglupta and Eucalyptus pellita using (i)dbh, (ii)crown width and (iii)bole height to the first
branch as indicators after thinning treatment, and 2) to report the tree density for each species after
similar treatment.
2. MATERIALS AND METHODS
Two samples plot 20 x 20 m, treated and untreated, were established each for E. deglupta and E. pellita.
The distance measurement was done using 50 meters measuring tape (Zing Line©, Nylon-clad Steel
Tape). Tree growth assessment was done by measuring the diameter at breast height (dbhapproximately 1.3m from the base/ground) using a dbh tape (Jackson MS©), to the nearest millimeter
(mm). Five field assistants were involved in measurements of individuals of trees. Bole heights, distance
from the base of the tree to the first live branch were measured using a measuring pole. The crown width
was measured perpendicular to the bole using a measuring tape (Zing Line©, Nylon-clad Steel Tape).
Method of thinning used in the study sites was direct felling and girdling where smaller trees were cut and
the larger ones were girdled. Blanket slashing had been done to prepare the surrounding trial plots before
any treatment. Thinning process was focused on poor performing trees, attacked and infected trees as
well as the undesirable form of trees (e.g. bended, coppice). In both plots, pest infestation was relatively
heavy (e.g. Ring bark borer; Lepidoptera). Thus, many tree individuals were removed in order to reduce
the affected trees. Removing infected trees will reduce the rate of spreading the disease and the
manipulation of insect abundance (Intachat, 2002) which in return will substantially improve the stand
health. This approach is particularly effective in controlling pests and pathogens with low dispersal ability
and slow development, such as root rot fungi and sap sucker bugs (Wainhouse, 2005). Therefore the
untreated category would have less trees removed compared to treated plots for both species.
The type of fertilizer used was Forest King with the ratio of 17N, 9P, 8K and 4MgO. Ten holes were
prepared around individual trees for manure purpose, with a distance approximately 1 and half ft from the
tree.
Thirteen gram of Forest King fertilizer was added into the prepared holes. The silviculture
treatments of thinning and fertilizer application were done simultaneously in 2010 when the trees were
about 3 years old.
The relationships between growth assessment and treatment (thinning –removed trees) for E. deglupta
and E. pellita were analyzed using the Least Squares Linear Regression analyses. The statistical
significance of these relationships was not tested as the plots established were not replicated because the
silviculture treatment was carried out in only one location each for both planted species.
3. RESULTS
A total of 117 trees were measured their stem diameter at breast height, bole height and crown width. The
dbh of E. deglupta ranged from 5.2cm to 27.8cm while for E. pellita from 4 cm to 19 cm,suggesting higher
dbh growth for E. deglupta. The mean values for the dbh are given in Table 1 below.
Table 1 The mean diameter, standard deviation (SD), standard error (SE) of the two species of
E.deglupta and E. pellitain treated and untreated plots.
Mean dbh
(cm)
SD
SE
E. deglupta (Untreated)
16.0
5.33
0.96
No.
of
standing
trees
31
E. deglupta (Treated)
18.39
3.75
0.84
20
E. pellitta (Untreated)
14.29
4.03
0.62
42
E. pellitta (Treated)
15.12
2.15
0.44
24
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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E. pellita plots have more residual trees in both treated and untreated plots resulting in higher tree density
(Figure 1). This suggests that E. pellita has higher resistance to pest compared to E. deglupta based on
the assumption that removal of trees was exclusively concentrated on infected and low bole quality trees.
edc=E. deglupta(untreated); edt=E. deglupta(treated); epc=E. pellita(untreated); ept= E. pellita(treated)
epc (Eucalyptus pellita –untreated) the highest value.
-1
Figure 1 Tree density (trees.ha ) in each treatment plot showing
A linear regression analysis was carried out to relate mean dbh of sample plot and the assumed trees
removed. A positive trend was observed between dbh and number of trees removed (Figure 2). This
suggests that the thinning treatment may have indeed decreased the competition among trees and gives
opportunity to residual trees to maintain a steady growth rate. In a uniform stand, thinning treatment is
agreeable in creating more space for optimum diameter growth (Medhurst et al., 2001; Ronggui et al.,
2003; Beadle, 2006).Between planted species, diameter growth performances of E. deglupta are higher
compared to E. pellita. The substantial increment in dbh of E .deglupta compared to E. pellita is suggested
due to the thinning and fertilizing programs.
Figure 2 Linear regression showing the relationship between mean dbh of stands and number of removed
trees (y = 0.10x + 13.59; R² = 0.80). Standard errors are denoted by vertical bars.
As expected, the crown width increased with the removal of trees. Mean crown width of four plots ranged
from 2.5m to 4.5m (Fig. 3). It showed that the thinning treatment slightly influenced the crown width with E.
deglupta showing more crown width compared to E. pellita. The removals of trees provide additional
space for the remaining trees for crown expansion. Thinning was best done after canopy closure to
maximize the growth response (Beadle, 2006).
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Figure 3 Linear regression showing the relationship between crown width of stands and number of trees
removed (y = 0.04x + 2.77; R² = 0.6). Standard errors are denoted by vertical bars.
The relationship between mean bole height and trees removed is as shown in Figure 4. There is very little
relationship between the bole height and the thinning treatment. The study showed that thinning did not
affect bole height. This may suggest that self pruning and crown development are consistent in both
treated and untreated plots.
Figure 4 Relationship between mean bole height of stands and number of removed trees (y = 0.01x +
9.28; R² = 0.01). Standard errors are denoted by vertical bars.
4. CONCLUSION AND RECOMMENDATION
-1
Eucalyptus spp in both trial plots were planted at high density (>1000 stems.ha ), with spacing distance at
3 x 3 m. Such high densities are not suitable for solid wood production (Beadle, 2006). Even though
higher stockings provide security for sufficient potential final trees in the stand (Beadle et al., 1994), it also
increases additional resources in managing these stocks.
One of the most important reasons for early thinning is to eliminate poor quality trees to maximize the
growth of the best trees within the stand for future harvest (Smith and Brennan, 2006). During the trial
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plots establishment, tree density has been reduced to 70% for E.deglupta and 83% for E. pellita. In this
particular study case, removals of trees do not consider the systematic spatial requirement of planted tree
species, in order to create a good gap. On the contrary, trees were removed non-systematically where the
thinning treatment was based on removal of infected, damaged and poorly formed trees.
Despite the fact that E. pellita showed some sort of better resistance to pest and resulting in higher tree
density compared to E. deglupta, E. pellita reacted poorly to the thinning treatment in terms of growth
performances. On the contrary, E. deglupta showed a higher increment in dbh which will result in this
species reaching the harvestable size much faster than E. pellita. This may reduce the cost and increase
the turnover rate. Though showing positive respond to thinning, E. deglupta is known to show higher
prevalence to pest (Marzuki and Ambrose, 2010).Tan (1987) reported that E. deglupta in Sabah
experienced periodic damage caused by wood borers (e.g. Endoclitahosei and Zuezeracoffeae).
Insufficient data compilation together with information not recorded systematically prevented
comprehensive statistical analysis. Inadequate of area being treated for thinning also limits the option for
sample plots replication. A more reliable statistical analysis would provide better information regarding the
growth performance of these species in relation to the thinning and fertilizer application that have been
carried out in the forest plantation. Appropriate silviculture practices will enhance production and therefore
will result in higher yield and profit on the long run.
In this preliminary exercise the mean dbh and crown width appear to be positively influenced by the
thinning treatment. However, the limited study sites are not sufficient to provide guideline to monitor trends
in silvicultural treatment. This preliminary study recommends that:
i.
Selection of growth indicator for regular monitoring and evaluation of growth performance
ii.
Good records/data management and set as high priority.
iii.
Higher thinning rate by preserving only well performing trees only. Forest plantation for a highvalue timber requires an optimum density of final crop trees in the range of 200-400 stems per
hectare(Ronggui et al., 2003; Donelly et al.,2003; Nolan et al., 2005)
iv.
Pruning of residual trees should also be carried out to produce a good stem form and wood quality
(McKenna and Woeste, 2006). However, further research on proper pruning approaches need to
be investigated for future consideration.
Acknowledgement
We are grateful to everyone involved in the assessment task, especially the plantation company who had
helped in organizing this rapid assessment trip to Ulu Mengiong, Kapit. We are also thankful to Peter Libu,
Lily Encharang, Maradi Tamin, Felix Uncham, Jeffry, and Pehin for providing assistance in the field.
Finally, we thank Miss Lucy Chong (DGM of AFSID, SFC) and Mr Julaihi Abdullah (Manager of Forest
Development Unit, SFC) for allowing us to conduct this study.
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Beadle, C. L., Turnbull, C. R. A. and McLeod, R. 1994.An assessment of growth and form for pruning to 6
metres in E. nitens plantations.Tasforest, 6, pp1-6.
Dickens, E. D., Moohead, D. J. and McElvany, B. 2003. Pine plantation fertilization. Better Crop 87 (1) pp
12-15.
Donnelly, R., Flynn, B. and Shield, E. 2003.The Global Eucalyptus Wood Products Industry: A Progress
Report on Achieving Higher value Utilization. Robert Flynn and Associates, Washington, 300pp.
Gerrand, A.M., Neilsen, W.A. and Medhurst, J.L. 1997.Thinning and Pruning Eucalypt Plantations for
Sawlog Production in Tasmania.Tasforest, 9, pp15-34
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Intachat, J. 2002. Plantation Forest: Insect Pests. A Manual for Forest Plantation Establishment in
Malaysia. Malaysian Forest Records. Forest Research Institute Malaysia, Kuala Lumpur. 286pp
Jactel, H., Branco, M., Gonzalez-Olabarria, J.R., Grodzki, W., δangstrom, B., εoreira, F., ……Vodde, F.
2011. Forest stands management and vulnerability to biotic and abiotic hazards. EFORWOOD
Tools for Sustainability Impact Assessment. 88pp
Marzuki, M. and Ambrose, A. 2010.Pest and disease assessment of Jayatiasa PF28 Mengiong, Kapit.In
Sabang, J. (Ed).Forest Health and Consultancy Field Reports, unpublished report.
McKenna, J. and Woeste, K. 2006. Fertilizing, Pruning, and thinning Hardwood Plantations. Planting and
Care of Fine Hardwood Seedlings, pp 1-8
Medhurst, J.L., Beadle, C.L. and Neilsen, W.A. 2001.Early-age and later-age thinning affects growth,
dominance and intraspecific competition in Eucalyptus nitens plantation.Canadian Journal Forest
Research, 31pp 187-197
Nolan, G., Greaves, B., Washusen, R., Parsons, M. and Jennings, S. 2005 Eucalypt Plantations for Solid
Wood Products in Australia: A Review. PN04.3002. Forest and Wood Products Research and
Development Corporation, Canberra, 130pp
Ronggui, Z., Baker, T. and Neilsen, W. 2003.Growth Response to Thinning in Young eucalyptus
Plantations in China and Australia. In Turnbull, J.W. (Ed) Eucalyptus in Asia, ACIAR Proceedings
No 111, 7-11 April, China, pp 169-173
Sabang, J. 2009. Assessment of sites and growth performance of tree species planted, Guanaco Maujaya
Plantation MengiongKapit. In Sabang, J. (Ed). Forest Health and Consultancy Field Reports,
unpublished report.
Sabang, J. 2010.Planted forest sites and trees species. Planted Forest Technical Workshop, 25 May
2010, Sarawak, Malaysia, pp 1-9
Smith, R. G. B. and Brennan, P. 2006. First thinning in sub-tropical eucalypt plantations grown for highvalue solid-wood products: Areview. Australian Forestry 69(4) pp305-312.
Tan, K.C. 1987. Exotic tree species in commercial plantations in Sabah, Malaysia.The Malaysian Forester
Vol. 50 (1) pp 62-71.
Wainhouse, D., 2005. Ecological Methods in Forest Pest Management. Oxford University Press, Oxford,
288pp.
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THE EFFECT OF RAINFALL AND SEDIMENT CHANGES ON THE POLYCHAETES DENSITY OF
SANTUBONG BEACH, SARAWAK
Diana Abd Rasani* and Shabdin Mohd Long
Department of Aquatic Science, Faculty of Resource Science and Technology, University Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak.
*Email: dianarasani@gmail.com
Abstract
A study was conducted at Santubong Beach to determine the correlation of rainfall and sediment on the
density of polychaetes. The samples were collected monthly from a fixed location during low tide for 12
months. The samples were sieved using 500 µm geological sieve. The result showed that the highest
2
density was recorded in September (659 individuals/m ) and the density of polychaetes during Northeast
2
monsoon season (November to March) (168.8 individuals/m ) was lower than the mean density during
2
Southwest monsoon (June to September) (401.2 individuals/m ). While during the inter-monsoon phase
2
from wet to dry season (April), the amount of density was 192 individuals/m and from dry to wet season
2
(October), the amount of density was 422 individuals/m . Pearson correlation showed moderate negative
correlation between density and rainfall and moderate positive correlation between density and sediment.
This study showed that the amount of rainfall and sediment structure influence the polychaetes’ density.
Keywords: Polychaetes, Santubong Beach, Northeast monsoon season, and Southwest monsoon season
1. INTRODUCTION
Polychaete is generally known as a worm with repeated segments forming a long longitudinal segmented
body and has at least one paired of parapodia. The word ‘polychaete’ is originated from δatin word which
means; poly-many and chaete-setae. Polychaete is studied widely in the world because they are
important to the ecology Class Polychaeta comprise more than 70 % of the species found in phylum
Annelida (Pechenik, 2000). About 9000 species of polychaetes are currently recognized with several
thousand more names in synonymy, and the overall systematic of the group remains unstable until now
(Rouse and Pleijel, 2003). Polychaetes play an important role in the processes happened in the sea floor.
This involves in mineralization, promote mixing of sediments and flux of oxygen into sediments, and
cycling of organic matter (Meksumpun and Meksumpun, 1999). They are also play great significance role
as a component in ocean food web (Sunil Kumar, 2002) and therefore, they were cultured extensive and
intensively as a live feed for fish aquaculture industry (Olive 1999). Recently, they were also commercially
cultured as bait to recreational fishing. Futhermore, since polychaetes have a direct contact with the water
and sediment, they have also becoming a good bioindicator for marine pollutions (Sakri et al., 2006).
Since polychaete is very significance to the marine environment sustainability, it has become a great
interest among the marine researchers worldwide (Wlodarska-Kowalczuk et al., 2007; Ramos et al., 2010;
Al-Hakim, 2001; Aswandy et al., 1991; Kastoro et al. 1991)
In the tropical countries, seasonal variation always refer to the monsoon changes that influenced by
monsoon rainfall (Shabdin and Othman, 2005). Direct addition of freshwater from the rainfall will affect the
salinity and the wind and wave action will affect the sediment. In Sarawak, the coastal areas experience a
rainfall regime of one maximum and one minimum. The maximum rainfall occurs during January and the
minimum rainfall occurs in September. Under this regime, much of the rainfall is received during the
northeast monsoon months of December to March. In fact, it accounts for more than half of the annual
rainfall received on the western part of Sarawak (Malaysia Meteorology Department, 2010).
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There were several studies had been done in Sarawak on the ecology of the polychaetes. For examples
in Kuching Bay, Balkhis (1999) and Rodrigo (1999), Blungi Bay by Mustapah (2001), Santubong Beach,
(Diana and Shabdin, 2009; Diana and Shabdin 2010). For this study, the main objective is to find the
seasonal correlation between the polychaetes density and the rainfall and changes of the sediment.
2. MATERIALS AND METHODS
Three transects had been placed from high tide to low tide level. Each transect were divided into stations
every 50 meter. Every first and last station was classified as high and low tide level. Samples were
collected from each stations and sieved using 500 µm. Samples retained on the sieve were preserved in
10 % formalin and brought back to Unimas laboratory for identification.
In the laboratory, the samples were poured into 500 µm sieve and rinsed by using tap water. The
polychaetes retained on the sieve were sorted out by using stereo microscope and identified at genus and
species level was done by using high power compound microscope model Olympus BX-05. Identification
was carried out based on Days (1967), Fauchald (2001), George and Hartmann-Schröder (1985) and
Pettibone (1970).
Replicate core of sediment samples were taken by hand corer with 4.3 cm diameter for particle size
analysis (PSA) and total Organic Matter (TOM) analysis. PSA were done according to Buchanan (1984)
and TOM was calculated in percentage by using formula proposed by Gresier and Faubel (1988).
3. RESULTS AND DISCUSSIONS
A total mean number of 866 individuals were collected during one year study from November to October
-2
2009 at Santubong beach. The average density was 288.7 individuals m per month. Figure 1 shows the
-2
mean density of polychaetes was highest in September (659 individuals m ) and lowest in November
-2
-2
2009 (32 individuals m ). While according to seasons, density of polychaetes (168.8 individuals m ) was
lower during Northeast monsoon season (November to March) than Southwest monsoon (June to
-2
September) (401.2 individuals m ). While during the inter-monsoon phase from wet to dry season (April),
-2
the amount of density was 192 individuals m and from dry to wet season (October), the amount of
-2
density was 422 individuals m . Rainfall (mm) was highest during January (1130.6 mm) and lowest during
September (133.4 mm). The density was observed higher during dry season and getting lower during wet
-2
season. Kastoro et al (1991) also found that average numerical density (individuals m ) of nearshore
macrobenthos was significantly higher in April which was during dry season.
Figure 1 Mean density and total amount of rainfall (mm) in Santubong Beach
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The were seven families of polychaetes were found (Nephtyidae, Glyceridae, Oweniidae, Onuphidae,
Aphroditidae, Lumbrineridae, and Spionidae). The most dominant species was Owenia fusiformis (Family
-2
Oweniidae), (2811 individuals m ) and the highest density was recorded in September (648 individuals m
2
). Sakri et al. (2006) reported that amount of macrofauna were higher during pre-monsoon (September to
October) compare to post-monsoon season (April to May) because during rainy season, seepage of
nutrients that flow to the sea will cause phytoplankton to increase. This might be the reason why Owenia
fusiformis had been dominant and highest in September because they are filter feeders and surface
deposit feeders (Fauchald and Jumars, 1979) where the plankton might be the source of food. Moreover,
during the rainy season, large amount of water from the river will flow towards the beach. The water will
bring a lot of nutrients from the land to be deposited to the beach.
The particle size analysis shows that the beach was dominated with sand throughout the year and there
were no variation of sediment type in every month of sampling found except in December (Table 1). Type
of sediment recorded in the monthly sampling area was very fine sand (diameter = 123 to 62 µm) except
for December which was fine sand (diameter = 250 to 125 µm). For total organic matter (TOM), the
percentage of the TOM was recorded highest in Mac (0.9768%) and lowest in April (0.7749 %).
Table 1 Percentage of sand, silt, clay and total organic matter (TOM)
Month
Sand (%)
Silt (%)
Clay (%)
TOM (%)
Nov
94.78
2.61
2.61
0.9713
Dec
95.98
2.68
1.34
0.9116
Jan
94.94
5.06
0.00
0.9471
Feb
99.95
0.04
0.00
0.9728
March
97.32
1.34
1.34
0.9768
April
96.18
2.58
1.27
0.7749
May
95.88
4.12
0.00
0.7759
June
96.16
2.56
1.28
0.9065
July
77.16
22.84
0.00
0.9344
Aug
97.38
2.62
0.00
0.8900
Sept
93.61
6.39
0.00
0.9622
October
98.69
1.32
0.00
0.9506
Seasonality of polychaetes is highly correlated to salinity and temperature while habitat preference and
abundance was substantially correlated with the sediment texture where the sediment is dependable with
the high percentage of sand content that mixed with a lot of detritus that originated from mangrove
(Kumar, 2002).
Table 2 showed that there were no significance difference (p>0.05) on the sediment and rainfall to the
density of the polychaetes for the 12-month period. However, sand, clay and rainfall were negatively
correlated with the density while the silt and TOM were positively correlated with the density presence in
every month.
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Table 2 Pearson correlation between density and sand,silt, clay, rainfall and total organic matter (TOM)
Pearson
Correlation
Density
Sig. (2tailed)
N
Sand
Silt
Clay
Rainfall
TOM
-.099
.138
-.292
-.294
.103
.310
.578
.519
.354
.750
12
12
12
12
12
4. CONCLUSION
Polychaetes were seasonally changed according to monsoon season. Rainfall and sediments like sand
and clay were negatively correlated with the density while the silt and organic matter were positively
correlated with the density.
ACKNOWLEDGEMENT
This research study is funded by the Universiti Malaysia Sarawak (UNIMAS) Fundamental Research
Grants; FRGS/06(02)/646/2007(11) and Scholarship for Postgraduates which was awarded to the first
author. We are thankful to UNIMAS and FRST staffs for the unconditional help, transportation and lab
facilities.
REFERENCES
Aswandy, I, Kastoro, W.W., Aziz, A., Hakim, I.A. and Mujino. 1991. Distribution, abundance and species
composition of macrobenthos in Seribu Island, Indonesia. Proceedings of Regional Symposium on
Living Resources in Coastal Areas. Manila, Philliphines
Balkis, T. S. 1999. Meiofauna at Kuching Bay. Sarawak: Unimas. Final Year Project Report. Unpublished.
Buchanan, J. (1984). Sediment analysis. In: Holme and McIntyre. 1984. Methods for the study of marine
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benthos. 2 ed. Oxford and Edinburg: Blacwell Scientific Publications
Days, J. (1967). A Monograph of the polychaetes of Southern Africa. Part 1 and Part London: British
Museum Natural History Publication.
Diana, A.R. and Shabdin, M.L. 2009. Horizontal study of polychaetes in Santubong Beach, Sarawak.
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Proceeding 3 Regional Conference on Natural Resources in the Tropics (NRTrops3).
Harnessing tropical natural resources through innovations and technologies. Unimas, Sarawak.
pp. 340-346
Diana, A.R. and Shabdin, M.L. 2010. Seasonal study of polychaetes in Santubong Beach, Sarawak.
Sarawak Biological Resources Forum 2010. Forest Department Sarawak, Kuching.
Fauchald, K., 1977. The polychaete worms. Definitions and keys to the orders, families and genera.
Natural History Museum of Los Angeles County, Science Series 28:1-190.
Fauchald, l. and Jumars, P. 1979. The diet of worms: A study of polychaetes feeding guilds. Oceanogr.
Mar. Biol. Ann. Rev. 17: 193 - 284
George, J.D. and Hartmann-Schröder, G. 1985. Polychaetes: British Amphonomida, Spintherida &
Eunicida. Keys and notes for the indentification of the species. Bath:
Hakim, A.I. (2009). Spatial distribution of Polychaeta (Annelida) of Natuna Island, South China Sea.
Oseanologi dan Limnologi di Indonesia, 35(3): 397 - 416
Kastoro, W.W., Aziz, A., Aswandy, I., Hakim, I.A., and Soedibjo, B.S., 1991. A study on the soft bottom
benthic community of a mangrove creek in Grajagan, East Java. Proceedings of Regional
Symposium on Living Resources in Coastal Areas. Manila, Philliphines
Meksumpun, C. and Meksumpun, S. 1998. Polychaete – Sediment relations in Rayong, Thailand.
Environmental pollution. 105: 447 – 456.
Mustapha Abdullah. 2001. Polychaeta dan Oligochaeta di Teluk Blugi. Sarawak: Unimas. Final Year
Project Report. Unpublished.
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Olive, P.J.W. 1999. Polychaete aquaculture and polychaete science: a mutual synergism. Hydrobiologia.
402: 175 - 183
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Pechenik, J.A. 2000. Biology of the invertebrates. 4 ed. New York: McGraw-Hill Higher Education. Pp
277- 322
Pettibone, M.H. 1970. Polychatea errantia of the Siboga-expedition. Part IV. Leiden: E.J. Brill
Ramos, J., San Martín, G., & Sikorski, A. 2010. Syllidae (Polychaeta) from the Arctic and sub-Arctic
regions. Journal of the Marine Biological Association of the United Kingdom, 90(5): 1041-1050.
Rodrigo, A. B., 1999. Macrofauna at tidal area of Kuching Bay. Sarawak: Unimas. Final Year Project
Report. Unpublished.
Rouse, G.W. and Pleijel, F. 2003. Problems in polychaetes systematics. Hydrobiologia. 496: 175 - 189
Sakri Ibrahim, Wan Mohd Rauhan Wan Hussin, Zaleha Kassim, Zuliatini Mohamad Joni1,Mohamad Zaidi
Zakaria, & Sukree Hajisamae. 2006. Seasonal Abundance of Benthic Communities in Coral Areas
of Karah Island, Terengganu, Malaysia. Turkish Journal of Fisheries and Aquatic Sciences, 6: 129
– 136
Shabdin, M.L. and Othman, B.H.R. 2005. Seasonal variations of marine nematode assemblages in
Sabah, Malaysia. Philippines Scientific, 42: 40 - 66
Kumar, S. 2002. Biomass, horizontal zonation and vertical stratification of polychaete fauna in the litoral
sediment of Cochin estuarine mangrove hábitat, south west coast of India. Indian Journal of
Marine Sciences. 31(2): 100 - 107
Wlodarska-Kowalczuk, M., Sicinski, J., Gromisz, S., Kendall, M.A., and Dahle, S., 2007. Similar softbottom polychaete diversity in Arctic and Antarctic marine inlets. Mar. Biol 151:607 – 616
Zurina Bolhi. 2001. Makrofauna di Teluk Blugi, Lundu. Sarawak: Unimas. Final Year
Project Report. Unpublished.
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THE DIVERSITY OF BACTERIAL ISOLATES FROM CMS AGROTECH COMPOST
Nur Azlan bin Yusuf* and Awang Ahmad Sallehin Awang Husaini
1,2
Department of Molecular Biology, Resource Science and Technology Faculty, Universiti Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak (Malaysia)
*Email: bannetscore@yahoo.com
Abstract
Open windrow composting is a practice done by CMS Agrotech at Sarawak, a traditional method to
produce quality compost. As bacteria are one of the main microorganisms in compost manufacturing
under suitable parameters, it is useful to collect data on various bacteria species present in the compost.
This enables us to determine types of bacteria that play major role in composting process as well as
providing a future path to study each of their functions. This involves sampling fresh compost from CMS
Agrotech and extraction of bacteria of the sample. Bacteria from the sample are analyzed by preparing
cultured plates of media by spread method to determine the bacteria population and streak method to
obtain the pure colony. Gram-staining is done on isolated pure colony of bacteria and later the possible
gram-negative bacteria are identified under API 20E kit, a biochemical test. The possible gram-positive
bacteria are identified molecularly using Polymerase Chain (PCR) colony where the PCR bands, after
undergoing electrophoresis, are sent for further sequencing. 3 types of bacteria have been identified, they
are;- Bacillus cereus, Pseudomonas plecoglossicida and Enterobacter cloacae.
Keywords: CMS Agrotech, compost, bacteria
1. INTRODUCTION
Composting involves a control decomposition of organic materials in a way to utilize waste products to be
converted into stable, humus-like product called compost. This is usually done with the help of microbes
such as bacteria and fungi. The composting product is preferred to be mixed with soil of crop site to
reduce the competition between microbes and plant roots for soil nitrogen as well as improving manure
handling, soil fertility, water holding capacity and tilth. A well management of these parameters like
moisture content, carbon-to-nitrogen ratio, oxygen supply, pH and temperature may encourage the activity
rate of the microbes. Different temperature phases during composting can provide diverse population of
predominantly aerobic microorganisms that grow in the sample (Peter Moon, 1997).
2. MATERIALS AND METHODS
Newly fresh matured compost is sampled from CMS Agrotech and is taken to Molecular Genetic
laboratory of Resource Science and Technology faculty (FRST) at University Malaysia Sarawak
(UNIMAS). About 5 to 10 gram of compost is resuspended into dilute pepton water in a flask and shakes
for 30 minutes in 150 rpm. 1 ml of the mixed solution is transferred into MB nutrient broth and left
-6
overnight to grow and enrich the bacteria. Serial dilution is made to 10 dilution in order to determine the
amount of bacteria in the sample by colony forming unit (CFU) and also to obtain the pure colony of
bacteria. By gram-staining, the isolated bacteria are characterized into gram-positive and gram-negative
types followed the identification of bacteria species through molecular and biochemical analysis. The
gram-positive bacteria are identified by colony polymerase chain reaction (PCR) while the gram-negative
bacteria are analyzed using API 20 E kit system.
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2.1 Colony forming unit
-1
In the serial dilution, I ml bacteria culture is transferred into 9 ml peptone water to make 10 dilution factor.
This is followed by transferring 1 ml solution from previous dilution to next tube containing 9 ml peptone
-2
water to make 10 dilution factor and so on. 0.1 ml solution of each dilution is transferred into 3 replicate
plates for greater accuracy in determining bacteria amount. Following the standard plate count method
principal, the colonies in a plate should be 30 to 300 colonies. The bacteria should be separated from
each other and produce a single discrete colony to be counted as ‘one colony’. There are 16 plates
labelled 1C, 2C and 3C for the triplicates of each dilution. Enumeration is begun after 24 hours of plate
incubation.
2.2 Morphology characterization and gram-staining
Morphology of bacteria is identified from a streaked plate. Some of the characterizations like form, size,
surface, texture, color, elevation and margin are recorded to differentiate the available types of bacteria
colony. All the various types of bacteria colony will be subjected to gram-staining to determine whether
they are gram-positive or gram-negative bacteria. By picking up a colony of bacteria, staining is done on a
microscope slide furthered by observation under 100X immersion oil of light microscope.
2.3 Biochemical characterization
API 20 E kit system is used to identify Enterobacteriaceae and other non-fastidious, the gram-negative
rods bacteria present in the compost. This is a standard system that applies 21 miniaturized biochemical
tests and database. The API 20 E strip is equipped with 20 microtubules containing dehydrated substrates
and the process starts when bacteria suspension is inoculated to each test media. Later, the strip is
incubated to allow the metabolism changes by observing color changes either by spontaneous or initiated
by addition of reagents. The species of bacteria is identified by referring to Identification Table after we
have calculated reading of results.
2.4 Molecular characterization
Polymerase chain reaction (PCR) technique is applied to generate enough amounts of DNA molecules for
sequencing and determines the species of bacteria. Colony PCR is one of PCR method by directly using
bacterial colony to do PCR instead of performing DNA extraction step. Selected colonies of characterized
bacteria will be picked by pipette tips from culture plate and inserted into eppendorf tubes by touching the
bottom of tube. 20 mL of sterile distilled water is mixed into the tube before all tubes are placed into water
o
bath for 2 minutes heating at 90 C. Then, we spun the tubes at 10 000 rpm to separate bacterial colony
cells (in form of pellet) from PCR template solution. After addition with PCR master mix creating a final
o
o
volume of 50 µL, PCR is done with given settings; 96 C for 5 minutes, 96 C for 30 seconds (30 cycles),
o
o
o
55 C for 1 minute (30 cycles), 72 C for 1 minute (30 cycles) and 72 C for 7 minutes. Later, the PCR
products will be purified before they are ready to be sent to 1st BASE for sequencing,
3. RESULTS
3.1 Bacteria enumeration
-6
The enumeration is done on plate with dilution factor 10 (Table 1.0). For the calculation, only two plates
are counted because one plate has exceeded the colony forming unit (CFU > 300).
-6
Table 1. Bacteria enumeration from 10 dilution factor
Replicate
Total Colony Forming Unit (CFU)
Enumeration (CFU/mL)
-6
9
-6
9
1
274
274/0.1 X 1/1 X 10 = 2.74 X 10
2
294
294/0.1 X 1/1 X 10 = 2.94 X 10
9
9
Average: [2.74 X 10 + 2.94 X 10 ] / 2 = 2.84 X 10
9
In a normal composting process, temperature is one of main factor that affects biology of composting. The
two temperature ranges are common to microbe growth, a mesophilic (mid-range) and thermophilic (high
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range). But thermophilic is reported to be more efficient in composting (Peter Moon, 1997). In determining
the overall sanitary quality of soil and water environments, coliform bacteria are the main indicator. This is
because it has higher frequencies than pathogens as well as safer and simple to detect. It is reported
there is decrease of average number of coliforms from beginning of composting to thermophilic phase due
to unfavourable environment established (Hassen et al., 2002). The presence of coliforms indicates
microbial activity is required for compost stability and maturity.
3.2 Bacteria Gram-staining
There are 5 types of bacteria colony recognized from the culture, which 3 are gram-positive bacteria and 2
are gram-negative bacteria (Table 2).
Table 2. The table shows morphology and gram-staining results of bacteria species A, B, C, D and E.
Bacteria
A
B
C
D
E
Morphology
punctiform/small
size/opaque/glistening
surface/moisture texture
circular/lobate/medium
size/translucent/dull surface/dry texture
circular/entire/medium
size/opaque/glistening surface/wet texture
irregular/undulate/medium
size/opaque/dull surface/dry texture
circular/undulate/medium
size/opaque/glistening surface/wet texture
Gram-staining
Purple and coccus shape with grape-like
cluster, indicates gram-positive bacteria
Pink-red and rod-shape, indicates gramnegative bacteria
Purple and rod-shape, indicates gram-positive
bacteria
Pink-red and rod-shape, indicates gramnegative bacteria
Purple and rod-shape, indicates gram-positive
bacteria
Bacteria were the most diverse group in composting as they secreted important enzymes to degrade
organic materials that fertilize the soil. Hence, most of the heat generation and initial decomposition of
compost were contributed by bacteria to ensure the end product of compost was accomplished (Rebollido
et al., 2008). Monitoring compost quality was important to ensure non-hazardous pathogens were
absence in compost product before they were sold to customers (Ashraf et al., 2007).
It was reported that Bacillus species was the second largest genus found in different mixture of compost
by estimating the total microbial isolates. This was related with wide temperature tolerance by Bacillus
abilities during hot composting stages (Ashraf et al., 2007). To understand more the variation of
population and how some microbes dominated in certain stage, we need to understand the flow of
composting. According to Hassen et al. (2002), selective role on evolution and succession of
microbiological communities were determined by temperature of the process. Generally, the operating
o
o
temperature will be higher than 55 C to maximize sanitation, 45-55 C to maximize biodegradation rate
o
and 35-40 C to maximize microbal diversity, Hence, three major steps of composting were mesophilico
heating phase, thermophilic phase and cooling phase. During mesophilic stage at temperature 40 C,
hydrogen-oxidizing, sulphur-oxidizing, nitrifying and nitrogen-fixing bacteria would be growing on topsoil.
They consume simple sugars and starches to overgrow and contributed most of microbes’ population and
heat release (Trautmann & Krasny, 1997). As the temperature exceeded and environment was not
suitable for mesophilic bacteria, thermogenic microbes would take over. Maintenance of compost
o
temperature below 60 C was important because most microbes’ activity would be affected if the heat rose
above the level. Bacillus species was the majority in the phase and if the condition was not suited for their
growth, they would form thick-walled endospores which were ubiquitous and extremely resistant to cold,
heat and dryness till they regain the activeness in favourable conditions (Trautmann & Krasny, 1997).
After thermophilic stage, composting would reach the next level where heat generated from pile was lost
and started to cool. The temperature dropped to indicate the low degradation activity and stabilization of
C/N ratio. The compost maturation constituted further decomposition of organic acids and decay-resistant
compounds, formation of humic substances and fixing of nitrate-nitrogen. Mesophilic bacteria returned to
grow in this cooling and curing level till the available carbon depleted and causing the microbes population
dropped (Graves & Hattemer, 2000).
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During mesophilic stage, gram-negative bacteria would contribute the abundance of microbes’ population.
It was important to ensure gram-negative bacteria were not present in matured compost as they have
endotoxins, toxic moleculs from family lipopolysaccharides (LPS). As the temperature of compost pile
increase during thermophilc, most of pathogenic gram-negative bacteria would be removed. Instead,
gram-positive bacteria population would increase to precede the next steps of composting (Drew et al.,
n.d.).
3.3 Biochemical characterization
Due to limited API 20E kit, we only can detect one gram-negative bacterium from the isolates. It is found
that the first gram-negative bacterium is Enterobacter cloacae, belongs to bacteria species D. It lives as
commensalism either in sewage, water, soil or in intestinal tracts of human and animals (Grimont &
Grimont, 2006). The result of the kit is described below (Figure 3.3 and Table 3.2).
Figure 1. The changes of color on API 20E kit
to characterize the biochemical activity of
bacteria
Table 3. Result score for a good identification of Enterobacter cloacae based on several tests
Test
Predict
Score
Result
Result
score
O
N
P
G
A
D
H
L
D
C
O
D
C
C H
I 2
T S
U
R
E
T
D
A
I
N
D
V
P
G
E
L
G M
L A
U N
I
N
O
S
O
R
R
H
A
S
A
C
M
E
L
A
M
Y
A
R
A
O
X
1
2
4
1
2
4
1
2
4
1
2
4
1
2
4
1
2
4
1
2
4
+
+
-
+
+
-
-
-
-
+
-
+
+
+
+
-
+
+
+
+
-
3
3
0
5
7
6
3
Enterobacter cloacae perform reaction towards 2-nitrophenyl-ßD-galactopyranoside (ONPG), L-arginine
(ADH), L-ornithine (ODC), trisodium citrate (CIT), sodium pyruvate (VP), D-glucose (GLU), D-mannitol
(MAN), inositol (INO), D-sorbitol (SOR), D-sucrose (SAC), D-melibiose (MEL), amygdalin (AMY) and Larabinose (ARA). This means it have most of these enzymes;- ß-galactosidase, arginine dihydrolase and
ornithine decarboxylase as well as undergoing these reactions;- citrate utilization, acetoin production,
fermentation or oxidation of glucose, mannitol, inositol, sorbitol, saccharose, melibiose, amygdalin and
arabinose. The predict scores are given according to positive results by monitoring color changing of the
each tubules.
The reason of Enterobacter cloacae availability in the compost may be referred to its role as phosphate
solubilising bacteria (Razikordmahalleh, 2006). It is important in plant growth-promoting bacteria to
increase plant yields by converting insoluble phosphorus to accessible one. Phosphorus is one of the
crucial macronutrient for plant growth but most of it present in certain form where plant cannot uptake it.
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Hence, Enterobacter cloacae would serve as efficient biofertilizer worker to enhance phosphate-nutrition
of most plants (Chen et al., 2005).
3.4 Molecular characterization
4 PCR products have been sent for sequencing and we got 3 similar results for Bacillus cereus
characterization and one for Pseudomonas plecoglossicida. After we got the single genetic sequence for
all bacteria isolated from the compost by merging of forward and primer sequence through EMBOSS
6.3.1., we undergo BLAST to obtain the similarity of the sequence from the bank database. Then, by
obtaining 5 possible sequences which have most similarity to the bacteria sequence we applied them to
create a phylogenetic tree to view the evolutionary relationship among the bacteria we have isolated
(Figure 3.2). The software we accessed to perform this is MEGA 4.0.2 while the tree-model we used is
Jukes and Cantor model.
Figure 2. The phylogenetic tree showing how the isolated bacteria (isolate-A, B, C and E) are related to
each other.
Bacteria A, C and E are belong to Bacillus cereus group which respectively from accession number
JN700144.1, HM449698.1 and JF728871.1 by according to maximal identity of the sequence. While
bacteria B is belong to Pseudomonas plecoglossicida with its posible accession number JF911370.1.
The condition of thermophilic stage may contribute the abundance of Bacillus cereus in compost
o
o
processing. The temperature range between 7 C to 49 C is reported suitable for Bacillus cereus growth
by also monitoring the other controls of environment (Tajkarimi, 2007). This is consistent with the
morphology and gram-staining results which are less different between each species. For the ‘coccus’
shape of bacteria colony A, this may related to error during obsevation where the bacillus shape is not
clear enough. However, molecular characterization indeed reveals the right species of the colony.
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Similar to Bacillus cereus which is easily found on environment, Pseudomonas plecoglossicida is
available in soil, wáter, plants and domestic places. It ables to degrade variety of chemical compounds,
fatty acids, insecticides and most of pollutant substances such as chromium, which is important
micronutrient for plant but toxic if excessive. Hence, Pseudomonas plecoglossicida is also a suitable
candidates for a bioremediation project (Poornima et al., 2010).
REFERENCES
Ashraf, R., Shahid, F. & Ali, T.A. (2007). Association of fungi, bacteria and actinomycetes with different
composts. Pakistan Journal Botany, 39(6), 2141-2151.
Chen, Y.P., Rekha, P.D., Arun, A.B., Shen, F.T., Lai, W.A. & Young, C.C. (2005). Phosphate solubilising
bacteria from subtropical soil and their tricalcium phosphate solubilising abilities. Applied Soil
Ecology, 34(2006), 33-41.
Drew, G.H., Deacon, L.J., Pankhurst, L., Pollard, S.J.T. & Tyrrel, S.F. (n.d.). Guidance on the evaluation
of bioaerosol risk assessments for composting facilities. Bristol: Environment Agency.
Graves, R.E. & Hattemer, G.M. (2000). Composting. National Engineering Handbook, 1-80.
Grimont, F. & Grimont, P.A. (2006). The Genus enterobacter. Prokaryotes, 6, 197-214.
Hassen, A., Belguith, K., Jedidi, N., Cherif, M. & Boudabous, A. (2002). Microbial characterization during
composting of municipal solid waste. Proceedings of International Symposium on Environmental
Pollution Control and Waste Management, 357-368.
Land Technologies (1997). Basic on-farm composting manual. Washington: Peter Moon, P.E.
Poornima, K., Karthik, L., Swadhini, S.P., Mythili, S. & Sathiavelu, A. (2010). Degradation of chromium by
using a novel strains of Pseudomonas species. Journal of Microbial and Biochemical Technology,
2(4), 95-99.
Razikordmahalleh, I. (2006). Enriching sugarcane bagasse compost by sulphur, nitrogen fixing
(Azotobacter chroochoccum) and phosphate solubilizing bacteria (Enterobacter cloacae) bagasse
decomposition and produced compost enrichment. Paper presented at the 18th World Congress
of Soil Science in Philadelphia, Pennsylvania.
Rebollido, A., Martinez, J., Aguilera, Y., Melchor, K., Koerner, I. & Stegmann, R. (2008). Mirobial
populations during composting process of organic fraction of municipal solid waste. Applied
Ecology and Environmental Research, 6(3), 61-67.
Tajkarimi,
M.
(2007).
Bacillus
cereus.
Retrieved
June
01,
2012,
from
http://www.cdfa.ca.gov/ahfss/Animal_Health/.../25007BcerMH__2_.pdf
Trautmann, N.M. & Krasny, M.E. (1997). Composting in the classroom: Scientific inquiry for high school
students. New York: PhotoSynthesis Productions.
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SUSTAINABILITY OF PLANTS WITH MEDICINAL PROPERTIES VIA DNA PROFILING
Rosmawati bt Saat*, Siti Izyan Kamarol, Zuliza Ahmad and Jaya Mania
Department of Molecular Biology, Faculty of Resoursce Science & Technology,
University Malaysia Sarawak, 94300 Kota Samarahan, Sarawak.
*E-mail: srosma@frst.unimas.my
Abstract
εalaysia is rich with varieties of plants and amongst them are the “hidden treasures”. These are the
plants with medicinal properties which have long been used by the locals as traditional medicines in
treating among others; diarrhea, pneumonia, spasm, fever, snake bite, insect bite and anti-malaria. These
plants were shown to have bioactive compounds against such as microbial activity, tumour activity,
diabetic activity and diarrheal activity which have the potential to be explored in the production of novel
drugs for a spectrum of pharmaceutical applications. Identification of these medicinal plants has been
primarily based on morphology and similar looking plants may not be distinguished. Profiling using DNA
markers provides an accurate tool in identification and authentication of the medicinal plants, enabling the
new species to be identified. Thus, allowing the sustainability of the plant species to be achieved.
Key words: Profiling, DNA markers, medicinal plants
1. ITRODUCTION
In various cultures worldwide, since ancient times, plants are used in the traditional medicine. Malaysia
with its warm climate condition provides a hub for the growth of a diverse plant species, particularly, the
plants with medicinal properties. Among them are the Asteraceae family which includes Elephantopus
scaber Linn and Ageratum conyzoides Linn that are commonly used by the locals in folk medicine.
Elephantopus scaber Linn is commonly known as prickly-leaved Elephant’s foot or in εalay, Tutup bumi
(Barn & Chan, 1990). This common wild plant grows in shady places in tropical/subtropical conditions can
easily be found by the roadsides, grass plots, grasslands, wasteland as well as in forest borders of altitude
as high as 1500 m (Ho et al, 2009; Hammer et al., 1993) and it can be cultivated in field (Hammer et al.,
1993). It is a short, stiff and tufted herb which its stem can reaches up to 30 cm in height. Leaves of E.
scaber L are white and hairy, variable in size and oblong shape (David & Luz 1990). The roots, leaves
and whole plant are commonly used in Asian countries such as China, India, Thailand, Malaysia, Japan,
Indonesia and The Philippines to treat among others; hepatitis, bronchitis, cough associated with
pneumonia, jaundice, chest pain, snake bite, insect bite, anti-malaria, diarrhea and various stomach
disease (Inta et al., 2008; Ichikawa et al., 2008; Singh et al., 2005; But et al., 1997). In Malaysia,
decoction of E.scaber L root has been used to accelerate contraction of abdominal area and to prevent
inflammation after birth (Ho et. al., 2009). Also, the whole plant is boiled with red bean to remove
flatulence (Ong and Nordiana, 1999a; Ong and Nordiana, 1999b).
A. conyzoides is widespread in tropical and subtropical regions. Common names for A. conyzoides
include goat weed, chick weed, tropical white weed, floss flower and to the locals in Malaysia it is known
as “rumput tahi ayam”. It is an annual branching plant which can grow up to 1 m in height (Shekhar and
Anju, 2012). The flowers are white or bluish lavender in colour (Motooka et al., 2003). It has a hairy stem.
In some countries, A. conyzoides L is considered as weeds due to their growths that are highly abundant
and difficult to control (Ming, 1999). Due to its bad odour, it is not eaten by man (Shekhar and Anju,
2012). Nevertheless, the whole plant is used only for medicinal purposes and has a long history in folk
medicine of varying countries. It is used in various parts of Africa, Asia and South Africa (Shekhar and
Anju, 2012). The applications of the plant include in treating diarrhea, pneumonia, spasm, fever, to heal
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burns and wound (Ming, 1999; Saichin et al., 2009). In Malaysia, it is used to treat dysentery, boil and
poultries (Tropical Database, 1999). It also has been used as a tea for diabetic patients and pneumonia
(Ming, 1999).
A number of studies have been conducted and major bioactivities of these plants based on their traditional
applications were confirmed.
Bioactive compounds of E. scaber L that were discovered include
chromenes,
benzofurans,
cumarins,
alkaloids,
flavonoids,
tannins,
deoxyelephantopin,
isodeoxyelephantopin and a germacranolide sesquiterpene lactone named scabertopin (But et al., 1997;
Liang & Min, 2002; Mohan et al., 2010; Quintero et al., 1999; Than et al., 2005; Xu et al., 2006). These
active chemical compounds were reported to have anti-tumour activity (Quintero et al., 1999; Xu et al.,
2006; Rajkapoor et al., 2002), anti-microbial activity (Avani & Neeta, 2005;Wiart, 2002), wound-healing
property (Singh et al., 2005) anti-diarrhoeal activity (Muthumani et al., 2010), anti-inflammatory activities
(Inta et al., 2008), anti-diabetic activity (Daisy et al., 2009) as well as cardio tonic activity (Muthumani et
al., 2010). A. conyzoides L is rich in polyoxygenated flavanoids which 21 of them were reported in the
whole plant (Shekhar and Anju, 2012). Other range of bioactive compounds that were found in the plant
includes chromenes, benzofurans, cumarins, alkaloids, tannins and isoflavone (Osho and Adentunji, 2011;
Saichin 2009). These compounds were shown to possess antibacterial property such as against
Staphylococcus aureus, Klebsiella pneumonia and Basillus subtilus (Osho and Adentunji, 2011),
fungicidal property (Ogbebor and Adekunle, 2005), antioxidant activity (Nyemb et al., 2009), wound
healing property (Oladejo et al., 2003), antihyperglycaemic (Nyemb et al., 2010).
Medicinal plants of such are promising alternative medicine resources (herbal medicine) which have the
potential to be developed as novel drugs for curing diseases as well as for pharmaceutical applications.
However, before any scientific research can be conducted, the plant needs to be identified. There is a
variety of medicinal plants/herbs available in the market and some of them may look similar which difficult
to be distinguished. Identification of these medicinal plants has been primarily based on morphology and
it may not be accurate due to the environmental factors that may have effects on growth and morphology
of the plant (Viruel et al., 2005). In addition, the lack of comprehensive references makes identification of
the plant species more difficult. Correct identification of the starting materials which are the plants/herbs is
essential pre-requisite to ensure the efficacy and safety of the herbal medicine. The medicinal plants may
be similar in morphology, yet with different active chemical compounds. The use of wrong plant in
medicinal treatment may create different effects; the treatment may not be effective or may be fatal (Sekar
et al., 2007). Profiling using DNA markers may provide an accurate tool in identification of the medicinal
plants. DNA markers are reliable for informative polymorphisms as the genetic composition is unique for
each species. Furthermore, the genetic composition of is not affected by age, physiological conditions and
environmental factors (Joshi et. al., 2004; Neale et al., 1992; Suslow & Bradford, 1999). DNA can be
extracted from fresh or dried plants; hence the physical form of the plant sample of assessment does not
restrict the detection (Joshi et. al., 2004). The DNA profiling is a technique whereby DNA is analysed to
reveal the repetition of a particular short nucleotide sequences throughout the genome (Montaldo & MezaHerrera, 1998). Different species will have a different DNA banding pattern. Thus, it can act as a
signature for a particular plant species which it can be utilised for identification and authentication of the
medicinal plants. Even more, level of variations within the plant species can be detected by simply looking
at the profile patterns (Shaw et al., 2002). Therefore, applications of DNA profiling could provide a
platform in achieving sustainability of the medicinal plant species.
Various types of DNA-based molecular techniques can be utilised in profiling and one of them is
polymerase chain reaction (PCR)-based method. Two types of PCR-based DNA marker systems which
are Inter-simple sequence repeat (ISSR)-PCR and Random Amplified Polymorphic DNA (RAPD)-PCR are
discussed in this paper. ISSR-PCR is a technique which based on amplification of DNA region present at
amplifiable distance in between two identical microsatellite repeat regions inversely oriented (Reddy et al.,
2002). This technique uses microsatellites as a single primer PCR reaction targeting multiple genomic
loci to amplify mainly the inter-simple sequence repeats (SSRs) motifs of different sizes (Reddy et al.,
2002). The microsatellite repeats used as primers can be in di, tri, tetra or penta nucleotides (Reddy et
al., 2002). It is a fast, simple, efficient technique and it has high reproducibility (Reddy et al., 2002). Other
advantages of ISSR-PCR include; it can easily be applied to any plant species and it may reveal a much
higher number of polymorphic fragments as the sequences that ISSR targets are abundant throughout the
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eukaryotic genome (Esselman et al., 1999; Fang & Roose, 1997). The technique has been demonstrated
to provide as an effective tool in identification of many plant species (Reddy et al., 2002). ISSR-PCR can
be used in the studies of interspecific and intraspecific relationships in plants (Bornet & Branchard, 2001).
RAPD, on the other hand, is a method that based on the amplification of random DNA segments using
single ten-base synthetic DNA arbitrary primers (Welsh et al. 1991). No prior knowledge of the organism’s
DNA sequences is required (Williams et al. 1990). It is simple, rapid, sensitive; require lower
concentration of DNA samples (Williams et al. 1990). This technique has been successfully used for
identification of plant species which include herbs and determination of the genetic variation level of plant
species (Li et al., 2004; Cheng et al.,2000).
2. MATERIALS AND METHODS
2.1 Plant Samples
Fresh leaves of the plant samples were collected, cleaned with water and kept at 4 ºC prior to DNA
extraction. Individual E. scaber L samples were collected from three different districts of Sarawak,
namely, Samarahan, Sematan and Tabuan Jaya. As for A. conyzoides L, individual plant samples were
obtained from four different locations in Sarawak; Rantau Panjang, Samarahan, Matang and Kuching.
2.2 DNA extraction
DNA extraction of the plant was performed using sodium dodecyl sulphate (SDS) method (Kikuchi et. al.,
1998) with some modifications (Saat et. al.,2011a; Saat et. al.,2011b). The leaves were freeze-dried
using liquid nitrogen and ground into a fine powder. Then, followed with the addition of 600 µl of preheated extraction buffer [0.1M Tris-HCl (pH 8.0), 0.01M EDTA (pH 8.0) and 0.01M NaCl; 10% SDS] and
0.2% (v/v) β-mercaptoethanol (was added to the extraction buffer just before used) and the mixture (in a
1.5 ml centrifuge tube) was thoroughly mixed by inversion. The mixture was then incubated at 65ºC for 30
minutes and mixed by inverting the tube every 15 minutes. It was left to cool at room temperature prior to
addition of 220 µl of 5 M ammonium acetate and again it was mixed thoroughly. Incubation was
0
performed at 4 C for 15 minutes and subsequently, centrifugation for 10 minutes at 5700 rpm. The
supernatant was then transferred into a new tube which contained 300 µl of cold isopropanol and
occasional inverted for 5 minutes to precipitate the DNA. Following this was centrifugation of the samples
for 10 minutes at 5700 rpm to form a DNA pellet. The DNA pellet was collected and air-dried at room
temperature. Washing of the pellet was conducted by adding 500 l of 70% ethanol and centrifuging for 10
minutes at 5700 rpm. The collected pellet was air dried at room temperature to remove any drops of
ethanol. The pellet was resuspended in 100 l of TE buffer and left to dissolve overnight at 4°C. Cellular
debris which was not dissolve was spun down at 500 rpm for 20 minutes. The collected supernatant was
stored at 4 ºC or -20°C prior to use.
2.3 Quantification and Purity determination of extracted DNA
The quantification and purity determination was determined via spectrophotometer reading. The
absorbance readings at the wavelengths of 260 nm and 280 nm were taken using spectrophotometer
®
(Ultrospec 1100 Pro, Amersham). The DNA concentration was calculated using the following formula:
DNA Concentration ( g/ml) = Measured A260 X Dilution factor X 50 g/ml. The measurement of the DNA
purity was performed by taking the ratio of measured A260 / measured A280.
2.4 ISSR-PCR
Extracted DNA samples of E. scaber L were subjected to ISSR-PCR analysis (Rosmawati et. al.,2011b).
Four ISSR primers with different sequences (Research Biolabs, Table 1) were used in generating the
DNA profiles. A total of 20 µl of PCR reaction mixture was prepared with the following components; 20 ng
of genomic plant DNA, 1X PCR buffer (Yeastern Biotech), 2.5 mM magnesium chloride (Fermentas), 0.4
mM dNTPs (Promega), 1 Unit of Taq DNA polymerase (Yeastern Biotech) and 1.0 µM of ISSR primer
(Saat et. al.,2011b). PCR amplification was performed (BIOER Little Genius Thermal Cycler) with the
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optimal thermocycling conditions as follows; initial denaturation at 94ºC for 5 minutes and followed by 45
cycles denaturation at 94ºC for 30 seconds, annealing temperature at 39ºC for 45 seconds, extension at
72ºC for 1.5 minutes, with an extended final extension at 72ºC for 7 minutes (Rosmawati et. al.,2011b).
Table 1. List of ISSR primers and their sequences
Primer
Sequence
Primer 1
5’ CACACACACACAAC 3’
Primer 2
5’CACACACACACAGG 3’
Primer 3
5’GTGGTGGTGGC3’
Primer 4
5’GAGAGAGAGAGAGG 3’
2.5 RAPD-PCR
Extracted DNA samples of E. scaber L and A. conyzoides L were subjected to RAPD-PCR analysis. Two
different sets of RAPD-PCR reaction were performed.
RAPD-PCR for E. scaber L
A total number of 18 decamer RAPD primers, namely, OPA (1 to 6), OPB (1 to 6), OPC2, OPC3, OPC5,
st
OPC7, OPC11, and OPC13 (1 Base, Table 2) were randomly chosen and used in the initial RAPD-PCR
screening of the plant DNA samples (Rosmawati et. al.,2011b). A total of 20 l PCR reaction mixture was
prepared with 25 ng of template DNA, 1x PCR buffer with 2 mM MgCl (Yeastern Biotech), 100mM of each
st
2
st
dNTPs (1 Base), 10 pmol of primer (1 Base), 1 Unit of Taq DNA polymerase (Rosmawati et. al.,2011b).
PCR amplification was performed (BIOER Little Genius Thermal Cycler) with the optimal thermocycling
conditions as follows; initial denaturation at 94ºC for 1 minute and followed by 45 cycles denaturation at
94ºC for 1 minute, annealing temperature at 33ºC for 1 minute, extension at 72ºC for 2 minutes, extension
at 72ºC for 2 minutes, with an extended final extension at 72°C at 7 minutes (Rosmawati et. al.,2011b).
RAPD-PCR for A. conyzoides L
st
Eight decamer primers (1 BASE Inc): OPA01, OPA6, OPA19, OPB04, OPB08, OPB16, OPC11 and
st
OPC19 (1 Base, Table 2) were used in the analysis (Rosmawati et. al.,2011a). PCR amplification was
conducted using the following components: 50 ng of genomic plant DNA, 2 µ1 of 10 X PCR buffer
(Yeastern Biotech), 3 mM magnesium chloride (Fermentas), 0.2 mM dNTP mix (Promega), 1U Taq DNA
polymerase (Yeastern Biotech), 1.0 µM of primer and sterile distilled water in a 20 µ1 reaction mixture
(Saat et. al.,2011a). PCR amplification was performed (BIOER Little Genius Thermal Cycler) with the
optimal thermocycling conditions as follows; 1 min initial denaturation at 94 °C and followed by 40 cycles
of 1 min denaturation at 92°C, 1 min annealing at 35°C and 2 min extension at 72°C, with an extended
final extension of 5 min at 72°C (Rosmawati et. al.,2011a).
Table 2 List of RAPD primers and their sequences
Primer
Sequence
Primer
OPA01
5’CAGGCCCTTC 3’
OPB04
OPA02
5’TGCCGAGCTG 3’
OPB05
OPA03
5’AGTCAGCCAC 3’
OPB06
OPA04
5’AATCGGGCTG 3’
OPB16
OPA05
5’AGGGGTCTTG 3’
OPC02
OPA06
5’GGTCCCTGAC 3’
OPC03
OPA19
5’-CAAACGTCGG-3’
OPC05
OPB01
5’ GTTTCGCTCC 3’
OPC07
OPB02
5’ TGATCCCTGG 3’
OPC11
OPB03
5’CATCCCCCTG 3’
OPC13
OPC19
287
Sequence
5’GGACTGGAGT 3’
5’TGCGCCCTTC 3’
5’TGCTCTGCCC 3’
5’-TTTGCCCGGA-3’
5’GTGAGGCGTC 3’
5’GGGGGTCTTT 3’
5’GATGACCGCC 3’
5’GTCCCGACGA 3’
5’AAAGCTGCGG 3’
5’AAGCCTCGTC 3’
5’-GTTGCCAGCC-3’
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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2.6 Detection of the ISSR-PCR and RAPD-PCR amplicons
PCR amplicons for each analysis were separated on a 1.5% (w/v), 1.6 % (w/v) and 2.0 %(w/v) agarose
gel (First Base Lab) at the voltage power of 100 and the gel was stained using ethidium bromide
(Sambrook et al. 1989) and documented using Kodak Gel Documentation System (BioRad).
3. RESULTS
An intact genomic DNA with relatively good quality in terms of purity and yield was successfully extracted
with most samples was found to be in the range of 1.8 to 2.0 for the ratio of measured A260 / measured
A280 (data not shown).
All ISSR primers except ISSR primer 3 had successfully generated DNA profiles for E. scaber L (Table 3).
Primer 3 failed to generate any band despite several optimisation of PCR conditions had been conducted.
Primer 1 had successfully generated a total of five different amplicons for samples obtained from
Samarahan. Samples obtained from Sematan and Tabuan Jaya both showed the same DNA profile with
Primer 1 which three amplicons were detected (Figure 1, Table 3). The size range of the amplicons was
between 400 bp and 1,400 bp (Figure 1). Again, samples collected from Sematan and Tabuan Jaya
shared the same DNA profile pattern with only two amplicons produced with Primer 2 (Figure 2, Table 3).
However, a total of nine amplicons were observed in the profile of samples obtained from Samarahan
(Figure 2, Table 3). The size of the amplicons detected using this primer was in the range of 300 bp and
1,200 bp (Figure 2). Both profiles of samples from Samarahan and Tabuan Jaya had the same profile
with a total of nine amplicons generated using Primer 4 and samples from Sematan in contrarily, showed
a different pattern of profile with only three amplicons being produced (Figure 3, Table 4). Primer 4 had
generated amplicons with the size range of between 300 bp and 1,200 bp as well (Figure 3).
Out of 18 RAPD primers screened, only OPA05 primer had successfully generated DNA profiles for all the
samples of E. scaber L obtained from Samarahan, with the size of amplicons ranging from 500 bp to 1200
bp (Figure 4). A number of four samples showed the same DNA profile patterns with a total of 6
amplicons observed and the rest of the samples had 7 amplicons in total (Figure 4, Table 3).
Only Primer OPB08 has successfully generated a DNA profile for Ageratum conyzoides L. Samples from
all the four locations showed the same DNA banding pattern. Three distinct bands were observed with
the sizes of 1000 bp, about 1200bp and 2000 bp (Figure 5).
(A)
(B)
Figure 1. DNA profiles generated using ISSR primer 1.; (A) A 2% ethidium bromide stained agarose gel
of DNA profiles, (B) A reverse picture of the ethidium bromide stained gel picture of DNA profiles, Lanes:
K - sample from Samarahan, S – sample from Sematan, and T – sample from Tabuan Jaya.
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(A)
(B)
Figure 2. DNA profiles generated using ISSR primer 2; (A) A 2% ethidium bromide stained agarose gel of
DNA profiles, (B) A reverse picture of the ethidium bromide stained gel picture of DNA profiles, Lanes: K sample from Samarahan, S – sample from Sematan, and T – sample from Tabuan Jaya.
(A)
(B)
Figure 3. DNA profiles generated using ISSR primer 3; (A) A 2% ethidium bromide stained agarose gel
picture of DNA profiles, (B) A reverse picture of the ethidium bromide stained gel picture of DNA profiles,
Lanes: K - sample from Samarahan, S – sample from Sematan, and T – sample from Tabuan Jaya.
Note: K - sample from Samarahan, S – sample from Sematan, and T – sample from Tabuan Jaya.
(A)
(B)
Figure 4. DNA profiles generated using RAPD primer OPA05; (A) A 1.6 % ethidium bromide stained
agarose gel picture of DNA profiles. (B) A reverse picture of the ethidium bromide stained gel picture of
DNA profiles, Lanes: L- 1kb ladder, B1 to B10 – individual samples from Kota Samarahan.
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Table 3 Summary of DNA profiles of E. scaber L samples obtained from Samarahan, Sematan and
Tabuan Jaya using 4 different ISSR primers.
Location of samples
collected
Primer 1
abcde
Primer 2
abcdefghi
Primer 3
abcdefghi
Primer 4
abcdefghi
Samarahan
11111
111111111
000000000
111111111
Sematan
01101
011000000
000000000
111000000
Tabuan Jaya
01101
011000000
000000000
111111111
Noteμ The ‘0’ and ‘1’ correspond to the absence and presence of a band detected on the agarose gel,
respectively. ‘a’ to ‘i’ refers to the band location on the ISSR profiles.
Table 4. Summary of DNA profiles of E. scaber Linn individual samples obtained from Samarahan using
RAPD Primer OPA05
Individual sample
Primer OPA05
abcdefg
Sample 1
Sample 2
Sample 3
Sample 4
Sample 5
Sample 6
Sample 7
Sample 8
Sample 9
Sample 10
1110111
1110111
1110111
1110111
1111111
1111111
1111111
1111111
1111111
1111111
Noteμ The ‘0’ and ‘1’ correspond to the absence and presence of a band detected on the agarose gel,
respectively. ‘a’ to ‘i’ refers to the band location on the RAPD profiles.
5. DISCUSSIONS
Generally, problems may be encountered in the extraction of a good quality DNA from a certain medicinal
and aromatic plant species, for example, degradation of DNA which due to endonucleases, co-isolation of
highly viscous polysaccharides and inhibitor compounds like polyphenols as well as other secondary
metabolites which directly or indirectly interfere with the extraction processes and subsequent analyses
(Matasyoh et al., 2008; Ribeiro & Lovato, 2007). Due to these reasons, a suitable extraction method is
required for medicinal plants such as E. scaber L and A. conyzoides L which contain compounds of
secondary metabolites (flavanoids, alkaloids, tannin and sesquiterpene lactones). Thus, DNA extraction
method using SDS was performed. The extraction method is rapid, simple, efficient and able to yield clean
DNA as well as PCR amplifiable (Kikuchi et al., 1998). Some modifications on the method, which include
high percentage of SDS (10%), were made as to obtain a good quality DNA from the plants. The genomic
DNA was extracted from young leaves as the cell walls are soft and it is easier to perform the extraction
process as compared to the matured leaves that become highly harden by structural tissues (Lokvam et
al., 2006). The extracted genomic DNA was subsequently subjected to ISSR-PCR and RAPD-PCR
analyses.
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ISSR-PCR had successfully generated DNA profiles of E.scaber L with all the di-nucleotide primers which
are Primer 1, Primer 2 and Primer 4 (Table 3) for all locations. However, DNA profile was failed to be
generated with tri-nucleotide primer, Primer 3 (Table 3). This observation is probably due to the fact that
tri-nucleotides are less abundant than di-nucleotides in plants (Reddy et al., 2002). In addition, primers
with (CA), (GA), (CT), (AC), (TC) and (GA) repeats generally show higher polymorphism than primer with
other di-, tri or tetra-nucleotide repeats (Reddy et al., 2002). Two different DNA profile patterns were
observed for each ISSR primer used that may indicate some level of variations within the plant species.
Some variations may be due to the mutation events (Reddy et al., 2002). However, further studies are
required in confirming the event. ISSR study which had been performed on eight natural E. scaber L
populations from South China also showed variations within the plant species and suggested that
fragmented local environments as well as human disturbance might play important roles in shaping the
population structure of E. scaber L (Wang et al., 2006). Apart from that ISSR-PCR analysis had been
used to distinguished between various cultivars of chrysanthemum (Wolf et. al., 1995), strawberry (Arnau
et al., 2002), potato (McGregor et al.2000) and cotton (Liu & Wendel, 2001) and maintenance of cocoa
collection (Charters & Wilkinson, 2000).
Sets of RAPD decamer primers OPA, OPB and OPC were initially screened in ten individual E. scaber L
plant samples obtained from Samarahan district area. However, only OPA05 primer was successfully
generated reproducible PCR multiple bandings. This OPA05 primer was then used in the second
screening of the plant DNA samples. Two DNA profiles were generated from these 10 individual plant
samples which a group of samples had all the seven bands and the rest had six bands (Figure 4, Table 4).
The loss of a band from the DNA profile may be caused by failure to prime a site in some individuals
which may due to changes in nucleotide sequences (Clark & Lanigan 1993). RAPD-PCR analysis had
also been performed on E. scaber L to identify the plant species in the Chinese drug retailed in Hong
Kong, Taiwan, Fujian and Macau markets (Chao et. al., 1996). Eight RAPD decamer primers were
screened in A. conyzoides L samples and only primer OPB08 had successfully generated a DNA profile
for the plant species. All the samples collected from the four locations showed the same DNA profile with
three distinct bands (Figure 5) which indicated that there is no variation within the plant species despite
different locations of the samples obtained. RAPD markers have been shown to be useful in
differentiating a list of plant species with medicinal properties collected from different geographical regions
(Joshi et al., 2004). It has been widely used in identification and determination of genetic variation level of
plant species (Cheng et al., 1999; Li et al., 2004).
As DNA markers derived from DNA profiling could be used as a tool to verify species identity, varieties as
well as hybrids, the markers could be utilised for plant breeding development as well as conservation.
Hence, allowing the sustainability of such plants with medicinal importance to be achieved. Apart from
identification and authentication, DNA profiling also provides a platform for mapping of markers associated
with the medicinal properties of the plant species (Joshi et. al., 2004). Prediction of the concentration of
active phytochemicals associated with a particular plant for quality control in the use of plant materials for
pharmaceutical purposes may be allowed with the data obtained from profiles generated using DNA
based markers as such (Joshi et. al., 2004). Thus, the development of novel drugs and commercialisation
of the herbal medicines could be realised.
6. CONCLUSION
Sustainability of the medicinal plants can be achieved via the applications of DNA profiling in various
fields.
Acknowlegdement
We like to thank Faculty of Resource Science and Technology, UNIMAS in providing us the facilities,
equipments and some chemicals for us to conduct this study successfully.
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GENETIC DIVERSITY OF Duabanga moluccana USING DOMINANT DNA MARKERS BASED ON
INTER-SIMPLE SEQUENCE REPEATS IN SARAWAK
1*
1
1
2
3
Ho, W.S. , Diyanah, M. J. , Liew, K.S. , Pang, S.L. , Ismail, J. & Julaihi, A.
2
1
Forest Genomics and Informatics Laboratory (fGiL), Department of Molecular Biology, Faculty of
Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak
2
Applied Forest Science and Industry Development (AFSID), Sarawak Forestry Corporation, 93250
Kuching, Sarawak
3
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak
*Email: wsho@frst.unimas.my
Abstract
Duabanga moluccana or locally known as Sawih is a widely known forest tree species for its multipurpose timber and other natural products such as fibers. Genetic diversity investigation of this species
can provide baseline information for indirect selection in tree improvement programme. This study has
employed Inter Simple Sequence Repeat (ISSR) technique to assess the genetic diversity and
relatedness within and between three populations of D. moluccana located in Sarawak, namely, Mukah,
Tatau (Bintulu) and Niah. A total of 151 loci from 90 individuals were successfully amplified with 6 selected
ISSR primers and the percentage of polymorphic loci was λ0.1%. The Shannon’s diversity index showed
that D. moluccana in Mukah natural forest (0.429) was the most diverse compared to Tatau (Bintulu)
(0.362) and Niah natural forest (0.387). Neighbour joining tree were constructed to show relationship
among the selected populations. The overall populations were completely clustered into three main
groups, according to their corresponding population. Based on these results, it implies that D. moluccana
trees are genetically diverse among populations.
Keywords: Inter simple sequence repeat, Duabanga moluccana, genetic diversity, genetic structure
1. INTRODUCTION
Establishment of forest tree plantations is becoming more crucial as forest resource reduction increases
exponentially due to human activities. Most losses are measured in square kilometers, but a more precise
loss of forest tree resources cannot be measured. As forests disappear, so do their genetic resources
(Arnold, 1991; Sedjo and Lyon, 1990). For this reason, forest tree plantation development is a necessity
rather than a choice to alleviate the problem arisen from forest degradation, to reinstate forest system
function and productivity (Kidd and Pimentel, 1992). Forest tree such as timber has a lot of benefits and
has been long used by human for many purposes. It has been used as sawn timber, construction
materials, fodder, fuel wood, shelter and medicine. Conventionally, forest tree plantations use planting
material from wild-type tree. Today, biotechnology allows selection of genetically good traits tree that
gives better quality and higher number of yields. Obviously, forest tree plantation has a major role to play
in the long term timber production strategy.
Inter simple sequence repeats (ISSR) are DNA fragments with length of about 100 to 3,000 bp located
between adjacent, oppositely oriented microsatellite regions (Zietkiewicz et al., 1994). ISSR marker is
chosen in the present study due to its main property that no sequence information is required, thus give a
strong advantage to this markers. Furthermore, ISSRs are randomly distributed throughout the genome. It
permits detection of polymorphism in microsatellites and inter microsatellites loci without previous
knowledge of the DNA sequence (Gupta et al., 1994). ISSR produces informative loci which is suitable to
discriminate closely related genotype variants (Roose et al., 1997). The ISSR technique uses primers that
are complimentary to a single SSR (Zietkiewicz et al., 1994). The amplicons generated consist of regions
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between neighbouring and inverted SSRs. As a result, the high complex banding pattern obtained will
often differ greatly between genotypes of the same species.
Duabanga moluccana or locally known as Sawih is one of the potential plantation tree species to be
planted in Sarawak due to its fast growing properties and high commercial value in wood industry. Apart
from making boxes and firewood, they are also numerously used for house and boat building. Additionally,
D. moluccana is suitable for interior paneling, matches, moulding and pulping (CIRAD, 2003). Plantation
of D. moluccana has become an important management strategy in rehabilitation activities such as the
planting of the trees in the attempt of re-establishing and enhancing forest structure and diversity.
Therefore, the objective of this study was to determine the genetic diversity and genetic relatedness of D.
moluccana from Niah, Tatau (Bintulu) and Mukah populations using ISSR markers.
2. MATERIALS AND METHODS
Leaf samples of Duabanga moluccana used in this study were collected and preserved using CTAB-NaCl
method from three locations in Sarawak namely, Mukah, Tatau (Bintulu) and Niah. Total genomic DNA
was extracted from leaf samples using a modified CTAB method (Doyle and Doyle 1990). The quality and
quantity of the extracted DNA were estimated spectrophotometrically and verified on a 0.8% agarose gel.
The DNA was then subjected to ISSR-PCR amplifications. Six microsatellite primers, namely, (AC) 10,
(ACC)6G, ACG(GT)7 TGG(AC)7, (GA)8C and (AG)8C were used in this study to amplify the ISSR region.
PCR was carried out using a Mastercycler Gradient PCR (eppendorf, Germany). DNA amplification was
carried out in 25µl reaction volume containing 1 x PCR buffer, 2.5 mM MgCl2, 0.2 mM of each dNTPs, 0.5
U of Taq DNA polymerase (Invitrogen, USA), 10.0 pmol of primer and 2 ng/µl of DNA. The thermal cycling
profile was programmed at 94°C for 2 minutes as initial denaturation step, 40 cycles of 30 seconds at
94°C, 30 seconds at optimum annealing temperatures for each primer. 1 minute at 72°C and final
extension step at 72°C for 10 minutes. The amplified products were then subjected to 2.0% agarose gel
electrophoresis.
Amplified DNA marker bands were scored in a binary manner as either present (1) or absent (0) and
entered into a binary data matrix. The genetic diversity of the populations was then estimated using
POPGENE version 1.32 software (Yeh et al., 1997). To determine the relationship between all D.
moluccana from the three natural populations, a consensus neighbour-joining tree was constructed based
on shared allele distance, DSA (Chakraborty and Jin 1993). The software PowerMarker 3.25 was used to
generate the shared alleles matrix while the neighbour-joining trees were generated using MEGA 4.0.2
(Tamura et al. 2007).
3. RESULTS AND DISCUSSION
The ISSR-PCR was successfully carried out using the six selected primers. All 30 samples from each
population which are Mukah, Niah and Tatau (Bintulu) were successfully screened using all the primers.
The primers revealed a total of 151 different loci in the range of 200 bp to 1,500 bp. The genetic diversity
estimation of the three selected populations from Mukah, Tatau (Bintulu) and Niah was primarily
determined by calculation of Shannon’s diversity index. In the overall analyses of genetic diversity among
the three populations, the mean Shannon’s diversity index was 0.3λ2. Estimation of the Nei’s genetic
diversity and percentage of polymorphic band were 0.256, and 90.1% respectively. In the analysis carried
out within the population, the results indicated that D. moluccana was most diverse in Mukah population
followed by Niah and Tatau (Bintulu) (Table 1).
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Table 1. Genetic diversity of Duabanga moluccana
Parameter
Population
Mukah
Tatau (Bintulu)
Niah
Total/ Average
Sample Size
30
30
30
90
I
0.429
0.362
0.387
0.392
h
0.283
0.233
0.251
0.256
P(%)
88.08
84.77
97.42
90.1
Gst
0.134
Note: I - Shannon’s diversity, h - Nei’s genetic diversity, P(%) - percentage of polymorphic band
In general, comparison to genetic diversity study from other species of tropical rainforest tree, the genetic
diversity measurements obtained for D. moluccana tree in this study were fairly at high level. The
Shannon’s diversity index, Nei’s genetic diversity, and P value in Dalbergia sissoo at species level were
0.418, 0.273 and 89.11%, respectively (Wang et al., 2011). Another result from genetic diversity study of
tropical forest tree, Sonneratia paracaseolaris showed lower genetic diversity indices for Shannon’s
diversity, Nei’s genetic diversity, and percentage of polymorphic band of 0.3501, 0.2241 and 81.37%
accordingly (Li and Guizhu, 2009).
According to Hamrick and Loveless (1989), tropical trees frequently express high levels of genetic
diversity. The genetic structure of a species is affected by a number of evolutionary factors. On the basis
of its life history features, D. moluccana, is a long-lived woody plant and is pollinated by bat which is
nocturnal mammal capable of sustained flight for an effective cross-pollination. Moreover, its seeds have
tails that aid the seed dispersal mechanisms by wind. These characteristics confer a higher possibility of
gene flow occurrences between populations, which is factor that capable of increasing genetic diversity in
natural population.
Figure 1 illustrates the neighbour-joining tree that was generated by MEGA 4.0.2 software to determine
the relationship between all D. moluccana from the three natural forests. In this analysis, each branch was
colour-coded according to which population they belong to. Three main groups were identified based on
the genetic relatedness of D. moluccana from three natural forests in Sarawak. The three groups formed
lines that naturally grouped by population or location, and this indicates that individuals from the same
population are genetically related to each other. This correlates well to the gene differentiation coefficient
(Gst = 0.134) obtained from the present study. If migrants move from their population of origin to another
population area of slightly different characteristics, they may not be well established to the new location
and thus may be less likely than the local individuals to pass their genes on to the next generation
(Cooper, 2000). The well-structured neighbour-joining tree which clearly established the clusters
according to their population supported this theory. The mating system plays a critical role for the
population genetic structure. Although ISSR markers can potentially distinguish many individuals, they
unfortunately do not provide direct information on the mating system due to their dominant inheritance
(Wolfe et al., 1998).
4. CONCLUSIONS
This study was able to provide data on the genetic diversity and distribution of D. moluccana in natural
populations. From the clustering patterns and the genetic relationship obtained, selection for breeding
programmes can be done by capturing genetic diversity from the available gene pool. The result obtained
from this study can be the preliminary information to aid in the conservation and tree improvement of D.
moluccana in the near future.
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Figure 1. Neighbour-joining tree of D.moluccana from three natural forests based on shared allele
distance implemented in the Powermarker program. D. moluccana samples from Mukah were coded in
blue colour, red colour for Tatau and green colour for Niah
Acknowledgments
The authors would like to thank all the lab assistants and foresters involved in this project for their
excellent field assistance in species identification and sample collection. This work is part of the joint
Industry-University Partnership Programme, a research programme funded by the Sarawak Forestry
Corporation (SFC) and the FRGS (MOHE) Grant No. FRGS/01(09)/680/2008(13).
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References
Arnold, M. (1991). Forestry Expansion: A study of technical, economic and ecological factors. Oxford
Forestry Institute Paper No 3. Oxford.
Chakraborty, R. & Jin, L. (1993). Determination of relatedness between individuals by DNA fingerprinting.
Human Biology 65: 875-895
CIRAD forestry Department, (2003). Duabanga. Retrieved August 22, 2010 from French Agriculture
Research Centre for International Development (CIRAD) website: http://tropix.cirad.fr/asia/
duabanga.pdf.
Cooper, M. L. (2000). Random amplified polymorphic DNA analysis of Southern Brown Bandicoot
(Isoodon obesulus) populations in Western Australia reveals genetic differentiation related to
environmental variables. Molecular Ecology, 9, 469–479.
Gupta, M., Chyisys, Romero, J. & Owen. J. L. (1994). Amplification of DNA Markers from evolutionarily
diverse genomes using single primers of simple sequence repeats. Theoretical and Applied
Genetic 89, 998–1006
Hamrick, J. L. & Loveless, M. D. (1989). The genetic structure of tropical tree populations: associations
with reproductive biology. In: Bock, J.H., Linhart, Y.B. (Eds.). The evolutionary ecology of plants.
Westview Press, Boulder, pp. 129–146.
Kidd, C. V. & Pimentel, D. (1992). Integrated resource management: Agroforestry for Development. C. V.
Kidd and D. Pimentel (eds.). Academic Press Inc.
Li, H. & Guizhu, C. (2009). Genetic variation within the endangered mangrove species Sonneratia
paracaseolaris (Sonneratiaceae) in China detected by inter-simple sequence repeats analysis.
Biochemical Systematics and Ecology 37, 260–265
Roose, M. L., Fang, D. Q., Federici, C. T. & Krueger R. R. (1997). Fingerprinting trifoliate orange germ
plasm accessions with isozymes, RFLPs and inter-simple sequence repeat markers. Theoretical
and Applied Genetic 95, 211–219.
Sedjo, R. A. & Lyon, K. S. (1990). The long-term adequacy of world timber supply. Resources for the
Future. Washington DC.
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis
(MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-159
Wang, B.Y., Shi, L., Ruan, Z. Y. & Deng, J. (2011). Genetic diversity and differentiation in Dalbergia
sissoo (Fabaceae) as revealed by RAPD. Genetics and Molecular Research 10 (1), 114-120
Wolfe, A.D., Xiang, Q.Y. & Kephart, S.R. (1998). Assessing hybridization in natural populations of
Penstemon (Scrophulariaceae) using hypervariable inter simple sequence repeat markers.
Molecular Ecology 7, 1107–1125.
Yeh, F. C., Yang, R. C., Boyle, T. B. J., Ye, Z. H. & Mao, J. X. (1997). POPGENE, the User-Friendly
Shareware for population genetic analysis. Molecular Biology and Biotechnology Centre,
University of Alberta, Canada.
Zietkiewicz, E., Rafalski, A. & Labuda, D. (1994). Genome fingerprinting by simple sequence repeat
(SSR)-anchored polymerase chain reaction amplification. Genomics 20,176-183.
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Candida tropicalis IN ETHANOL FERMENTATION AT HIGH TEMPERATURE
1
1
2
1
Nurul Faseeha Binti Zulkiffli, *Cirilo Nolasco-Hipolito, Octavio Carvajal-Zarrabal, Ming Gim Lim,
3
3
1
1
Kohei Mizuno, Yui Morishita, Shafri Bin Semawi and Kopli Bin Bujang
1
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan,
2
Sarawak, MALAYSIA. Biochemical and Nutrition Chemistry Area, University of Veracruz, SS Juan Pablo
3
II s/n, Boca del Río, CP 94294 Veracruz, Mexico. Department of Materials Science and Chemical
Engineering, Kitakyushu National College of Technology, 5-20-1 Shii, Kokuraminami-ku, Kitakyushu 8020985, Japan
*Email: hcnolasco@frst.unimas.my
Abstract
Candida tropicalis ATCCa isolated from rotted pineapple has been shown to grow at temperatures higher
than that reported for this strain (40°C). The objectives of this study were to find-out the effects of the
temperature on the growth and the ability to produce ethanol from this strain. Results showed that the
strain grows well at temperatures between 36 - 42°C in batch fermentation and it was able to survive until
51°C, substantiating that this is a thermotolerant microorganism. SEM studies demonstrated that the
yeast reproduces by multilateral budding, commonly found in the genera Candida. Further experiments in
repeated batch fermentation were performed to investigate the production of ethanol at 36°C. It was
found that C. tropicalis ATCCa can produce 45 g/L ethanol using 100 g/L glucose. In conclusion, C.
tropicalis ATCCa has potential applications for industrial production of ethanol due to its ability to grow at
high temperatures compared to conventional yeast.
High temperatures minimized problems of
contamination maximize the use of substrates and avert the use of chiller systems.
Keywords: Candida tropicalis, thermotolerant yeasts, ethanol, batch and repeated-batch fermentation.
1. INTRODUCTION
Interest in thermophilic or thermotolerant microorganisms has been developed a long time ago. This
mainly due to the increase in most reaction rates, product yield and final product resistance to
degeneration at higher temperatures, therefore these microorganism are very useful for certain industrial
processes (Banat et al, 1998 & Kadam & Schmidt 1997). A lot of research has been done to develop
thermotolerant yeasts which involve various techniques like temperature adaptation, protoplast fusion,
mutagenesis techniques, molecular biology techniques and screening of existing yeast strains. However,
success usings this route was usually limited and the most successful technique is isolation and selection
of strains from nature (Banat et al., 1998). The strains are selected based on the capability of growth at
elevated temperatures while producing ethanol. The use of natural thermotolerant microorganisms brings
a number of advantages in fermentation technology especially in the field of industrial ethanol production.
Candida tropicalis is one of the species of yeast in the genus Candida and has been used mainly for
ethanol production. Previous studies performed by Jefries et al., (1981), Hahn-Hägerdal et al., (1989),
Jamai et al., (2006), Patle and Lal (2008) and Oberoi et al., (2010), involved the use of Candida tropicalis
o
o
o
o
o
at fermentation mild temperatures of 25 C, 30 C, 37 C, 30 C and 35 C, respectively.
In this study, a strain of Candida tropicalis which was naturally isolated from decomposing pineapple has
been used as sample due to its ability to grow at high temperature which is above those previously
reported for this yeast. Based on previous studies done in the Universiti Malaysia Sarawak (UNIMAS
o
o
shows that this strain can grow well up to 42 C and still able to grow at 51 C. The ability of this strain to
grow at elevated temperature has become the main reason to performed more detailed research on
ethanol productivity of this strain.
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The main objective of this study is to investigate the best performance of this Candida tropicalis strain in
the production of ethanol based on the regulated temperature, product concentration, substrate
concentration and time. The main objective is reached through other specific objective which is to
determine maximum ethanol production efficiency of this strain.
This Candida tropicalis ATCCa could bring a number of process advantages in ethanol industrial
production. As claimed by Banat et al., (1998), the advantages of thermotolerant yeast are energy savings
through reduced cooling costs, higher saccharification and fermentation rates, continuous ethanol removal
and reduced contamination. Furthermore, the use of these kinds of microorganisms could increase the
consumption rate of substrate and consequently the product yield. Thus, these advantages induce many
efforts and build interest to seek or develop thermotolerant yeast strains (Gera et al., 1997; Kiran et al.,
2000). On the other hand and from an industrial point of view by using the called repeated batch
fermentation mode the fermentation time process can be shortened, compared to standard fed-batch or
batch processes resulting in a significant increase of the final product yield (Russ et al. 2007).
2. MATERIALS AND METHODS
2.1 Microorganism and culture conditions
Candida tropicalis ATCCa strain was used in the study. This strain was naturally isolated from
decomposing pineapple in UNIMAS, Kota Samarahan, Sarawak. The strain was grown on 20 g/L glucose,
o
5 g/L yeast extract medium added with 1.5% agar at 33 C. Subculture is done every two weeks.
2.2 Inoculum preparation
Active culture for inoculation is obtained in 250 ml of Erlenmeyer flasks with 200 ml of growth medium
containing 40 g/L of glucose and 5 g/L of yeast extract. The pre-culture is grown on a rotary shaker at 150
o
rpm for 12 hours at 33 C. After 12 hours, the pre-culture has been centrifuged at 8000 rpm for 10 minutes
to harvest the cells.
2.3 Fermentation medium
The fermentation medium consisted of autoclaved tap water, glucose and yeast extract. All the medium
were prepared in different bottles. Autoclaved tap water was prepared in 10 Litre bottle, meanwhile
glucose medium was prepared in 5 Litre bottle with the concentration of 300g/L. Yeast extract medium
was prepared with the concentration of 5g/L in 250mL bottle.
2.4 Feeding system
Glucose and autoclaved tap water are pumped into the fermentor for each cycle starting from cycle two.
Medium are prepared initially in the fermentor for the first cycle with the volume of 1600mL. The remaining
volume are for the inoculum (20%) which is 400mL. Density for glucose are calculated to know the exact
volume needed to pump into the fermentor. Feeding for glucose concentration medium has been set for
each cycle which are 30g/L for the first cycle until third cycle, 60g/L for the fourth cycle until seventh cycle,
and 100g/L for cycle eight until cycle ten.
2.5 Repeated-batch fermentations system
Repeated-batch fermentations are performed in a 3 L fermentor. The working total volume of the
o
o
fermentation is 2 L. The temperature was regulated at 42 C for the first three hour and 36 C for the next
hours. The fermentations are carried out without pH control. The agitation rate is fixed at 200 rpm. The
optical density, the temperature, the agitation, the pH, the carbon dioxide is monitored on line by
computer. The initial optical density (OD) is standardized at 0.04.
During the fermentation, 20 ml of broth sample is removed every 3 hours in order to determine colony
forming unit (CFU/ml), glucose consumption and ethanol concentration. The samples are centrifuged at
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8000 rpm for 10 minutes to remove cells. The supernatant were filtered through 0.45µm filter membrane.
o
The crude filtrate will be kept at 4 C until further analysis. Sampling also being done at five different OD
which are 0.1, 0.2, 0.3, 0.4 and 0.5 to determine dry cell weight (DCW). For DCW, 30ml sample are taken.
Standard curve for dry cell weight is plotted based on the data obtained.
2.6 Analytical procedures
Ethanol and glucose residual concentrations in the culture broth have been analysed by HPLC system.
The production of carbon dioxide is measured by an online standard mass flow-meter Mode; 3660 Series
(Kofloc, Tokyo, Japan). Dry cell weight is calculated by reference to a standard curve of cell mass versus
absorbance. The time course of ethanol production, residual glucose, pH changes and cell population
growth has been determined. The parameters of temperature, pH, OD, gas production, and agitation have
been monitored on-line to determine the effect of the temperature on ethanol production by Candida
tropicalis. DNS method also is used to check for the residual glucose concentration at each sampling
hour.
2.7 Statistical analysis
The results are subjected to statistical analysis using ANOVA to compare whether there are significance
differences or not between the experimental treatments. SPSS software is used to analyse all the data.
Excel also will be used to perform kinetic analysis and plotting graph.
3. RESULTS AND DISCUSSION
3.1 Effects of temperature on growth of Candida tropicalis ATCCa
Figure 1 showed the effects of temperature on biomass production of Candida tropicalis ATCCa strain. As
the temperature increased, length of fermentation also increased. The vast literature on yeasts shows that
temperature is one of the important physical parameter which influence yeast growth. Hence, the vast
different of maximal fermentation time is due to the effects of fermentation temperature where higher
temperature will reduce the fermentation efficiency. As stated by Shin et al., (2001), the usual optimum
growth temperature for most yeast is ranging from 25-30°C. However, this strain of Candida tropicalis was
able to grow at non-conventional temperature which could contribute to some advantages to the industry
o
of alcohol production. According to the results obtained, the ability of this strain to grow at 42 C and able
to ferment sugar to produce ethanol can be considered as thermotolerant yeast (Slapack & Ingledew,
1988).
Figure 1 Effects of fermentation
temperature on biomass production.
Simbols: () 33°C, () 36°C, ()
39°C, () 42°C, 5°C.
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Figure 2 is the graph of specific growth rate plotted to express the growth rate, substrate consumption rate
o
and product formation rate. Since fermentation at 42 C showed the one of best figure for specific growth
o
rate, fermentation temperature of 42 C was chosen for repeated-batch fermentation to test for the ethanol
productivity. It was observed that strain is able to grow at different temperature although the growing was
affected by decreasing the specific growth rate.
Figure 2 Specific growth rate at
different temperature. Simbols: ()
33°C, () 36°C, () 39°C, ()
42°C, 5°C.
3.2 Biomass and ethanol productivity of Candida tropicalis ATCCa
The biomass production during the repeated batch fermentation (RBF) is showed in the Figure 3. It was
observed from the figure 3, that the biomass production is not affected using 42°C as in the cycle 1, and
36°C as from the cycle 3 to 10. Therefore, there is a good margin to play with the temperature to control
the contamination of unwanted microorganism. As the fermentation period increased, biomass also
increased concordantly with the consumption rate of glucose. Hence, different concentration of glucose
had been applied at different cycles. Initially, low concentration of glucose 30 g/L had been applied in the
RBF (1st, 2nd, 3rd, and 4th cycles of fermentation). The glucose concentration increase into 40 g/L,
50g/L, 60 g/L, 90 g/L during 5th, 7th, 6th, and 8th cycles of fermentation respectively. Next, the
concentration of glucose increases into 100 g/L for the 9th and 10th cycles of fermentation.
Figure 3 Biomass production
during
the
Repeated
Batch
Fermentation using hydrolyzed
sago starch.
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The initial glucose, residual glucose, ethanol production and other by-product concentration were
recorded (Table 1). Increasing the biomass concentration of Candida tropicalis can be achieved by
lowering the temperature of fermentation. At this point, fermentation temperature had been decreased
from 42°C into 36°C during 3rd cycle and the subsequent cycles of fermentation. As showed in the table
1, the glucose uptake efficiency increased from the 3rd cycle of fermentation until 9th cycle of
was obviously decreased up to 0.03 hefficiency 89%. Similar result also performed by Ton & Lee, 2011 in which the glucose uptake rate are
high for the immobilized yeast increase for the first several cycles and started to reduce at the end of their
RBF cycle (10th cycles).
The fermentation performed at 36°C, still is considered as thermophilic process since very few yeast have
the ability to grow and even produce ethanol. This way could reduce the risk of contamination and
increase the substrate consumption efficiency. RBF has been successfully performed until cycle 10 with
100g/L glucose concentration. Lower glucose concentration applied for the first 3 cycle which was 30g/L.
This is done to induce the increment of cells density and subsequently increase the productivity. One mole
of glucose was converted into 2 mole of ethanol and 2 mole of carbon dioxide (CO 2). The CO2 dissolved in
the liquid made the liquid fizzy. However, other by-products were produced concomitantly with ethanol
and CO2. During anaerobic fermentation, the major products were essentially ethanol following with other
minor products such as lactic acid, formic acid, glycerol and acetic acid. HPLC analysis revealed that
mainly 2 types of by-products which are lactic acid and acetic acid were table 1. Energy also released
from the ethanol fermentation in form of heat. Another by-product during the ethanol fermentation is
biomass of Candida tropicalis.
Table 1. Productivity of the repeated batch fermentation during ten cycles using Candida tripicalis in
hydrolyzed sago starch. The data are the results obtained at the end of each fermentation cycle.
Cycle
Time
(h)
DCW
(g/L)
Glucose
(g/L)
Ethanol
(g/L)
Lactic Acid
(g/L)
Acetic Acid
(g/L)
(h )
1
2
3
4
5
6
7
8
9
10
0
25
73
121
144
163
189
213
247
284
0.7±0.23
1.1±0.01
1.7±0.01
2.3±0.23
2.6±0.20
4.3±0.16
5.6±0.10
7.5±0.57
8.2±0.17
6.7±0.71
4.36±0.96
22.32±0.11
2.50±0.08
0.42±0.084
0.08±0.00
0.54±0.03
0.14±0.03
0.88±0.68
3.12±0.11
11.64±0.4
4.27
3.12
11.39
10.07
15.73
26.44
20.38
40.61
45.31
41.09
0.727
0.33
2.55
2.30
2.50
3.21
2.42
4.68
4.26
5.05
0.00
0.00
0.97
0.62
0.62
0.49
0.44
0.62
0.59
0.68
0.560
0.130
0.100
0.061
0.240
0.051
0.043
0.045
0.048
0.030
max
-1
The production of lactic acid during the repeated batch fermentation was increasing with the cycles
performed as the biomass concentration increased. The maximum lactic acid produced was 45 g/l. This
concentration of lactic acid was achieved when the initial glucose concentration was 106 g/l. These data
suggested that this strain could be able to stand high glucose concentration to metabolize it at a
temperature as high as 36°C.
Increasing the biomass concentration of Candida tropicalis can be achieved by lowering the temperature
of fermentation (Milkesa, 2009). At this point, fermentation temperature had been decreased from 42°C
into 36°C during 3rd cycle and the subsequent cycles of fermentation. As showed in the table 1, the
glucose uptake efficiency (as ethanol production) increased from the 3rd cycle of fermentation until 9th
strain was obviously decreased up to 0.03 hglucose
uptake efficiency 89%. Similar result also performed by Ton & Lee, 2011 in which the glucose uptake rate
are high for the immobilized yeast increase for the first several cycles and started to reduce at the end of
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their RBF cycle (10th cycles). Under the observation from the microscope, most of the Candida tropicalis
showed brownish and thick on its membrane due to ageing. Furthermore, some of the Candida tropicalis
was subjected to cytolysis to exposure its organelles such as mitochondrion, nucleus and so on. Based on
the observation, we can conclude that Candida tropicalis at batch fermentation can repeat 9 times
continually without loss of any fermentation ability.
4. CONCLUSION
In conclusion, Candida tropicalis ATCCa has potential applications for industrial production of ethanol due
to its ability to grow at high temperatures compared to conventional yeast. High temperatures minimized
problems of contamination maximize the use of substrates and avert the use of chiller systems.
References
Banat, I. M., Nigam, P., Singh, D., Marchant, R., & Mchale, A. P. (1998). Review: Ethanol production at
elevated temperatures and alcohol concentrations: Part I- Yeasts in general. World Journal of
Microbiology & Biotechnology, 14, 809-821.
Gera, R., Dhamija, S. S., Gera, T., & Singh, D. (1997). Intergenic ethanol producing hybrids of
thermotolerant Kluyveromyces and non–thermotolerant Saccharomyces cerevisiae. Biotechnology
Letters, 19, 189–193.
Hahn- Hägerdal, B., Lohmeier-Vogel, E., Skoog, K., & Vogel, H. (1989). Nuclear magnetic resonance
study of the effect of azide on xylose fermentation by Candida tropicalis. Applied and
Environmental Microbiology, 55,8, 1974-1980.
Jamai, L., Ehayebi, K., Yamani, J. E., & Ehayebi, M. (2006). Production of ethanol from starch by free and
immobilized Candida tropicalis in the presence of α-amylase. Bioresources technology, 98, 2765–
2770.
Jeffries, T. W. (1981). Conversion of xylose to ethanol under aerobic conditions by Candida tropicalis.
Biotechnology Letters, 5, 213–218.
Kadam, K. L., & Schmidt, S. L. (1997). Evaluation of Candida acidothermophilum in ethanol production
from lignocellulosic biomass. Applied Microbiology Biotechnology, 48, 709-713.
Kiran, S. N., Sridhar, M., Suresh, K., Banat, I. M., & Venkateswar, L. (2000). Isolation of thermotolerant,
osmotolerant, flocculating Saccharomyces cerevisiae for ethanol production. Bioresource
Technology, 72, 43 – 46.
εilkesa, T. (200λ). Evaluation of yeast biomass production using molasses and supplements. (εaster’s
thesis). Retrieved from Faculty of Science, Addis Ababa University.
Oberoi, H. S., Vadlani, P. V., Briwaji, K., Bhargav, V. K., & Patil, R. T. (2010). Enhanced ethanol
production via fermentation of rice straw with hydrolysate-adapted Candida tropicalis ATCC
13803. Process Biochemistry, 45, 1299–1306.
Patle, S., & Lal, B. (2008). Investigation of the potential of agro-industrial material as low cost substrate for
ethanol production by using Candida tropicalis & Zymomonas mobilis. Biomass Bioenergy, 32,
596–602.
Russ, K., Schlenke, P., Huchler, H., Hofer, H., Kuchenbecker, M., & Fehrenbach, R. (2007). Repeated
batch fermentation using a viafuge centrifuge. Cell Technology for Cells Products, 10, 523-526.
Ton, N.M.N., & Lee, V. V. M. (2011). Application of immobilized yeast in bacterial cellulose to the repeated
batch fermentation in wine-making. International Food Research Journal. 18(3), 983-987.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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REMOVAL OF STARCH FROM STARCH SOLUTIONS BY TANGENTIAL FLOW FILTRATION
1
1
2
1
Samantha Siong Ling-Chee, *Cirilo Nolasco-Hipolito, Octavio Carvajal-Zarrabal, Kopli Bujang
3
and Esaki Shoji
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan,
2
Sarawak, MALAYSIA. Biochemical and Nutrition Chemistry Area, University of Veracruz, SS Juan Pablo
II s/n, Boca del Río, CP 94294 Veracruz, Mexico; Kurume National College of Technology, 1-1-1
Komorino, Kurume-shi, Fukuoka 830-8555 Japan
*Email: hcnolasco@frst.unimas.my
Abstract
In the sago industries, the effluent is discharged into nearby waterways and eventually resulting water
pollution, and this has become problematic due to the absence of proper water treatment in this industry.
Tangential flow filtration is an attractive alternative for treatment of sago effluent. The aim of this study is
to clean the sago effluent by tangential flow filtration and to improve performance of the membrane by
reducing its fouling. Preliminary studies using 1.0 % sago starch solution as model effluent was tested for
2
starch removal using polysulfone membrane filter cassettes (0.45µm; 0.1 or 0.2m ). Fifty litres of starch
solution was filtered and concentrated to a factor of 3.3 and 5 (final volume at 15 and 10L, respectively).
2
2
Increasing the filtration area (from 0.1 to 0.2m ) improved the membrane performance from 35.4 L/h.m to
2
446.7 L/h.m and the process time was reduced from 11 to 0.5 h. The permeate obtained was free of
sedimentable and total suspended solids and turbidity was not detected. The COD showed a significant
difference (p < 0.05) before and after treatments. The next step in this research will be to test the sago
effluent from sago mills in situ.
Keywords: Sago starch, sago effluent, tangential-flow filtration, filtration flux, back-flush
1. INTRODUCTION
Sago Palm (Metroxylon spp.) is an essential source of starch and it provides a staple diet for the
populations in Sarawak, Malaysia (Phang et al., 2000). Sarawak is the world’s largest sago exporter
because Anually Sarawak exports up to 40,000 tons sago per year due to the largest swamps and
peatlands with estimated area of 19,270 hectares for sago palm in Sarawak (Abd-Aziz, 2002; W.
Mohamad Daud et al., 2010). Eventually, large amount of sago waste produced from the sago starch
factory is discharged into the river nearby (Petrus et al., 2009). The sago effluent discharged from the
factory is likely to cause contamination of our water supply as well as its effect to the environment (Savitha
et al., 2009). There are several reports on treating the sago wastewater by using several methods such
as anaerobic treatment (Savitha et al., 2009), aerobic (Rashid et al., 2010), supplement for media
formulation (Senthilkumar et al., 2011), conversion to biodiesel by algae cultivation (Phang et al., 2000),
and also used as animal feedstock (Chanjula & Ngampongsai, 2009).
The technologies of membrane filtration such as microfiltration (MF), ultrafiltration (UF), nanofiltration (NF)
and reverse osmosis (RO) have been widely used in dairy and food industry as well as in industrial
wastewater treatment since the past four decades (Mappelli et al., 1977; Glover, 1985; Bouhabila et al.,
1998; Sima et al., 2011). Microfiltration like tangential flow filtration (TFF) has been practically used in
separating the cell protein (Reis et al., 1991), plankton (Giovannoni et al., 1990), RNA (Eon-Duval et al.,
2003) and organic colloid (Gueguen et al., 2002). However, membrane fouling has become the main
problem faced by most researchers because of the increasing of maintenance and operating costs.
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Many studies on membrane fouling by sorts of chemical compounds such as starch (Singh & Cheryan,
1997; Shukla et al., 2000), protein (Chan & Chen, 2004), organic and inorganic matters (Pierre et al.,
2006), and also microorganisms like bacteria (Meng et al., 2006) have been done. As a result, several
fouling control techniques have been discovered by the researchers to overcome the problem. So far,
there is no report on treatment of the sago effluent by using tangential flow filtration process. Furthermore,
there is no wastewater treatment plant has been installed in factory producing sago. Consequently, the
river is exposed to high risk of health hazard due to serious water contamination. Therefore, it is more
beneficial if converting some wastes into some useful products such as clean water and animal feeds
instead of discharging the effluents without treatment into the water body.
The purpose of this study is to remove the starch compound from a starch solution using tangential flow
filtration process, aimed at reducing the turbidity, total suspended solids (TSS) and chemical oxygen
demand (COD) in water.
2. MATERIALS AND METHODS
2.1 Model Solution
Working volume of 50 L of 1.0% and 3.0% (w/v) starch solutions were used to test the performance of the
membrane to remove the starch in the solution.
2.2 Tangential Flow Filtration (TFF)
The starch solution was drawn using a peristaltic pump Masterflex I/P precision brushless pump system
which provides 8 LPM maximum flow (Figure 1). The polysulfone membrane filter cassettes with pore size
2
2
of 0.45 µm and the area of 0.1 m or 0.2 m was used in this filtration system. The speed of pump system
used was 50% while 30% for back-flushing.
3
TFF
1
50 L starch
solution
Pump
motor
2
Permeate
Key:
Valve
Figure 1 Tangential flow filtration system setup. Number one ( ) dominates feed route, number two (
indicates permeate route and number 3 ( ) dominates retentate route.
)
As shown in Fig. 1, the starch solution was pumped via route 1 and filtered by tangential flow filtration
(TFF) system. The retentate containing starch compound which unable to cross the membrane pores in
time during the filtration was flowed back to the original 50 L tank through route 3. Permeate produced
from TFF run though route 2 was collected in a clean tank.
For back-flush process, the permeate was used as clean water to wash the membrane by reversing the
flow in terms of maintaining the performance for filtering.
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2.3 Experimental Design
2.3.1 Experiment A
2
In the first experiment, 3.0% starch solution was filtered using a 0.1m cassette membrane. The solution
o
was well-agitated at room temperature (25±1 C). The filtration process with and without back-flushing
were conducted. The flow was reversed every 30 minutes. The starch in the storage tank was
concentrated until 20-30% of the original volume.
2.3.2 Experiment B
2
Second experiment was involved 0.2m of membrane area to filter 1.0% starch solution. As previously
o
described, 50 L of working volume was agitated well at room temperature (25±1 C). The filtration process
without back-flushing was tested. The starch in the storage tank was concentrated until 20-30% of the
original volume.
2.4 Membrane Flux Tests
The performance of membranes in Experiment A and B were evaluated by measuring the volume of
permeate produced in total process time of filtration. The total surface area of membrane filter cassette
was considered. Filtrate fluxes were acquired by using the formula shown below (Adham et al., 2006). The
2
unit of filtrate flux is δ/h•m .
Where,
2
J = filtrate flux (δ/h•m );
Q = filtrate flow (L/h);
2
S = membrane surface area (m ).
2.5 Water Analysis
The water samples were collected, measured and analyzed before and after the treatments as well as the
samples from clean water produced for dissolved oxygen (DO), pH, temperature, turbidity, total
suspended solids (TSS) and chemical oxygen demand (COD) in accordance with Standard Methods
(APHA, 2005). Water analyses were done by triplicate.
2.6 Statistical Analysis
The means of data were compared using analysis of variance (ANOVA) to test the significant differences
(p = 0.05) between treatments. All data analysis was conducted using SPSS (PASW) statistics version
19.0.
3. RESULTS AND DISCUSSION
3.1 Filtrate Fluxes
The results of the effect of wastewater treatment using different operating conditions for the experiments A
and B, such as initial starch concentration, membrane surface area and back-flushing is shown in Table 1.
Generally, an increase in starch concentrations in a solution results in a decrease in the permeate flux.
2
The initial flux rate in the Experiment A (without back-flush) at 0 minute was 8.02±5.82 L/min•m and the
2
flux declined up to 2.49±0.64 L/min•m . Previous tangential flow filtration studies also have demostrated
that permeate flux decreased sharply within 30 minutes during filtration (Choi et al., 2005). The flux
decreased over time was mainly due to the starch compounds gradually clogged and deposited in the
membrane pores during operation. Thus, membrane permeability decreased during operation as the
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filtration process proceeded (Ognier et al., 2002). In addition, membrane fouling also resulted in a gradual
increasing in the pump pressure. However, the filtration flux was improved slightly after the application of
back-flushing. The results in Experiment A showed that at least an improvement of 45% on filtration flux
2
2
was obtained when operated with back-flush, that is from 24.3 δ/h•m to 35.4 δ/h•m (Table 1). There
were no significant different at p > 0.05 between the means of fluxes for each condition of Experiment A.
Even filtrate flux on the first 30 minutes after back-flushed had showed slightly improvement, that is from
2
2
2.49±0.64 L/min•m increased to 2.85±0.45 L/min•m .
Besides that, the process time had also been reduced to 11 hours at constant concentration factor, 3.3 if
compared to Experiment A without back-flush which the total process time taken was 16 hours. This was
because the starch particles blocked in the membrane pores were removed partially by flowing backwards
from the membrane and hence, increased the efficiency of water filtration. Seventy percent (70%) of water
was able to be removed from the solution after the final treatment.
Table 1 Comparisons of filtrate fluxes of Experiment A and B under different operating conditions.
Experiment
A
B
Operating Condition
Not back-flushed
Back-flushed
Non back-flushed
3.0
3.0
1.0
Membrane surface area (m )
0.1
0.1
0.2
Final volume (L)
15
15
10
16
11
0.5
Filtration flux (δ/h•m )
24.3
35,4
446.7
Concentration factor
3.3
3.3
5
Water removal (%)
70
70
80
Initial starch concentration (%)
2
Process time (h)
2
2
In contrast, Experiment B produced much higher flux (446.7 δ/h•m ) than Experiment A which is only 242
35 δ/h•m by at most 13-fold (Table 1) while the permeate fluxes at 0 and first 30 minutes were 8.86
2
2
L/min•m and 6.66 L/min•m , respectively, which is higher than Experiment A as previously described. The
means of fluxes of Experiment A and B were significant different (p > 0.05). This was due to the larger
2
membrane surface area (0.2 m ) and also lower initial concentration of starch (1.0%). Starch with low
concentration resulted in a lower fouled membrane when compared to 3.0% starch concentration.
Moreover, increased of membrane surface area tend to create more volume of clean water. These may be
the reasons why 80% of water had been removed from 50 L starch solution within 30 minutes and the
concentration factor was 5 (Table 1).
These results showed that rapid removal of large amount of water from a solution within a shortest time
and capable to compact the starch content could reduce the starch transporting costs as well as the
operating costs by saving energy. In addition, reduced in filtration process time also would avoid heavy
starch fermentation. However, further investigations on water treatment using membrane filter cassette
2
with 0.1m of surface area for filtering 1.0% starch solution is required. On the other hand, the filtration flux
in Experiment B could be improved by increasing the transmembrane pressure which could results in less
problem of membrane fouling.
3.2 Water Analyses
In both experiments, the most variables were turbidity, total suspended solids (TSS) and chemical oxygen
demand (COD). Dissolved oxygen (DO) values for both experiments fall in the range of 6-8 mg/L and pH
values were within the range of 6.5-7. The mean difference of temperature, pH and DO between before
and after treatment including permeate were not significant at 0.05 level.
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Table 2 Turbidity, TSS and COD values
mean ± S.D. of triplicates.
Experiment
A treatment
No back-flush
Operating
Before
Condition
Permeate
(0 min)
Turbidity
2876.67
5.17±0.38
(NTU)
±105.04
31683.33
TSS (mg/L)
0
±423.03
33806.12
COD (mg/L)
0
±451.38
of water samples in Experiment A and B. Data are expressed as
Back-flush
Before
(0 min)
3283.33
±254.23
23760
±243.31
25351.92
±259.61
Permeate
5.40±1.7
0
0
B treatment
No back-flush
Before
After
(0 min)
(30 min)
592.67
3110
±15.28
±236.43
9445
40633.33
±351.71
±448.84
11082.57
47691.34
±412.74
±524.28
Permeate
5.20±0.1
0
0
In Experiment A, before the starch solution was treated without back-flushing, the turbidity of the model
solution was 2876.67±105.04 NTU; total suspended solids (TSS) was 31683.33±423.03 mg/L; and
chemical oxygen demand (COD) value was 33806.12±451.38 mg/L (Table 2). After the solution was
filtered, the turbidity of water had decreased to 5.17±0.38 NTU. Whereas for back-flush condition,
turbidity, TSS and COD values obtained were 3283.33±254.23 NTU, 23760±243.31 mg/L and
25351.92±259.61 mg/L, respectively (Table 2). Meanwhile, the cloudiness of clean water produced was
significantly low (5.40 ±1.7 NTU) as compared with water before treatment.
For Experiment B, the turbidity of the solution before treatment was 592.67±15.28 NTU; TSS was
9445±351.71 mg/L; and the COD value was 11082.57±412.74 mg/L (Table 2). The results showed that
these values increased drastically after the final treatment (turbidity: 3110±236.43 NTU; TSS:
40633.33±448.84 mg/L; COD: 47691.34±524.28 mg/L) (Table 2) due to high removal of water at 80%
(Table 1) and hence, increased the concentration of starch in the final volume of solution. Theoretically,
increases in starch concentration could lead to an increase in COD value due to the its degradation.
Therefore, the final COD reading was significantly high, that is 47691.34±524.28 mg/L. However, the
starch was separated from solution and thus, reduced the turbidity of water to 5.20±0.1 NTU.
In the meantime, zero values were obtained for TSS and COD in both experiments, regardless the
operating condition. These results proved that the starch had been removed effectively from the solution
after the water was treated. There were highly significant difference (p < 0.05) of turbidity, TSS and COD
values between before and after treatment including permeate.
4. CONCLUSIONS
The present study demonstrated that the starch compound could be removed from starch solution by
using tangential flow filtration (TFF) system. The results showed that the treated water (permeate) had a
very low turbidity and none for TSS and COD values. Low filtration flux due to decreased of membrane
permeability could be improved by applying back-flushing in the membranes. Furthermore, the flux also
could be improved with increased of membrane surface area. Shorten the filtration process time would
save maintenance, operating and transport costs. It is also recommended to increase the speed of pump
system if the initial pressure is low so that high volume of permeate could be produced and therefore,
increases the filtrate flux within a shorter time and also reduces the membrane fouling.
References
Abd-Aziz, S. (2002). Sago starch and its utilisation Journal of Bioscience and Bioengineering. 94(6): 526529.
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Wastewater (21 ed.). Washington DC: American Public Health Association.
Adham, S., Chiu, K.P., Lehman, G., Mysore, C. & Clouet, J. (2006). Optimization of Membrane Treatment
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Bouhabila, E.H., Aim, R.B. & Buisson, H. (1998). Microfiltration of activated sludge using submerged
membrane with air bubbling (application to wastewater treatment). Desalination. 118(1-3): 315322.
Chan, R. & Chen, V. (2004). Characterization of protein fouling on membranes: Opportunities and
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Chanjula, P. & Ngampongsai, W. (2009). Effects of sago palm pith as replacement for corn grain on
intake, rumen fermentation characteristics and microbial N supply of cattle fed paspalum
plicatulum hay. Asianaustralasian Journal of Animal Sciences. 22(3): 378-387.
Choi, H., Zhang, K., Dionysiou, D.D. Oerther, D.B. & Sorial, G.A. (2005). Effect of permeate flux and
tangential flow on membrane fouling for wastewater treatment. Separation and Purification
Technology. 45(1): 68-78.
Eon-Duval, A., MacDuff, R.H., Fisher, C.A., Harris, M.J. & Brook, C. (2003). Removal of RNA impurities by
tangential flow filtration in an RNase-free plasmid DNA purification process. Analytical
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Giovannoni, S.J., DeLong, E.F., Schmidt, T.M. & Pace, N.R. (1990). Tangential flow filtration and
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Glover, F.A. (1985). Ultrafiltration and Reverse Osmosis for the Dairy Industry. National Institute of
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Gueguen, C., Belin, C. & Dominik, J. (2002). Organic colloid separation in contrasting aquatic
environments with tangential flow filtration. Water Research. 36(7): 1677-1684.
Meng, F., Zhang, H., Yang, F., Li, Y., Xiao, J. & Zhang, X. (2006). Effect of filamentous bacteria on
membrane fouling in submerged membrane bioreactor. Journal of Membrane Science. 272(1-2):
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Mappelli, P., Santori, M., Chiolle, A. & Gianotti, G. (1977). Membrane processes used for the treatment of
industrial effluents. Desalination. 24(1-3): 155-173.
Ognier, S., Wisniewski, C. & Grasmick, A. (2002). Characterisation and modelling of fouling in membrane
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Petrus, A.C., Ahmed, O.H., Muhamad, Ab M.N., Nasir, H.M., Jiwan, M. & Banta, M.G. (2009). Chemical
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Phang, S.M., Miah, M.S., Yeoh, B.G. & Hashim, M.A. (2000). Spirulina cultivation in digested sago starch
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Pierre, L.C. Chen, V. & Fane, T.A.G. (2006). Fouling in membrane bioreactors used in wastewater
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FATTY ALCOHOLS: BIOMARKER OF ORGANIC MATTER IN PORT DICKSON, NEGERI SEMBILAN
Norfariza Humrawali, Siti Norzulaiha bt Mat Jusoh & Masni Mohd Ali*
School of Environment and Natural Resources Sciences, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor
*Email: masni@ukm.my
Abstract
This paper presents the composition of the fatty alcohols in surface sediments obtained from Port Dickson,
Negeri Sembilan. A total of 18 surface sediment samples were extracted and analysed using Gas
Chromatography-Mass Spectrometry (CG-MS). The results show that by using quantification of fatty
alcohols, it clearly defined that the study area was dominated by marine derived compounds. Short-chain
fatty alcohols constitute 63% of total fatty alcohols, followed by long-chain compounds (25%) while the
least of all was branched compounds (12%). Other than that, the samples were also found to have high
concentrations of phytol even though the compound can originate from both terrestrial plants and marine
phytoplankton. The results obtained based on the short-chain/long-chain fatty alcohol ratio and Alcohol
Source Index (ASI) calculated revealed the high abundance of marine derived fatty alcohols. Therefore,
phytol that was present at the study area might have originated from marine sources. It can be concluded
that the evaluation of fatty alcohols suggests the input of various sources in the study area, but was
mainly dominated by marine sources.
Keywords: fatty alcohol, phytol, surface sediment
1. INTRODUCTION
This paper describes fatty alcohols as biomarkers of organic matter in the aquatic environments. Fatty
alcohol is one of the lipid compounds that are normally used as lipid biomarkers to differentiate the input
of organic matter derived from various sources especially from the marine, terrestrial and bacterial activity
(Mudge & Norris 1997;Mudge & Seguel 1999). Fatty alcohol is a hydrophobic compound, thus it tends to
be adsorbed by sediments with higher organic matter content (Froehner et al. 2008). The compound has a
long residence time in the environment and does not degrade over a short period of time (Mudge & Duce
2005). These characteristics therefore endow it as a promising biomarker in the environment.
Generally, fatty alcohols can be categorized into three main groups, namely short chain fatty alcohols
(≤C20), long chain fatty alcohols (≥C21) and branched chain fatty alcohols (-iso and –anteiso). Each
category represents different main sources. Short chain fatty alcohols are significant with input derived
from marine organisms such as plankton (Mudge & Norris 1997; Treignier et al. 2006; Volkman et al.
1999). In contrast, long chain fatty alcohols can be derived from terrestrial plants (Bechtel & Schubert
2009; Mudge & Norris 1997; Treignier et al. 2006), while the branched compounds are formed from
bacterial activities toward the straight chain fatty alcohols (Mudge & Duce 2005; Mudge & Norris 1997).
There is one more compound, namely phytol, which is also used as lipid biomarker, and grouped together
with the long chain fatty alcohols as the sources of both compounds are the same (Mudge et al. 2008).
The aims of this study are 1) to quantify the type of fatty alcohols, 2) to determine the concentrations of
these compounds present in the samples taken from Port Dickson and 3) to figure out the most probable
sources of each compound in the study area.
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2. METHODOLOGY
2.1 Study area
A total of eighteen sampling stations were established along the coastal area of Port Dickson, Negeri
Sembilan (Figure 1 and Table 1). Surface sediment was collected from each of these sampling stations
using a PONAR grab, and samples were stores in a freezer at <0°C whilst awaiting further analysis.
Figure 1 Sampling stations at the study area
Table 1. Coordinate of the sampling stations
Station
Coordinate
PD1
N 02º 32.160’
E 101º46.78λ’
PD2
N 02º 31.714’
E 101º47.084’
PD3
N 02º 31.201’
E 101º47.648’
PD4
N 02º 31.020’
E 101º47.48λ’
PD5
N 02º 30.λ85’
E 101º4λ.121’
PD6
N 02º 30.628’
E 101º48.858’
PD7
N 02º 2λ.3λ0’
E 101º50.260’
PD8
N 02º 2λ.061’
E 101º4λ.λ58’
PD9
N 02º 28.3λ3’
E 101º50.663’
Station
PD10
PD11
PD12
PD13
PD14
PD15
PD16
PD17
PD18
Coordinate
N 02º 28.182’
N 02º 27.367’
N 02º 27.300’
N 02º 25.243’
N 02º 24.λ83’
N 02º 24.λ53’
N 02º 23.678’
N 02º 23.100’
N 02º 23.531’
E 101º50.373’
E 101º51.122’
E 101º50.83λ’
E 101º54.310’
E 101º54.624’
E 101º56.516’
E 101º56.257’
E 101º57.274’
E 101º58.251’
2.2 Fatty alcohol extraction
The methods used for preparation and analysis followed the extraction procedures given in the literature
of Mudge & Norris (1997) and Masni & Mudge (2006). Approximately 30-40 g wet weight of sediment was
hydrolyzed with 50 mLl of 6% potassium hydroxide in methanol. The samples were refluxed for 4 hrs and
centrifuged at 2500 r.p.m for 5 minutes. The supernatant was then funneled into a separating flask.
Non-polar lipids were extracted from the supernatant by liquid-liquid separation. A total of 20 mL of
hexane and 10 mL of double distilled water were added to the supernatant. The mixture was then shaken
vigorously. After being shaken, the cap of the separating flask was loosened to release the pressure
inside. The non-polar fraction was collected and transferred into a florentine flask. The whole procedure
was repeated to ensure maximum extraction. Samples were evaporated at 40˚C in a rotary evaporator,
redissolved in 2-3 mL of hexane and then transferred into a 14 mL vial. Anhydrous sodium sulphate was
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added to remove any water and polar compounds left in the samples. The remaining solution was filtered
through filter paper and blow-dried under oxygen free nitrogen (OFN).
Sample derivatisation had to be undertaken in order to permit the analysis of compounds with the Gas
Chromatograph (GC). Approximately 2-3 drops of bis-(trimethylsilyl) triflouroacetamide (BSTFA) were
added to the samples and then heated in a heating block for 10 minutes at 60˚C. Finally, they were
evaporated to dry under OFN and then redissolved in 1 mLof hexane.
A computerized gas chromatography-mass spectrometry (GC-MS) (Perkin Elmer Clarus 500) was used to
analyze the fatty alcohols in the samples. The programmed temperature started at 80˚C, increasing at
-1
-1
15˚C min to 300˚C, then at 5˚C min to a maximum of 350˚C for 10 minutes. Calibration was carried out
using an octadecanol-TMS solution in order to quantify the peaks obtained from the analysis. All results
are relative to bulk sediment and are expressed on a dry-weight basis.
3. RESULTS and DISCUSSION
The results of the fatty alcohol analysis are presented in Table 2. Total fatty alcohol concentrations of the
surface sediment analysed ranged from 0.49 to 2184.67 ng/g dry weight of sediment, consisting of
eighteen fatty alcohol compounds (C12-C24). The surface sediment samples reveal a predominance of
short chain fatty alcohols (C12-C20) which accounted for 63% of the total fatty alcohols. This is followed by
long chain fatty alcohols (25%) and branched chain fatty alcohols (12%) (Figure 2).
Figure 2. Percentage of fatty alcohols
Besides assessment using fatty alcohol categories, there are a few ratios and index that can be calculated
to strengthen the result obtained. The most applied fatty alcohol ratios are the short chain/long chain fatty
alcohol ratio and Alcohol Source Index (ASI). Both of these ratio and index have been widely used to
assess the predominance of marine or terrestrial contributions (Mudge & Norris 1997; Treignier et al.
2006). Figure 3 illustrates the short chain/long chain fatty alcohol ratio whilst Figure 4 and Figure 5
illustrate the ASI. The value of 1 indicates an equal amount of short chain and long chain fatty alcohols,
values >1 indicate a greater concentration of short chain compounds and values <1 indicate that long
chain compounds are dominant which is usually common in the freshwater systems.
As expected, the study area was dominated by input from marine sources which were represented by the
short chain compounds as Port Dickson’s coastal area did not receive any input from any major riverine
system. Even based on the short chain/long chain fatty alcohol ratio calculated, most of the sampling
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Figure 3. Short chain/long chain fatty alcohol ratio
Figure 4. Alcohol Source Index (ASI)
Figure 5. Alcohol Source Index (ASI)
stations have the value >1 which represents the high amount of short chain compounds. These
compounds indicated input from marine organisms especially phytoplankton and zooplankton (Seguel et
al. 2001; Treignier et al. 2006). Of all the short chain fatty alcohol compounds detected, C 16 compound
was of the highest abundance. According to Volkman et al. (1999), C16 compound can be derived from
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algae and bacteria, and according to Lü & Zhai (2006) based on the study done by Ogura et al. (1990),
this compound is the main component in phytoplankton.
Even though the short chain fatty alcohols were dominant in the study area, the long chain compounds
were also detected; this indicated input from terrestrial plants (Seguel et al. 2001; Treignier et al. 2006).
This might be due to particularly by transportation via coastal currents and deposition of the compound.
The ASI, which was calculated using C22 and C24 that represented terrestrial sources and C14 and C16
which represented marine sources, showed C22 was more dominant than C14 but C16 was the most
dominant (Figure 4) while only a few sampling stations were dominated by C24 rather than C14 and once
again C16 was found to be the most dominant (Figure 5). Both ratio and ASI value for station PD14
differed from other sampling stations; this might be due to the influence of the riverine system nearby.
Together with the long chain compounds, phytol which is also an indicator for terestrial plants, recorded
high concentrations in all the samples. Generally, phytol concentration is much higher than any other fatty
alcohol compounds quantified. Other than being derived from terrestrial plants, phytol and the long chain
fatty alcohols can also be derived from phytoplanton or any aquatic plants (Sun et al. 1998; Tolosa et al.
2008). According to Mudge et al. (2008), surface sediments normally receive high amount of phytol that is
contributed from diatom, waste from zooplankton that consumes phytoplankton and input from terrestrial
plants.
4. CONCLUSION
This study infers that the fatty alcohols quantified from the surface sediment samples of the study area
show a mixture of various main sources. However, it can be concluded that the study area was dominated
by marine derived fatty alcohols, indicated by the high amount of short chain fatty alcohols.
Acknowledgment
This work was supported by the Sciencefund grant No. 04-01-02-SF0698. The authors gratefully
acknowledge the support provided by the Ministry of Science, Technology and Innovation (MOSTI) and
Universiti Kebangsaan Malaysia.
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Mudge, S.M. & Seguel, C.G. 1999. Organic contamination of San Vicente Bay Chile. Marine Pollution
Bulletin 38: 1011-1021.
Ogura, K., Machihara, T. & Takada, H. 1990. Diagenesis of biomarkers in Biwa Lake sediments over 1
million years. Organic Geochemistry 16: 805-813.
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Seguel, C.G., Mudge, S.M., Salgado, C. & Toleda, M. 2001. Tracing sewage in the marine environment:
Altered signatures in Concepción Bay, Chile. Water Research 17: 4166-4174.
Tolosa, I., Miquel, J.-C., Gasser, B., Raimbault, P., Goyet, C. & Claustre, H. 2008. Distribution of lipid
biomarkers and carbon isotope fractionation in contrasting trophic environments of the South East
Pacific. Biogeosciences 5: 949-968.
Treignier, C., Derenne, S. & Saliot, A. 2006. Terrestrial and marine n-alcohol inputs and degradation
processes relating to a sudden turbidity current in the Zaire Canyon. Organic Geochemistry 37:
1170-1184.
Volkman, J.K., Barrett, S.M. & Blackburn, S.I. 1999. Eustigmatophyte microalgae are potential sources of
C29 sterols, C22-C28 n-alcohols and C28-C32 n-alkyl diols in freshwater environments. Organic
Geochemistry 30: 307-318.
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Table 2 Fatty alcohol concentration
Sampling
station
Fatty alcohol concentration (ng/g dry weight sediment)
C12
C13
C14
iC15
aC15
C15
C16
iC17
aC17
C17
C18
C19
C20
C21
C22
C23
C24
Phytol
PD1
14.71
0.72
137.79
63.87
25.53
56.71
294.36
1.74
127.29
216.07
262.98
3.01
445.21
483.74
62.76
350.20
3.10
220.54
PD2
8.71
5.23
23.95
40.12
5.02
96.20
603.40
2.95
40.70
9.83
226.47
2.23
0.49
18.15
76.69
21.27
25.23
220.35
PD3
2.83
3.84
29.77
41.37
13.57
35.32
155.70
5.63
11.71
19.95
93.53
3.82
26.56
32.23
26.57
25.96
7.97
573.63
PD4
8.34
3.54
56.57
96.93
29.48
90.81
252.85
13.98
25.49
22.99
274.72
7.59
50.49
24.17
104.11
51.19
21.12
1194.58
PD5
5.93
1.14
43.88
74.95
24.88
113.70
219.48
14.39
44.86
19.86
139.78
8.80
22.03
12.27
140.37
36.83
45.95
955.95
PD6
4.71
1.30
16.54
27.94
7.38
26.95
179.92
11.30
18.18
11.44
72.37
3.57
2.20
12.44
85.16
60.05
100.70
412.23
PD7
12.78
8.17
59.43
62.41
15.07
64.52
190.56
17.99
39.53
15.00
211.28
6.85
59.23
71.70
229.37
63.54
102.88
2184.67
PD8
4.66
0.57
3.98
7.78
2.15
9.10
33.30
0.76
0.51
3.52
39.32
0.85
1.12
9.81
9.51
0.50
7.41
74.36
PD9
2.33
2.29
16.24
19.56
4.60
14.36
74.92
6.72
5.60
2.08
45.73
0.72
9.87
8.76
31.98
13.82
14.07
381.38
PD10
2.86
1.05
5.17
4.83
2.11
5.07
22.32
0.52
0.52
1.21
20.98
1.48
7.86
10.28
12.26
16.28
2.48
71.60
PD11
3.97
5.30
17.85
17.70
5.53
18.47
62.06
0.56
3.27
4.10
34.05
0.57
13.55
10.17
24.20
16.69
5.81
155.33
PD12
5.22
6.97
23.48
23.29
7.28
24.30
81.66
0.74
4.30
5.39
44.80
0.75
17.83
13.38
31.85
21.96
7.65
204.38
PD13
3.55
0.69
26.25
30.58
10.65
29.47
175.40
5.38
4.70
9.52
55.47
2.02
9.84
8.23
8.18
7.29
14.22
416.77
PD14
16.08
2.22
3.70
1.48
2.16
5.90
14.68
3.40
7.20
11.10
98.17
4.67
40.96
64.91
44.87
67.14
31.16
11.03
PD15
24.53
5.90
143.57
154.06
45.72
114.76
338.45
31.57
21.06
30.22
183.86
6.13
114.24
43.92
44.69
65.54
7.18
1553.04
PD16
2.90
4.93
21.28
40.11
12.91
37.25
123.96
0.52
7.94
10.26
121.73
3.51
23.69
13.38
120.20
27.76
32.24
436.35
PD17
5.51
3.21
38.99
82.89
24.83
66.86
222.39
11.21
17.70
8.52
181.75
5.07
35.17
24.59
45.76
36.79
36.20
1062.93
PD18
3.86
0.74
4.03
1.70
1.01
25.57
5.90
11.16
6.77
39.87
126.04
3.98
24.27
28.30
1.80
34.34
1.47
76.40
i-iso; a-anteiso
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GREEN PRODUCTION OF ECO MAGICAL PEPPER CRYSTALS
1*
1
2*
Nurasyikin Abdul Rahman , Norashikin Irdawaty Abdul Rahman , Zainab Ngaini , Vannessa Lawai
2
1
Lembaga Lada Malaysia, P.O. Box 1653, 93916, Kuching Sarawak
Faculty of Resources Science and Technology, 94350, Kota Samarahan, Sarawak
2
*Email: nurasyikin@mpb.gov.my & nzainab@frst.unimas.my
Abstract
Pepper waste, particularly pepper stalk was chemically treated at room temperature to afford magical
black pepper crystals (MPC) The proposed method not only increased the porosity but most
importantly preservation of the nutrient content and other important compounds in the MPC. These
low cost activated carbon crystals offer various applications such as dye removal from textiles industry
or methylene blue (MB) in the range up to 90 %, which comparable to commercially available
activated carbon. The MPC is performed efficiently as an adsorbent in removing heavy metals such
as Cr (III) and Cu (II) in aqueous solution.
Keywords: Pepper, activated carbon, adsorbent, heavy metals, plant growth enhancer
1. INTRODUCTION
Production of Piper nigrum or known as pepper is increasing every year. Every production of pepper
produced an average of 10 to 18 tonnes of waste per year. The wastes were mostly pepper stalks,
pinhead, light berries and pepper dusk which were obtained during dry and wet cleaning process.
Some of the pepper wastes such as the stalks are used for mosquito repellent or organic fertilizer.
Although there are many researches on agricultural waste, little scientific investigation has been done
to produce activated carbon from pepper waste.
Production of activated carbon from agricultural wastes such as wood sawdust, wheat straw, orange
peel, banana pitch, peanut hull, rice husk and others are more economical because they are
structured by cellulose compare to almond shells, peach stones and coconut shell. Pyrolysis of
cellulosic structure is not necessarily (Tsai et al., 1997; Hu et al., 2009). Adsorption of heavy metal by
activated carbon has been widely studied for removing heavy metal from waste water. Heavy metal
has contributed to environment contamination. Chromium (VI), for instance is 100 to 1000 times more
toxic to organisms than Cr (III). Thus, removal of Cr (VI) is a mandatory (Low et al., 2002).
In this study, pepper waste which consisted of pepper stalks, pinhead, light berries and pepper dusk
was subjected to chemical treatment to afford activated carbon which was then applied as heavy
metal and dye absorber.
2. MATERIAL AND METHOD
2.1 Preparation of Eco Magical Pepper Crystals (MPC)
Pepper wastes were collected from Malaysian Pepper Board (MPB), Sarawak factory and used
without bleaching or sieving. The wastes were soaked in acid for 24 h at room temperature and
0
neutralized in alkali. The products were filter and dried for 24h at 100 C.
2.2 Absorption Study on Methylene Blue (MB)
The suspension of activated carbons and MB were mixed with different concentration at room
0
temperature (27 C) in 250ml conical flask in a shaker at 150rpm. The adsorbent of dye solution were
determined using spectrophotometry GeneQant model 1300 at 663nm. The amounts of MB adsorbed
were calculated before and after adsorption.
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2.3 Heavy Metal Adsorption Study: Effect of Initial Concentration
Seven different initial concentrations of heavy metal solutions were prepared at 5, 10, 15, 20, 25, 30
and 35 mg/L. 1.0 g of adsorbent was added into 100 ml of the prepared concentration, followed by
agitation of the solution on a rotary shaker at 150rpm for 24h. The samples were withdrawn from the
shaker after agitation and the solution was separated by gravity filtration. The concentration of the
residual heavy metal solution was determined using Atomic Absorption Spectrometer (AAS). Removal
efficiency was calculated using the following equation:
Removal (%) =
x 100 %
Where,
Co- the initial concentration (mg/L)
Ce - the final concentration (mg/L).
3. RESULTS AND DISCUSSION
Preparation of magical pepper crystals (MPC) has been successfully performed under low
concentration of acid at room temperature. The Infrared spectroscopy of MPC and untreated pepper
waste (PW) is shown in Figure 1. In comparison to the commercially activated carbon (ACC), both PW
-1
and MPC showed the presence of strong adsorption band at 3200-3550 cm which attributed to OH
-1
stretching vibration, whereas the C=O groups is shown at 1690 cm . The infrared spectra of both PW
and MPC also showed the presence of CH2 symmetric and asymmetric stretching vibrations at 2920–
_1
2850 cm .
(a)
(b)
(c)
Figure 1. The spectra of (a) untreated pepper waste (PW), (b) MPC and (c) ACC
The comparison of the morphology of MPC with PW is shown in Figure 2. MPC (b) showed more
porosity in the surface area compared to PW (a). The treatment of cellulosic material with dilute acid
at low temperature would alter the crystalline structure, thus making the substrate spongy by
penetration of water molecules into the cellulose crystals thereby expanding the specific surface area
(Camacho et al., 1996).
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a
c
b
Figure 2. Scanning Electron Microscope image of (a) Untreated Pepper Waste, (b) MPC, dan (c) ACC
3.2 Absorption Study on Methylene Blue (MB)
MB Absorption mg/g
The adsorption capacity of activated carbons is shown in Figure 3. Eight different concentration of MB
were examined. The weight of MPC and pH values was the same. The results showed that PW has
the lowest ability to absorb MB, while MPC and ACC showed significant adsorption with high
concentration of MB. The linear graph indicated that the adsorption of MPC is comparable with ACC.
Concentration of MB, mg/L
Figure 2. Absorption Study of PW, MPC and ACC on Methylene Blue (MB)
Micro porous of pores on the carbon surface has increased the adsorption MB. In comparison
between εPC and ACC, the present of moisture on the εPC’s pores might be the reason of lower
adsorption of MB into MPC compared to ACC.
3.3 MPC as Heavy Metal Remover: Effect of Initial Concentration
The adsorption experiments were carried out at various initial concentrations which were at 5, 10, 15,
20, 25, 30 and 35 mg/L. The removal efficiency of Cu (II) and Cr (IV) for MPC, ACC, and PW were
higher (>85%) at low concentration after 24h adsorption. MPC gave an adsorption up to 100% of Cu
(II) compared to ACC, and PW (Figure 4). As the initial concentration increased, the removal efficiency
was decreased for ACC and PW in removing Cu and Cr metal ions. It is due to the complete utilization
of adsorption surface (Babu & Gupta, 2008) which caused saturation of the active binding sites
(Amuda et al., 2007). Both Figure 4 and Figure 5 indicated that MPC showed excellent Cu (II) and Cr
(IV) removal compared to ACC. The presence of functional group has improved the efficiency of MPC
since the metal ions can be selectively coordinated by the functional groups (e.g. C=O or -OH).
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Figure 4. The effect of initial concentration on the removal efficiency of ACC, MPC, and PW on Cu (II)
Figure 5. The effect of initial concentration on the removal efficiency of ACC, MPC, and PW on Cr (IV)
4. CONCLUSION
MPC, an activated carbon from pepper waste was successfully prepared at room temperature. MPC
can be used as an alternative absorbing material towards implementing green and low cost dye
removal and metal adsorbent.
ACKNOWLEDGEMENT
Authors express their sincere thanks to Malaysian Pepper Board and University Malaysia Sarawak for
financial supports of the research.
References
Alzaydien, A. S. 2009. Adsorption of Methylene Blue from Aqueous Solution onto a Low Cost Natural
Jordanian Tripoli. American Journal of Environmental Sciences 5[3]:197-208
Amuda, O.S., Giwa, A.A., & Bello, I.A. (2007). Removal of Heavy Metal from Industrial
Babu, B.V. & Gupta, S. (2008). Adsorption of Cr (VI) Using Activated Neem Leaves: Kinetic Studies.
Adsorption, 14, 85-92.
Camacho, F., Gonzalez-Tello, P., Jurado, E. & Robles, A. 1996. Microcrystalline-Cellulose Hydrolysis
with Concentration Sulphuric Acid. J. Chem. Tech. Biotechnol. 350-356.
Hu, C.W., Li, J.L., Zhou, Y., Li, Mei., Xue, F.,& Li, H. 2009. Enhanced Removal of Methylene Blue
from Aqueous Solution By Pummelo Peel Pretreated with Sodium Hydroxide. Journal of
Health Science 55[4]:619-624.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Low, K. S, Lee, C.K. & Lee, T.S. 2002. Acid-activated Spent Bleaching Earth As A Sorbent For
Chromium (VI) in Aqueous Solution. Environmental Technology, 24:197-204
Tsai, W.T, Chang, C. Y. & Lee, S.L. 1997. Preparation and Characterization of Activated Carbons
from Corn Cob. Environmental Sciences. 1198-1200.
Wastewater Using Modified Activated Coconut Shell Carbon. Biochemical Engineering
Journal, 36, 174-181.
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CYTOXICITY EFFECTS OF GINGER AQUEOUS EXTRACT ON LARYNGEAL CARCINOMA
( HEP-2 ) CELLS
Hemaniswarri Dewadas, Nor Hazwani Ahmad, Ruzita Ahmad*, Ishak Mat
Advanced Medical and Dental Institute, Universiti Sains Malaysia,
13200 Kepala Batas, Pulau Pinang, MALAYSIA
*Email: ruzita@amdi.usm.edu.my
Abstract
Ginger (Zingiber officinale Roscoe, Zingiberaceae) has been traditionally used as medicinal herbs for
a number of disorders, including cancer. However, the in vitro and in vivo cytotoxicity mechanisms
exhibited by the extract remain unclear. Hence, the aim of the present study is to examine the effect of
aqueous extract of ginger on laryngeal cancer ( Hep-2 ) cell line. The effect of the ginger extract on
the proliferative responses of Hep-2 cells was assessed by MTS assay at various concentrations from
1000µg/ml to 1.95 µg/ml and incubation times of 24, 48, and 72 hr.. Annexin/PI staining by flow
cytometric analysis was performed to confirm the induction of apoptosis on the cells. Ginger extract
showed significant growth inhibitory activity on the cells with IC 50 values of 416.67 µg/ml and 458.33
µg/ml at 24 and 48 hours, respectively. Moreover, flow cytometric analysis revealed the inhibition of
the proliferation was due to apoptosis especially at 24 hours, where the percentages of apoptotic
cells were 8.76% and 7.04% respectively for treated and untreated samples. Since the ginger extract
exhibited inhibition of cell proliferation through apoptosis, the results suggest that this extract may
have a potential cytotoxicity activity against laryngeal carcinoma cells.
Keywords: ginger, aqueous extract, apoptosis, Hep-2 cell line, laryngeal carcinoma
1. INTRODUCTION
According to Malaysian Society of Otorhinolaryngology and Head Neck Surgeons, laryngeal cancer
ranks the 4th most common head and neck cancer where in 2006, 216 cases of laryngeal cancer were
reported in Peninsula Malaysia (Marioni, 2006). In addition, the high consumption of tobacco products
and alcohol can affect the risk of developing laryngeal cancer. According to WHO (2002), 50% of men
and 30% of adolescent boys smokes in Malaysia, causing smoking related disease to increase. The
combined effect of smoking and alcohol consumption is estimated to account for 89% of laryngeal
cancers (Hashibe et al., 2009).
Currently, conventional strategies such as surgery, chemotherapy and radiotherapy may not eradicate
cancer cells completely in late stage of cancer. These treatments alone may not be the best option for
late-cancer patients. Some even become radio-resistant and chemo-resistant to the treatment.
Furthermore, side effects are sometimes left and interfere with patients’ daily live (Boon et al., 2000;
Morris et al.,2000; Morrow et al., 1996; Sparber et al., 2000). Thus, traditional/complementary
medicine is extremely essential to complement standard approach (conventional treatment) in late
cancer patients. Several diet-derived agents are evaluated clinically as cytotoxic agents for major
cancer targets including breast, prostate, colon and lung cancers (Kellof et al., 2005).
Ginger (Zingiber officinale Roscoe) from Zingiberaceae family is a well known plant in Southeast Asia
and a number of its species are being used in traditional medicine. It was found to be quite effective in
the treatment of several diseases (Afzal etal., 2007). There are several pungent compounds identified
in ginger such as gingerols,shogaols, paradols, and gingerdiols. Some phenolic substances found in
ginger generally possess strong anti-inflammatory and anti-oxidative properties, and exert substantial
anti-carcinogenic and anti-mutagenic activities as well (Surh, 2002; Surh,1999; Surh et al., 1998).
Therefore, the cytotoxicity effects of the ginger extract on laryngeal cancer cell line, Hep-2 cells, were
evaluated. Our data demonstrated that the ginger extracts inhibited cell proliferation through the
induction of apoptosis. Our data also revealed the potential of this extract to induce cytotoxicity effect
on the Hep-2 cells, suggesting that this extract has high potential to be used for the treatment of
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laryngeal carcinoma.
2. MATERIALS AND METHOD
2.1 Preparation of extract
The fresh ginger was collected and dried in the oven at 60°C. After 4 days, the dried ginger was
ground. 100g of the ground ginger was mixed with 1 L of ultra pure water. The mixture was placed in
shaking water bath at 28°C for 24 hours. After 24 hours, the ginger mixture was centrifuged at 21,000
rpm for 15 minutes. The supernantant obtained was filtered through Whatman No.1 filter paper and
freeze-dried. The stock concentration of the extract was prepared by dissolving 100 mg/ml of the
freeze-dried powder with 100 ml of ultra pure water and stored to be used for the subsequent
experiments. The method was modified from Yasser and Hemat (2010).
2.2 Preparation of cells
Hep-2 cells were detached from cell culture flask using trypsin solution and centrifuged at 1000rpm for
10 minutes. Following centrifugation, the pellet obtained was resuspended with RPMI-1640 (Sigma,
USA) media supplemented with 1 % Penicillin-Streptomycin (v/v) (Gibco, USA), 1 % L-glutamine (v/v)
(Gibco, USA) and 10 % Foetal Calf serum (v/v) (Gibco, USA). The cells were counted and prepared at
4
a concentration of 1 10 cells/ml.
2.3 MTS assay
The method was performed as described in the MTS kit manual (Promega, USA). The cells were
double diluted at concentrations of 500 g/ml, 250 g/ml, 125 g/ml, 62.5 g/ml, 31.25 g/ml, 15.625
g/ml, 7.813 g/ml,3.λ06 g/ml, and 1.λ53 g/ml. The untreated cells were used as a control. The
cells were incubated for 24, 48 and 72 hours. Following each of the incubation time, the MTS/PMS [3(4,5-dimethylthiazol-2-yl)-5-(3-carbonxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium/phenazine
metho sulfate] solution was added into the wells with a ratio of 20/1. Cells viability was determined by
ELISA microplate reader at 490nm excitation wavelength. The IC50 values were calculated according
to the formula below:
Cell viability (%) = Absorbance of treated cells
100
Absorbance of untreated cells
2.4 Annexin/PI staining
The cells were plated on a 6-well plate at a density of 1 10 cells/ml, and exposed to the IC50 value
obtained from the εTS assay which was approximately 500 g/ml ginger extract and incubated at 6,
12 and 24 hours. The cells treated with 4 M of camptothecin (Sigma, USA) were used as a positive
control. After each of the incubation time, the cells were harvested and centrifuged at 1000rpm for
5minutes. The cells were washed by resuspending in 1X binding buffer and the concentration was
5
adjusted at 1 10 cells per tube. The cells were then stained with FITC-labeled annexin V and
propidium iodide (PI) and incubated in the dark for 15 minutes at room temperature and subsequently
analyzed by flow cytometry.
4
2.5 Statistical analysis
The data were expressed as mean ± SD by using Student’s t-test.
significance was established at P <0.05.
The level of statistical
3. RESULTS AND DISCUSSION
3.1 Ginger aqueous extract inhibited the proliferation of Hep-2 cells
As illustrated in Figure 1, growth inhibitory activity of Hep-2 cells was observed with IC50 values of
416.67µg/ml and 458.33 µg/ml at 24 and 48 hours, respectively. The growth inhibition was correlated
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with morphological observation of cells under microscope (Figure2) which showed decrease in the
number of cells at higher concentration under treatment periods of 24 hours, 48 hours and 72 hours.
Figure 1. Cytotoxic effect of the ginger extract on Hep-2 cells. The Hep-2 cells were treated with
ginger extract at various concentrations and incubated at 24, 48 and 72 hours. The IC 50 values were
416.67 µg/ml and 458.33 µg/ml at 24 and 48 hours, respectively. Error bars represent the Mean S.D.
24hr
48hr
A
72hr
B
C
Control
D
E
F
500µg/ml
Figure 2. Morphology of the Hep-2 cells. The cells were either treated with (D, E, F) or without (A, B,
C) the extract at a concentration of 500 g/ml at 24, 48 and 72 hours of incubations
3.2 Ginger aqueous extract induced cell death through apoptosis
The mechanism of apoptosis was observed with the positive cells with the annexin conjugated with
+
+
+
FITC (Annexin /PI ) for early apoptosis and the cells positive with annexin and PI (Annexin /PI ) for
late apoptosis. Representative histograms show that there was an increase in percentage of apoptotic
ginger-treated cells for early and late apoptosis compared to the control, especially at 6 and 24 hours
with 6.52% versus 5.60% and 8.76% versus 7.04% (Figure 3). The cells treated with camptothecin
remarkably increased the number of apoptotic cells, compared to the untreated and the cells treated
with the ginger extract.
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Cell
Count
Control
Cells + Camptothecin
Cells + Ginger
6 hr
12 hr
24hr
Annexin–FITC
Figure 3. The percentage of apoptotic cells by Annexin/PI staining. The Hep-2 cells were treated with
extract and camptothecin at 6, 12 and 24 h of incubation times and analyzed by flow cytometer. The
untreated cells were used as a control. The histogram is overlaid with the unstained cells (green line).
The present study showed that the ginger aqueous extract demonstrated cytotoxicity effects on Hep-2
cells through the induction of apoptosis mechanism. This might be due to the presence of natural
active constituents within the extract. Previous research reported by Jolad et al. (2004) found
approximately 63compounds in organically grown fresh ginger. In addition, 31 compounds had been
previously reported as constituents of ginger while 20 were hitherto unknown compounds. The
identified compounds include gingerols,shogaols, 3-dihydroshogaols, paradols, dihydroparadols,
acetyl derivatives of gingerols,gingerdiols, mono- and di-acetyl derivatives of gingerdiols, 1dehydrogingerdiones,diarylheptanoids, and methyl ether derivatives of some of these compounds.
Our data exhibited similar cytotoxicity effect as the study done by Lee and Surh (1998) which showed
that the compounds in ginger which are, 6-paradol and 6-gingerol induced the apoptosis of HL-60
cells. Moreover, a study conducted by Wei et al (2005) showed that many diarlyheptanoids and
gingerol-related compounds obtained from the rhizome of ginger(Zingiberofficinale) has significant
anti-proliferation activity on HL-60 cells through the mechanism of apoptosis.
Other ginger related study was conducted by Bode et al., (2001) which showed that [6]-paradol, one of
the active compounds in ginger exerted its primary inhibitory effect on cell transformation through the
induction of apoptosis. [6]-Paradol and other structurally related derivatives induced apoptosis in oral
squamous carcinoma cell line in a dose dependent manner through a caspase-3-dependent
mechanism (Keum at al., 2002).Caspases are crucial mediators of programmed cell death (apoptosis).
Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage
ofmany key cellular proteins leading to apoptosis (Porter and Janicke, 1999).
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4. CONCLUSION
Ginger extract showed cytotoxicity effect on laryngeal carcinoma cells. Significant growth inhibitory
activity on the Hep-2 cells was observed with IC50 values of 416.67 µg/ml and 458.33 µg/ml ginger
extarct at 24 and 48 hours, respectively. Flow cytometric analysis revealed the inhibition of the
proliferation was due to apoptosis especially at 24 hours.
References
Afzal, M., Al-Hadidi, D., Menon, M., Pesek, J., Dhami, M.S. 2001. Ginger: and ethnomedical, chemical
and pharmacological review. Drug Metabolism. Drug Interaction. 18, 159–190.
Bode, A.M., Ma, W.Y., Surh, Y.J. & Dong, Z. 2001. Inhibition of epidermal activation by [6]gingerol.Cancer Research, 61, 850-853.
Boon, H., Stewart, M., Kennard, M.A., Gray, R., Sawka, C., Brown, J.B., McWilliam, C., Gavin, A.,
Baron, R.A., Aaron, D. & Haines-Kamka, T. 2000. Use of complementary/ alternative medicine by
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IN VITRO PROPAGATION OF CRYPTOCORYNE FERRUGINEA ENGLER
Chen, M.Y.*, Sani, H.B. and Ipor, I.B.
1
Plant Tissue Culture Laboratory, Department of Plant Science and Environmental Ecology,
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan,
Sarawak
*Email: myin.chen@gmail.com
Abstract
Cryptocoryne ferruginea Engler (Araceae) is an endemic species of Borneo. Many Cryptocoryne
species are popular aquarium plants but there is limited information on successful commercialization
of any species from Malaysia. Surface sterilization using 70% ethanol for 1 minute, 15% (v/v) Clorox®
for 15 minutes and rinsed thrice with distilled water produced the highest percentage of contaminationfree runner explants at 58% for the first 2 weeks of culture initiation in Murashige & Skoog (MS)
medium. Only 4 explants (4.17%) explants remained uncontaminated after 4 weeks. Incorporation of
Plant Preservative εixture (PPε™) into εS medium reduced contamination up to 100% for runner
explants but only 5 explants (5.21%) were viable for shoot regeneration. The surface sterilization
method requires further refinement to improve its efficiency. Multiple shoot, leaf and root regeneration
used combinations of 6-benzylaminopurine (BAP) at 0, 0.5, 1.0, 2.0 mg/L with indole-butyric-acid (IBA)
at 0 or 0.1 mg/L. Highest mean number of shoots (6.93) and leaves (3.33) per explants was observed
in medium supplemented with 1.0mg/L BAP and 0.1mg/L IBA. Addition of 0.1mg/L IBA alone induced
the highest mean number of roots (2.93) per explant. Presence of both BAP and IBA was significant
for the development of leaves and roots. Different medium (solid or liquid) with or without 0.1mg/L
NAA was used for root induction. Highest mean number of roots (4.25) per explant was induced in
liquid medium supplemented with 0.1mg/L NAA.
Keywords: Araceae, Cryptocoryne ferruginea, tissue culture, surface sterilization, shoot proliferation
1. INTRODUCTION
The genus Cryptocoryne (Araceae) is locally known as tropical-bog, kiambang batu (Sarawak Malay),
teron anum (Melanau) and kelatai (Iban), keladi laut or bakong (Samarahan Malay) and hati-hati paya
(Peninsular Malaysia) (Simon, Ipor, & Tawan, 2008). Cryptocoryne ferruginea Engler is an endemic
species of Borneo. This plant was initially found in slow running rivers and streams in the inner part of
the tidal zone in deep shade (Jacobsen 1985). C. ferruginea is abundant in Sarawak but has limited
distribution (Ipor, Tawan, Seng, Saupi, & Abai, 2007).
Many species of Cryptocoryne are popular aquarium plants and both local and international demands
for these plants are met through mass collection from natural habitats which often leads to significant
decrease of their populations. However, the supply of these plants from exporting countries were also
reported to be unreliable and there were considerable losses during transportation because of the leaf
decomposition condition called “Cryptocoryne melt down” (Bryan, 1λλ0, as cited in Kane, Gilman,
Jenks, & Sheehan, 1990).
Propagation of Cryptocoryne using rhizome in nurseries was reported as slow with limited scale of
production. In addition, most Cryptocoryne plants require artificial pollination of flowers for seed
production (Mansor & Masnadi, 1994). Ipor et al. (2007) further explained that it was very difficult to
maintain a sustainable population of living collections in the greenhouse for many years without proper
techniques of cultivation. In vitro plant propagation through tissue culture is increasingly used for rapid
and mass production of commercial freshwater aquarium plants. This method has decreased
problems related to over-collection and loses to water-transmitted diseases.
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Successful establishment of in vitro cultures was reported for Cryptocoryne wendtii, C. nevilli, C.
becketii, C. bogneri, C. wallisii, C. willsii, and C. thwaitesii (Staritski, 1977, as cited in Kane et al.,
1990; Dissanayake, Hettiarachchi, & Iqbal, 2007; Herath, Krishnarajah, & Wijesundara, 2008). Other
species included the C. lucens and C. cordata from Peninsular Malaysia (Sahidin, Othman, & Khalid,
2007; Amirrudin, Ipor, & Aziz, 2007). At present, there are limited reports on the in vitro propagation of
Cryptocoryne species originated from Sarawak. This experiment was conducted to develop an in vitro
propagation method for the micropropagation of Cryptocoryne ferruginea. In this study, the objectives
were to investigate the effects of different (1) sterilization methods (2) growth regulators and (3) type
of culture media for the micropropagation of this endemic species.
2. MATERIAL AND METHODS
2.1 Culture establishment
2.1.1 Effects of different concentrations of Clorox© concentrations and exposure time
Runner explants were collected from healthy growing stock plants of C. ferruginea in the greenhouse
of Universiti Malaysia Sarawak. These explants were washed well with water and rinsed under running
tap water for 30 minutes. Next, they were disinfected with 70% (v/v) ethanol for one minute and
Clorox© solution at concentrations of 10, 15 and 20% for 12 or 15 minutes. The explants were rinsed
well with sterile distilled water thrice at 5 minutes each time.
The runner explants were cut into 1 cm length fragments and four explants were placed horizontally
on half strength Murashige and Skoog (MS) (1962) medium with 3% sucrose and solidified using 3.0 g
of Gelrite. There were 3 replicates with 4 explants in each replicate. The experiment was repeated
once. The medium was adjusted to pH 5.79-5.80 and autoclaved at 121 °C at 1.06 kg/cm³ for 20
minutes. The number of contamination-free (axenic), contaminated and damaged explants were
recorded after 2 weeks of culture initiation.
2.1.2 Effects of different concentrations of Plant Preservative Mixture (PPM™)
The treatment in experiment 2.1.1 with the highest mean number of axenic explants will be considered
as the relatively better treatment and used to sterilize the runner explants. Then, four explants were
cultured in half strength εS medium added with PPε™ at concentrations of 1, 2, 3 and 4 ml/δ. There
were 3 replicates and 4 explants per replicate. The experiment was repeated once. The number of
axenic, contaminated and damaged explants was recorded after 2 weeks of culture initiation.
2.2 Shoot multiplication
To develop a suitable medium for shoot multiplication, shoots regenerated from runner explants in
medium without growth regulators were excised and five regenerated shoots were cultured on MS
medium supplemented with 0, 0.5, 1.0, and 2.0 mg/L of 6 benzylaminopurine (BAP) in combination
with or without 0.1 mg/L indole butyric acid (IBA). Each culture bottle with 40 ml medium was
considered a replicate and there were three replicates for each treatment. MS medium without growth
regulators was the control (Table 1). After four weeks, the number of shoots, leaves and roots for each
replicate and explants was recorded.
2.3 Root induction
To select a suitable media for root induction, two types of basal media were tested which were Gelrite
solidified and liquid half strength MS medium supplemented with or without 0.1 mg/L 1naphthaleneacetic acid (NAA). Shoots of 1 cm in height were cultured in these media. Each treatment
had four replicates and each culture bottle had two shoots. After three weeks, the number of roots in
each replicate and explants was recorded.
2.4 Statistical analysis
The experiment was arranged in a Completely Randomized Design (CRD). Statistical analyses of data
were performed using Statistical Package for the Social Sciences (SPSS) version 17.0 for Windows.
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Data were subjected to analysis of variance (ANOVA) and mean was compared using Tukey’s
Honestly Significant Difference (HSD) Test at 5% significance level.
3. RESULTS
3.1 Culture establishment
3.1.1 Effects of different concentrations of Clorox© concentrations and exposure time
Establishment of contamination-free (axenic) cultures were difficult at all treatments using combination
of different concentration of Clorox© and exposure time. The highest mean number of axenic explants
(2.33) was observed in surface sterilization using 15% Clorox© concentration for 12 minutes (Table 1).
Contamination was also found to be higher when exposure time was lengthen or when higher
concentration of Clorox© (20%) was used. However, a two way ANOVA showed that the interaction
effect of concentration and time was insignificant (p>0.05).
Table 1. Effects of different Clorox© concentrations and exposure time
Treatment
Condition
10%
12 min
10%
15 min
Axenic
1.33*
1.33
Contaminated
2.67
2.67
Damaged
0
0
15%
12 min
2.33
(58.33%)
1.67
0
15%
15 min
20%
12 min
20%
15 min
2.00
1.00
1.00
4.00
3.00
3.00
0
0
0
*Mean number of 6 replicates, 4 explants per replicate
3.1.2 Effects of different concentrations of Plant Preservative Mixture™
Contamination was fully suppressed or contamination-free in all cultures on MS medium added with 2,
3 and 4 ml/δ of PPε™. No visible contamination was observed during the initial 2 weeks after culture
initiation. However, when transferred onto shoot induction medium, only 5 explants sterilized with
PPε™ had regenerated shoots while the remainder were considered internally damaged due to the
lack of visible damage symptoms such as colour change. A one way ANOVA showed that the effects
of different concentrations were insignificant (p>0.05).
3.2 Shoot multiplication
During initial shoot induction, multiple shoots were successfully induced in medium without growth
regulators although they were no leaf and root formation and some shoots showed signs of
translucency or were less healthy. When transferred into medium supplemented with combinations of
BAP and IBA, healthy shoots was formed and leaf and root induction was successful in several
treatments. The highest mean number of shoots (6.93) and leaves (3.33) per explant was observed in
medium supplemented with 1.0 mg/L in combination with 0.1 mg/L IBA.
Highest mean number of roots (2.93) per explant was recorded in medium with 0.1 mg/L IBA alone. In
the control medium without growth regulators, no leaves were formed and the mean number of shoots
was the lowest among the treatments. In high BAP concentration at 2.0 mg/L BAP in combination with
0.1 mg/L IBA, no root were formed and root formation is generally lower when IBA was added to the
culture medium in combination with BAP when compared to BAP alone or IBA alone.
A two way interaction effect of BAP and IBA showed that the presence of both growth regulators was
significant (p<0.05) for leaf and root induction but insignificant for shoot development. Tukey’s HSD
test showed that 0.5 and 2.0 mg/L BAP was not significantly different in the number of leaves but 0
and 1.0 mg/L had significant results. Meanwhile, 0 mg/L BAP was significantly different for root
induction. Post-hoc tests could not be computed for IBA because there were fewer than three groups.
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Table 2. Effects of BAP (mg/L) and IBA (mg/L) on in vitro shoot multiplication of C. ferruginea at 4
weeks after culture establishment
Treatment
BAP
(mg/L)
IBA
(mg/L)
1
2
3
4
0
0.5
1.0
2.0
5
6
Mean and standard error of mean per replicate
Shoots
Leaves
Roots
0
0
0
0
9.00 ± 2.51*
21.00 ± 5.13
12.67 ± 3.76
11.33 ± 1.33
0.00 ± 0.00
11.00 ± 3.60
4.67 ± 0.67
9.33 ± 1.33
0
0.1
12.67 ± 0.33
8.33 ± 0.33
0.5
0.1
7.67 ± 2.60
4.00 ± 1.73
2.00 ± 1.52
1.33 ± 1.33
14.67 ± 0.88
(2.93)
0.33 ± 0.33
14.33 ± 5.84
6.33 ± 2.33
34.67 ± 13.32
16.67 ± 3.17
7
1.0
0.1
1.00 ± 1.00
(6.93)
(3.33)
8
2.0
0.1
16.67 ± 2.84
5.67 ± 2.19
0.00 ± 0.00
* Mean number of 3 replicates with 5 explants per replicate, Figures in parentheses is mean number
per explant
A
B
C
D
Figure 1 In vitro propagation of Cryptocoryne ferruginea (A) Shoot proliferation in runner explant (B)
Branching in shoot tips (C) Development of whole leaves after four weeks culture on MS medium
supplemented with 2.0 mg/L BAP (D) Multiple roots induction after 3 weeks in solid medium
supplemented with 1.0 mg/L BAP in combination with 0.1 mg/L IBA; Scale bar = 0.5 cm
3.2 Root induction
Highest mean number of roots (4.25) per explants was recorded in liquid MS medium supplemented
with 0.1 mg/L NAA. Mean number of roots per explant was observed to be lower when NAA was not
added into the culture medium. The lowest mean number of roots at 1.63 per explant was observed in
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solid MS medium without NAA while mean number of roots was also low at 2.50 per explant in liquid
medium without NAA. A two-way ANOVA between different media and NAA showed that there were
no significant differences between the numbers of roots in the explants. Visual observation showed
that several leaves in explants cultured in liquid medium both with and without NAA had symptoms of
‘Cryptococoryne melt’ or a condition which results in development of translucent leaf tips. The
regenerated roots were also observed to be covered in white hairs.
Table 3. Effects of different media and NAA (mg/L) on multiple root induction at 3 weeks after culture
initiation
NAA
(mg/L)
Mean and standard error of
mean of roots per replicate
Mean of roots per
explant
Solid
0
3.25 ± 1.11*
1.63
Solid
0.1
6.25 ± 2.14
3.13
3
Liquid
0
5.00 ± 2.97
2.50
4
Liquid
0.1
8.50 ± 1.32
4.25
Treatment
Medium type
1
2
* Mean of 4 replicates, 2 explants per replicate
4. DISCUSSION
The results of this study indicated that establishment of contamination-free cultures in Cryptocoryne
ferruginea was difficult due to high rates of contamination by both fungi and bacteria. Meanwhile,
addition of PPε™ exerted toxicity and was damaging to the runner explants as shown in the reduction
in shoot regeneration ability. Full removal of contaminants could not be performed using the surface
sterilization methods in this study and similar results were also recorded in several in vitro cultures of
Cryptocoryne and water plant species. Micropropagation of Cryptocoryne wendtii and Echinodorus
cordifolius by Dissanayake et al. (2007) reported that the use of 0.1% (v/v) mercuric chloride in
surface sterilization yielded 65% and 60% contamination-free rhizome explants of C. wendtii and E.
cordifolius respectively. When mercuric chloride was absent, axenic explants decreased to 35% and
30% respectively. Contact of aquatic plants to water can cause higher number of microbes within the
plants especially in warm tropical weathers.
In addition, it was monitored during culture establishment that the length of time for contamination by
microorganisms to manifest in the explants of C. ferruginea varied from within the first to the fourth
week (in several cases) of culture initiation which indicated possible presence of systemic or
endogenous contaminants in the plant tissues. Comparable contamination over 19 days in shining
club moss (Huperzia lucidula) and suggested that dormant fungal spores and bacterial endospores
slowly germinated when exposed to the high moisture environment during in vitro culture (Waegel,
2003).
Supplementation of growth regulators had positive effects on the growth of C. ferruginea. Shoot and
leaf development was the lowest when no growth regulators to added. Highest shoot and leaf
proliferation was obtained in MS medium supplemented with 1.0 mg/L BAP and 0.1 mg/L IBA. Herath
et al. (2008) reported that when cytokinin level was higher than the auxin level, shoot development will
be promoted. This was reflected in this study in which high levels of shoot and leaf development was
observed when both auxin and cytokinin were present in the culture medium and when their ratio was
low. In comparison, highest mean number of shoots for Cryptocoryne becketti (43.0) and C. bogneri
(51.8) per single shoot explant was observed by Herath et al. (2008) in full strength MS medium
supplemented with 5.0 mg/L BAP in combination with 0.1 mg/L IAA. However, shoot proliferation in
both species was reduced when the medium was supplemented with a high level of BAP (10 mg/L) in
combination with 0.1mg/L indole-3-acetic acid (IAA).
Maximum shoot proliferation in C. wendtii was recorded in 20 µM BAP alone (Kane et al., 1999).
Hongrat et al. (2005) observed that C. cordata cultured in medium supplemented with 1 mg/L BAP
alone produced the highest average number of shoots. Sahidin et al. (2007) had similar results in
which addition of 1.0 µM BAP alone had the optimum number of shoots per explants in C. lucens. In
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C. tonkinensis, MS medium containing 1mg/L BAP alone was the optimum amount to increase the
number of new shoots (Pongchawee et al., 1999, as cited in Pradissan et al., 2005). These results
concluded that addition of growth regulators especially BAP is essential for shoot proliferation in
Cryptocoryne plants.
Liquid medium to induce roots in this study was insignificantly different to the results of solid medium
although the highest mean number of roots was found in liquid medium. Herath et al. (2008) reported
that the use of a soil-based medium saturated with half-strength liquid MS medium supplemented with
0.1 mg/L NAA had significantly high root and shoot growth for C. bogneri and C. beckettii. The lowest
root length and shoot height of C. bogneri were observed in agar solidified full strength MS medium
without adding growth regulators. Herath et al. (2008) further suggested that the use of a soil-based
medium had efficient results for root induction and shoot growth and can be an alternative low cost
propagation protocol.
Another observation in explants of C. ferruginea cultured in liquid medium was signs of slight
Cryptocoryne melt in the leaves of several explants. Vitrification which is a physiological condition with
symptoms of translucency, waterlogging and glassiness can be caused by the use of liquid medium
(Pierik, 1997). A lower sensitivity for this condition can sometimes be prevented or minimized by the
use of a biphasic medium which features a first layer of solid medium into which explants are
inoculated and a second layer of liquid medium on top of the previous layer (Pierik, 1997). The use of
liquid or biphasic medium which imitates the natural and aquarium conditions could potentially provide
potential benefits during acclimatization such as better adaptation when planted out in the field.
5. CONCLUSION
A suitable surfaces sterilization protocol could not be established in this study. Optimum shoot and
leaf proliferation in C. ferruginea was obtained in MS medium supplemented with 1.0 mg/L BAP in
combination with 0.1 mg/L IBA. The use of a biphasic medium or a soil-based medium is
recommended for root induction.
References
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the Tropics: Development and Commercialization of Tropical Natural Resources.
Sarawak, Kuching: Lee Miing Press Sdn. Bhd.
Dissanayake, C., Hettiarachchi, M., & Iqbal, M.C.M. (2007). Sustainable use of Cryptocoryne
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micropropagation. Sri Lanka Journal of Aquatic Science, 12, 89-101.
Herath, H.M.I., Krishnarajah, S.A., & Wijesundara, D.S.A. (2008). Micropropagation of two endemic
threathened Cryptocoryne species of Sri Lanka. Tropical Agricultural Research & Extension,
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Ipor, I.B., Tawan, C.S., Seng, H.W., Saupi, N., & Abai, J. (2007). Proceedings from Conference on
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Mansor, M., & Masnadi, M. (1994). Cryptocoryne elliptica, an endangered amphibious plant in Pondok
Tanjung Forest Reserve, Peninsular Malaysia. Aquatic Botany, 47, 91.
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Publishers.
Pradissan, N. & Pongchawee K. (2005). In vitro multiplication of Laceleaf plant, Aponogeton
madagascariensis (Mirbel) Van Burggen, 1968. Proceedings from 31st Congress on Science
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Sahidin, N., Othman, R.Y., & Khalid, N. (2007). In vitro plant regeneration of exotic water
trumpet, Cryptocoryne lucens de Wit. Proceedings from Asia Pacific Conference on Plant
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Sarawak, Malaysia. Journal of Bioscience, 19 (1), 45-79.
Stanly, C., Bhatt, A., & Chan, L.K. (2011). An efficient in vitro plantlet regeneration of Cryptocoryne
wendtii and Cryptocoryne becketii through shoot tip culture. Acta Physiologiae Plantarum, 33,
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Waegel, A.S. (2003). Stepwise disinfestations reduces contamination of Huperzia lucidula shoot-tips
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MICROPROPAGATION OF Curcuma caesia Roxb. (KUNYIT HITAM)
Fong, Y.M.*, Sani, H.B., Zul Helmey, M.S. and Chen, M.Y.
Department of Plant Science and Environmental Ecology
University of Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
*Email: yin_mei1989@hotmail.com
Abstract
Curcuma caesia Roxb. is one of the Curcuma species belongs to family of Zingiberaceae. It is also
called as ‘Kunyit Hitam’ (εalay), Black Zedoary (English) and Nilkantha (Bengal). This plant is widely
used as medicinal herbs. The present study was to establish an efficient surface sterilization technique
and protocol for C. caesia. Rhizome buds were used as explants. These explants were surface
sterilized by using 70% of ethanol (1 minute), followed by 40% (v/v) of Clorox added with a few drops
of Tween-20 and agitated for 20 minutes. The rhizome buds were rinsed thoroughly with sterile
distilled water. Then, the explants were inoculated in ½ MS (Murashige and Skoogs) basal media
containing 30% g/L sucrose and 3g/L Gelrite for two weeks. The percentage of axenic explants
obtained after surface sterilization was 28%. After that, the axenic explants were cultured in half MS
supplemented with 1.0, 3.0 and 5.0 mg/L of 6-benzylaminopurine (BAP) either alone or with 0.5 mg/L
of indole-3-butyric acid (IBA) for multiple shoot induction. The explants produced multiple shoots after
2 weeks of culture. Rooting and callus formation had also been recorded. Higher percentage of axenic
explants had been recorded when medium was supplemented with 4 mg/L of plant preservative
mixture (PPM) and 4 mg/L of tetracycline (TET). The highest multiple shoot proliferations were
recorded in half MS supplemented with 1.0 mg/L BAP alone.
Keywords: Curcuma caesia, rhizome buds, BAP
1. INTRODUCTION
C. caesia is a rhizomatous herb plant species in the Zingiberaceae family. This species is originated
from India and can be found mostly in West Bengal, Himalaya region, north-Eastern Central India and
Nepal as indigenous plants. The habitat of this species is in the plains and hills (Velayudhan et al.,
1999).
The chemical compounds in this species are curcuminoids, flavonoids, phenolics, amino acids,
protein, volatile oil, and also alkaloid (Sarangthem and Haokip, 2010). The curcuminoids available in
this plant is important as anti-flammatory, anti-microbial, anti-coagulant, wound-healing, and
hypoglycemia (Maheshwari, Singh, Gaddipati & Smimal, 2006). As for the flavonoids and phenolics
component, they are crucial as antioxidant, and anti-flammatory (Miller, 1996); analgesic, locomotor
depressant, anticonvulsant and muscle relaxants effect (Karmakar et al., 2011).
It is used to treat diarrhea and cough (Kala, 2005); piles, leprosy, bronchitis, cancer, epilepsy, fever,
wounds, impotency, fertility, menstrual disorder, toothache, and vomiting (Raju, n.d.); relief rheumatic
pain (Sarangthem & Haokip, 2010); as diuretic, stimulant, and carminative, asthma, and allergic
eruption (Hussain & Hore, 2006); blood diseases, animals and insects bites, congestions and scabies
(Velayudhan et al., 1999).
The rate of propagating C. caesia through rhizome parts is very slow. So, in vitro plant tissue culture
can be used as an alternative way to accelerate the plant multiplication rate. Besides that, there are
limited plant tissue culture reports on this species. Moreover, there is a report concluded that C.
caesia has the possibility to become important in the economic aspects because of it phenolic
compound that higher than C. amada (Katalinic, Milos, Kulisic and Jukie, 2006). So, this technique is
also offered an opportunity to produce mass number of superior clones in limited time and space
(Shukla, Shukla, Vijaya, & Mishra, 2006). This study is initiated with objectives to develop a surface
sterilization technique and multiplication protocol for C. caesia species.
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2. MATERIALS AND METHODS
2.1 Surface sterilization
In this study, rhizome buds were used as explants. At first, the rhizome buds were cleaned and
washed with detergent solution for 5 minutes. Then, the explants were left under running water for
around 30 minutes. Rhizome buds was sterilized with 70% (v/v) ethanol for 1 minute. After that, they
were further sterilized with 40 % of Clorox® for 20 minutes. A few drops of Tween-20 were added into
the Clorox®. Then, the explants were rinsed with 3 times with sterile distilled water. The outer part of
the rhizome buds was peeled off before cultured them.
2.2 Establishment of axenic (free- contamination) culture
The rhizome buds that had been surface sterilized were cultured into half MS medium inoculated with
TM
or without 2 or 4 mg/L of PPM in combination with 4 mg/L of TET to obtained axenic explants.
The medium used in this study was ½ MS (Murashige and Skoog, 1962) medium which was consisted
30 g/ L of sucrose and 3 g/L of Gelrite. The cultures were kept under white fluorescent tubes
providing irradiance of 50µmol m-2 s-1 for 16-h photoperiod. The temperature in the culture room was
maintained around 25±2 ⁰C.
2.3 Shoot induction and multiplication
For shoot induction and multiplication, the axenic explants were transferred into ½ MS medium
incorporated with different concentrations of BAP (1, 3 and 5 mg/L) in combination with 0.5 mg/L of
IBA after 14 days.
3. RESULTS AND DISCUSSION
3.1 Establishment of axenic (free-contamination) culture
At the day 14, only 28 % of axenic rhizome buds were obtained from the ½ MS medium without 2 or 4
TM
mg/L of PPM in combination with 4 mg/L of TET. Most of the explants were contaminated with
bacterial infections. As for the rhizome buds that had cultured into medium inoculated with 2 mg/L of
TM
PPM in combination with 4 mg/L of TET, 60 % of axenic explants where obtained. For 4 mg/L of
TM
PPM in combination with 4 mg/L of TET, 100% of the axenic rhizome buds where obtained.
TM
However, when the rhizome buds were transferred into half MS medium without PPM and TET, the
TM
contaminations would still appear. This showed that PPM and TET were only able to suppress the
contaminants and not eliminate them totally.
3.2 Shoot induction and multiplication
Two weeks old axenic rhizome buds were used as explants for the shoot induction and multiplication.
Multiple shoots were observed in the second to third weeks after cultured into half MS medium
supplemented with different concentrations of BAP (1, 3 and 5 mg/L) in combination with 0.5 mg/L of
IBA.
The highest mean number of multiple shoots was obtained in 1.0 mg/L of BAP culture medium with
the mean of 7.40 shoots per explants. This was followed by mean of 6.4 and 1.0 shoots per explants
which recorded in 3.0 and 5.0 mg/L of BAP. Similar studies had been reported by Palee and
Dheeranupattana (2005) where MS medium supplemented with 1 mg/L of BAP could produce shoot
from the rhizome buds of Curcuma aeruginosa Roxb. However, it was slightly different from other
Curcuma species. For example, the shoot multiplication of C. aromatica, 5.0 mg/L was found to be the
best BAP concentration (Nayak, 2000). The Zingiber officinale also found to multiple the best in 4.0
mg/L BAP (Bhagyalakshmi & Singh, 1988).
On the other hand, the combinations of BAP and IBA showed lower number of multiple shoot
formation. For example, 1.0 mg/L of BAP in combination with 0.5 mg/L of IBA had only recorded mean
of 2.0 multiple shoots per explants. However, Loc et al. (2005) had reported that 3.0 mg/ml of BAP
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and 0.5 mg/L of IBA promoted shoot multiplication highly which was totally different from this study
results.
Combinations of BAP and IBA were observed to influence the shoot length. The highest mean of
shoot length was obtained in 1.0 mg/L of BAP with 0.5 mg/L of IBA (10.84 cm). This followed by the
mean of 10.00 cm for 3.0 mg/L mg/L of BAP with 0.5 mg/L of IBA medium and 5.2 cm for 5.0 mg/L of
BAP with 0.5 mg/L of IBA supplemented medium. However, medium of BAP alone did not provide the
highest mean in shoot length. For example, 1.0 mg/L of BAP alone (Fig. 4.2.1 (a)) was only able to
obtained mean of 4.54 cm. However, Loc et al., (2005) said that addition of 0.5 mg/L of IBA or NAA to
3.0 mg/L of BAP for C. zedoria increased the number of shoots per explants and reduced shoot
length. Kambaska and Santilata (2009) said that addition of any auxin such as NAA or IBA or IAA
could increase number of species towards shoot growth.
Rooting was also quite easy for this species because many roots were observed to emerge
spontaneously from the explants as they were inoculated in BAP alone or in combination with IBA.
The highest mean of roots was recorded in 1.0 mg/L of BAP with 0.5 mg/L of IBA with mean number
of roots about 14.6 roots. The roots in this medium were healthy, strong and larger in diameter which
was about 0.5 cm to 1.5 cm. The colour of the roots was yellowish in colour and coated with slightly
greenish in colour. In the medium which incorporated with BAP alone, the highest mean number of
roots recorded was in 1.0 mg/L of BAP with 9.60 roots per explants. The roots were smaller in in
diameter or more fibrous. The colour of the root was yellowish with some shade of green colour. In
micropropagation of Z. officinale, shoots and roots were also formed together in 2.0 mg/L of BAP
(Jasrai et al., 2000). According to Mello et al. (2001) and Stanly and Chan (2007), MS medium without
auxin was found to be effective in inducing roots in Curcuma zedoria Roscoe and Zingiber zerumbet
Smith. Sit and Tiwari (1998) had reported that the best rooting of turmeric was found in medium
incorporated with 0.5 mg/L of IBA.
Table 1 Mean number of shoots, shoot length (cm) and roots after eight weeks of culture on MS
medium supplemented with different concentrations of BAP alone at 1.0, 3.0 and 5.0 (mg/L) or in
combination with IBA (0.5 mg/L)
Conc. Of BAP
(mg/L)
Conc. Of IBA
(mg/L)
No. of multiple
shoots per explants
Shoot length (cm)
per explants
No of roots
per explants
0
1.0
3.0
5.0
1.0
3.0
5.0
0
0
0
0
0.5
0.5
0.5
0.0 ±0.0
7.40±2.16
6.40±2.23
1.00±0.55
2.00±0.45
4.00±1.26
0.60±0.40
2.70±0.46
4.54±0.74
4.80±1.21
4.50±0.45
10.84±1.61
10.00±1.50
5.20±1.55
0.60±0.40
9.60±1.36
6.40±3.83
1.20±0.49
14.6±5.74
11.20±3.98
7.80±5.61
Figure 1. (a) Shoot multiplication on MS medium supplemented with 1 mg/L of BAP, (b) Shoot
multiplication on MS medium supplemented with 3 mg/L of BAP, (c) Shoot multiplication on MS
medium supplemented with 5 mg/L of BAP
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Figure 2. (a) Shoot multiplication on MS medium supplemented with 1 mg/L of BAP and 0.5
mg/L of IBA, (b) Shoot multiplication on MS medium supplemented with 3 mg/L of BAP and 0.5
mg/L of IBA, (c) Shoot multiplication on MS medium supplemented with 5 mg/L of BAP and 0.5
mg/L of IBA
4. CONCLUSIONS
For rhizome buds, the protocol for the establishment of axenic explants has not been developed yet.
These biocide and antibiotics were not really effective towards contaminants in this study. The
recommended BAP concentrations are 1.0 mg/L of BAP alone for shoot multiplication. As for optimum
shoot length, 1.0 mg/L of BAP in combination with 0.5 mg/L of IBA is recommended to be used. For
healthy and bigger roots, 1.0 mg/L of BAP in combination with 0.5 mg/L of IBA is recommended.
ACKNOWLEDGEMENTS
I would like to take this opportunity to express my sincere appreciation and gratitude to my supervisor,
Prof. Dr. Hamsawi bin Sani for his constant and continuous support, patient guidance and invaluable
suggestion throughout preparation and completing this project. My appreciation also goes to Mr. Zul
Helmey Mohamad Sabdin, Master of Science in Biotechnology for his guidance that helped me to
improve my skills, experience and knowledge in this project. My greatest gratitude also goes to my
family and friends.
References
Balachandran, S. M., Bhat, S. R., & Chandel, K. P. S. (1990). In vitro clonal multiplication of Turmeric
(Curcuma spp.) and ginger (Zingiber officinale Rosc.). Plant Cell Reports. 8:521-524.
Bhagyalakshmi & Singh, R.S. (1988). Meristem culture and micropropagation of a variety of ginger
(Zingiber officinale Rosc.) with a high yield of oleoresin. Journal of Horticultural Science, 63(2):
321-327.
Hussain, S., & Hore, D. K. (2006). Collection and conservation of major medicinal plants of Darjeeling
and Sikkim Himalayas. Indian Journal of Traditional Knowledge, 6(2):352-357.
Jasrai, Y. T., Patel, K. G., & George,, M. M. (2000). Micropropagation of Zingiber officinale Rosc. and
Curcuma amada Roxb. In Centennial Conference in
Spices and
Aromatic Plants. pp.
52-54.
Kala, P. C. (2005). Ethnomedicinal botany of the Apatani in the Eastern Himalaya Region of India.
Journal of Ethnobiology and Ethnomedicine, 1(11).
Kambaska, K. B., & Santilata, S. (2009). Effect of plant growth regulator on micropropagation of ginger
(Zingiber officinale Rosc.) cv-Suprava and Suruchi. Journal of Agricultural Techology,
5(2):271-280.
Karmakar, I., Saha, P., Sarkar, N., Bhattacharya, S., & Haldar, P. K. (2011). Neuropharmacological
assessment of Curcuma caesia rhizome in experimental animal models.Oriental Pharmacy
and Experimental Medicine, 11(4): 251-255.
Katalinic, M., Milos, M., Kulisic, T., & Jukie, M. (2006). Screening of 70 medicinal plants extracts for
antioxidant capacity and total phenols. Food Chemistry, 94:550- 557.
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Loc, N. H., Duc, D. T., Kwon, T. H., & Yang, M. S. (2005). Micropropagation of zedoary (Curcuma
zedoria Roscoe) - a valuable medicinal plant. Plant Cell Tissue Organ Culure, 81: 119-122.
Maheshwari, R. K., Singh, A. K., Gaddipati, J., & Smimal, R. C. (2006). Multiple biological activities of
Curcumin: A short review. Life Science, 78(18): 208-217.
Miller, A. L. (1996). Antioxidant flavonoids: Structure, functions and clinical Usage. Alternate Medicine
Review, 1: 103-111.
Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiologia Plantarum, 15: 431-497.
Nayak, S. (2000). In vitro multiplication and microrhizome induction in Curcuma aromatica Salisb.
Plant Growth Regulation, 32: 41-47.
st
Palee, J., & Dheeranupattana, S. (2005). In vitro culture of Curcuma aeruginosa Roxb.H:31
Congress in Science and Technology of Thailand, 18-20 October 2005, Suranaree University
of Technology, Thailand.
Raju,
R.
(n.d.).
Black
Tumeric.
Retrieved
on
June
30,
2011
on
http://www.flowersofindia.in/catalog/slides/Black%20Turmeric.html.
Sarangthem, K., & Haokip, M. J. (2010). Bioactive components in Curcuma Caesia Roxb. Grown in
Manipur. An International Quarterly Journal of Life Sciences, 5(1): 113-115.
Shukla, S. K., Shukla, S., Koche, V., & Mishra, S.K. (2006). In vitro propagation of Tikhur (Curcuma
angustifolia Roxb.): A starch yielding plant. Indian Journal of Biotechnology, 6: 274-276.
Sit, A. K. & Tiwari, R. S. (1998). Micropropagation of turmeric (Curcuma longa L.). Recent Horticulture,
4: 145-148.
Stanly, C., & Chan, L. K. (2007). Micropropagation of Curcuma zedoria Roscoe and Zingiber zerumbet
Smith. Biotechnology, 6(4):555-560.
Velayudhan, K. C., Muralidharan, V. K., Amalraj, V.A., Gautam, P.L., Mandal, S., &Dinesh, K. (1999).
Curcuma genetic resources. Scientific Monograph No. 4. New Delhi:National Bureau of Plant
Genetic Resources.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
CHARACTERIZING SOILS UNDER VARIOUS CASH CROPS FARMING IN THE UPLAND AREAS
OF SARAWAK- A CASE STUDY AT KRUIN AREA, SABAL, SERIAN
S.Y. Ho*, M. E. Wasli*, H. Nahrawi and A. Said
Department of Plant Science and Environemntal Ecology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
*Emails: sherylsooying@yahoo.com, wmeffendi@frst.unimas.my
Abstract
Population expansion and the need of cash income result in intensified upland agricultural practices
globally. Even though efforts are progressing towards sustaining the agricultural practices in upland
areas, however, limited studies discuss on the present condition of upland agricultural practice. This
study discusses present soil fertility status and condition under various kinds of perennial cash crops
farming (rubber, Hevea brasiliensis; pepper, Piper nigrum; oil palm, Elaeis guineensis; vegetables and
fruits) in an upland area of Sarawak, Malaysia towards providing fundamental information and future
perspective of the current farming practice. The study was conducted at Kruin village in Sabal, Serian.
Soil samples were collected in each type of agricultural lands; pepper garden, oil palm field, rubber
field, fruit orchard and vegetable garden at the depths of 0-10cm and 30-40cm for soil
physicochemical analysis. The results showed that the soils are sandy in texture with acidic nature
which results in high Al saturation. The clay content is low with approximately 13.8% at the study
area. At different agricultural lands, soil exchangeable bases and total C were low and did not varied
widely although some sites especially in the home garden showed significantly high level of
exchangeable Ca. High level of exchangeable Ca in home garden probably due to the ash effect from
addition of burnt materials and left over unburned materials such as charcoal from household waste.
At pepper farm, bulk density and soil hardness were high, indicating regular farming practices such as
harvesting and weeding have resulted in considerable soil compaction. Based on the soil analysis, it
can be concluded that current farming practice does not show much impacts to the soil, the chemicals
levels in the soils were low, indicating little use of agrochemicals in the farming practice. However,
technologies such as intercropping and crop rotation should be integrated into current farming practice
to conserve soil fertility towards a more sustainable upland agriculture in the future.
Keywords: cash crop farming, upland agriculture, soil physicochemical properties.
1. INTRODUCTION
Recently, one of the crucial problems faced globally is food crisis where the food produced is not
sufficient for the growing population throughout the world (εung’ong’o, 2000)Furthermore, increase in
population pressure and influence of commercialism leads to the increasing of human impact of
agricultural use on naturally forested areas in humid tropics and subtropics (Funakawa et al., 1997).
Although shifting cultivation was formerly the central agricultural activity, however, due to the changes
in socio-economic with industrialism development, such agricultural practices have changed into a
more diversified upland farming system (Crumb, 1993). This involves the shortening of fallow cycles
and several years of continuous cropping with significant fertilizer application (Boonyanuphap et al.,
2007).
In Sarawak, agricultural lands cover three different types of areas namely coastal lowland, central
lowland and interior upland areas (Teng, 2003). As reported by Aminuddin ey al., (1990), 1.81 million
hectares of interior upland areas in Sarawak are marginally suitable for agricultural practices which,
including steep slopes. However, due to population expansion and depletion in available land
resources for agricultural purposes in Sarawak, the traditional form of landuse practices have slowly
being abandoned by the local farmers (Lim & Douglas, 2000). In the meantime, these areas are most
likely to decline due to gradual changes in various aspects such as the social structure, political and
national policies, environmental and cultural aspects (FAO, 2003). These have much influenced the
modification of swidden traditional land use practices towards a sedentary form of agricultural
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practices involving use of agrochemicals. Without proper management of agrochemicals applications
and its interventions, such cause may lead to environmental degradation (Padoch et al., 2001).
Various studies have been conducted mainly on the effect of shifting cultivation to soil ecosystems,
physical properties and fertility in the upland areas in the tropics (Cramb, 1989; Watanabe et al., 2004;
Kendawang et al, 2004; Hattori et al., 2005; Tanaka et al., 2009). Regarding site suitability for
agriculture as well as vegetation condition in fallow lands, limited studies have been carried out in the
interior upland areas of Sarawak and fallow shifting cultivation land (Bruun et al., 2006; de Neergaard
et al., 2008; Wasli et al., 2009). Limited attention has been paid to the current situation of intensive
upland agricultural practices in the Sarawak. Moreover, although government paid attention to the
need to sustain upland agricultural practices, still, limited information is available on the outcome of
such scenario in the current upland farming system.
Without information and evidence on the current conditions in the upland farming systems, the
sustainability of agricultural practices in the upland areas is at stake. Environmental problems still exist
if proper management is not implemented, and this goes against the mission in sustaining the upland
agriculture. The objective of this study is to characterize soils under different types of perennial cash
crops farming which are rubber (Hevea brasiliensis), pepper (Piper nigrum), oil palm (Elaeis
guineensis), vegetables and fruits in order to provide fundamental information and perspectives about
upland agricultural practices of Sarawak, using a case scenario of Kruin area, Sabal. Such information
is vital as a guideline for research purposes in the future to improve the farming systems towards
sustainable upland farming system in the upland areas of Sarawak where the economic, environment,
social and cultural values are to be well-managed.
2. MATERIALS AND METHODS
2.1 Study Area
This study was conducted from November 2011 to June 2012 at Sabal area (N 01°03’, E110°55’),
Kuching-Samarahan Division in Sarawak, Malaysia. The study site is located about 100km southeast
of Kuching city and is accessible by the Kuching-Sri Aman road. Climate is classified into AA’r on the
Thornthwaite classification system (Thronthwaite, 1948). Mean annual temperature was about 25°C
with little variation monthly (Meteorological Department, 2010). The average mean precipitation was
4000 mm at Sungai Pinang rainfall station near Sabal Forest Reserve (Department of Irrigation and
Drainage, 2010).
Soil in Sabal is derived and originated from non-calcareous sedimentary rock consisting of fine and
whitish sandstone during the mid tertiary period (Butt, 1983). The soil is classified into Oxyaquic or
Spodic Quartzipsamments based on the USDA Classification System (Soil Survey Staff, 1999). Kruin
village is located at the border between Samarahan and Sri Aman district. The village consisted of
approximately 80 families, of Iban origin. The main activities of the villager are subsistence farming
such as upland rice cultivation, cultivation of vegetables and fruits in home garden as well as cash
crop farming i.e. pepper and rubber. In addition, few farmers in the village also cultivate smallholder
based oil palm cultivation for their livelihood.
2.2 Data Collection and Soil Sampling Methods
Based from the local information on the land use history in Kruin village, all farming lands were
established in secondary forests which were formed after series of shifting cultivation practices as well
as previous cash crop farming. Before the study sites were selected, some local villagers were
interviewed on their current farming practices and crop management practices in their land, including
the land use history of their current farming lands.
Later, the soil samplings in the desirable sites were conducted through selection from the landowners
that were previously interviewed. Farmers’ selection as respondent is based on their years of farming
experiences and availability of crops such as rubber, pepper, oil palm, fruit orchard and vegetables
home garden. On the other hand, site selection is based on the types of cash crop planted and
accessibility to the study sites. Most of the farmers owned few different cash crop sites, they will
manage the crops with preference to higher market price.
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th
th
Soil sampling and collection was carried out from 19 January 2012 till 29 January 2012 at Kruin
Village, Sabal. Soil samples were collected from pepper sites ranging from 1 year old to 4 years
old, rubber sites ranging from 6 years old to 23 years old, oil palm sites ranging from 1 year old to
5 years old, fruit farm sites ranging from 8 years old to 29 years o ld and vegetable home garden
sites ranging from 3 years old to 26 years old.
Table 1 Information on study sites at Kruin Village, Sabal
Landuse
No. of sites
o
Slope Degree ( )
Years of cultivation (years)
Rubber (Ru)
6
between 20° to 35°
>10
Pepper (Pe)
5
between 30° to 45°
3 – >10
Oil Palm (OP)
6
between 15° to 25°
1–5
Fruit (Fr)
6
between 5° to 20°
8 – >10
Home Garden* (HG)
6
less than 5°
3 – >10
* All study sites located behind the house, RU; Rubber farm, PE; Pepper garden, OP; Oil palm
field, FR; Fruit orchard, HG; Home garden
Soil samples were collected at five different sites planted with pepper, rubber garden, vegetable
garden, fruit orchard, and oil palm field. Soil samples were collected at a quadrate square of 10m x
10m or 20m x 20m and 20m x 20m or 40m x 40m respectively at all study sites, depends on the size
of the farmlands. Composite soil samples were collected at depths of 0-10cm and 30–40cm from three
random points within the quadrates using hoe. Additional soil samples were also collected from 0–
10cm depth at the fertilizer point of pepper vines and oil palm. Soil was collected at the intersection of
the diagonal lines between four adjacent plants (center point) where the soil structure is undisturbed.
The soil samples that obtain in three replicates were mixed well to obtain a single composite sample. It
should be noted that at all study sites, undisturbed soil samples were collected at the depth of 0 – 10
cm and 30 – 40 cm using 100cc core sampler for determination of soil physical properties. Except for
vegetable gardens, undisturbed soil sample were collected at 0 – 10 cm depth only due to the existing
rocky subsoil.
2.2.1 Procedures for Soil Physicochemical Analysis
Bulk density was determined using fresh samples collected in 100cc core samplers, followed by drying
undisturbed soil samples at 105°C overnight. Soil hardness was measured directly in the field during
soil sampling, using Yamanaka-type push cone penetrometer. Soil particle size analysis is determined
through pipette method to separate the inorganic soil particle into sand silt and clay fractions. Soil
organic matter was measured using the loss in ignition method. Soil pH was determined in water and
1 M KCl in a soil to solution ratio of 1:5 using glass electrodes. Electrical conductivity (EC) was
measured before pH measurement using EC meter. Electrical conductivity is important to determine
the soil particle sizes and texture by determining the amount of soil moisture hold by soil particles.
After the pH and EC measurement, the filtrate is for Al and H measurement. Exchangeable Al was
extracted with 1M of KCl and the concentration was measured by the titration with 0.01M NaOH. The
contents of exchangeable bases were determined by atomic absorption spectrophotometer (AAS) for
Ca, Mg, K and Na.
2.3 Data Analysis
All results of soil analysis will be expressed on an oven dry basis. All statistical analysis will be
performed using SPSS. For comparison of soil properties between sites under different crops,
Tukey’s test of homogeneity of variance will be performed.
3. RESULTS
3.1 Landuse and Farming Practices by Farmers in Kruin Village, Sabal
According to the farmers, the total land allocated for the villagers in Kruin area, Sabal is 1,400
hectares with consist of mainly Native Customary Rights (NCR) lands. Most of the land that
covered Kruin Village is considered as upland areas with the topography of undulating or sloping
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upland areas. These lands were abandoned and developed into mature secondary forests. Due to
the limited availability of flat lands, farmers have no choice but to cultivate the cash crops on
undulating land or slopes. Land areas that were accessible, especially those located nearby the
roadside were selected by farmers as agricultural land. Occasio nally, farmlands thathas difficulty
in accessibility often being abandoned, forming secondary forests that surround the cash crop
farming areas.
Our findings revealed that cash crops cultivation such as rubber, pepper and oil palm cultivation is
important to their livelihood of the farmers in Kruin Village, Sabal for their monetary income.
Several farmers noted that cocoa was ever once planted by the villagers but at present, were mostly
abandoned due to disease infestation which damaged the cocoa trees. Furthermore, the farmers
indicated that cash crops cultivation at the Kruin village mostly determined by the current market
price of the cash crops. In addition to this, it is also noted that farmers cultivated cash crops with
less maintenance at the farmlands that are far away for the purpose of land security.
Our findings showed that most of the farmers apply fertilizers at various rate on their cash cro p
farmlands. As reported by Capistrano & Marten (1986), fertilizer application is necessary in order
to provide additional nutrients to support the growth of cash crops. Pesticides and herbicides were
also applied in the current farming practices and these agrochemicals are mainly obtained from
subsidy by local authority i.e. Agriculture Department, Malaysia Pepper Board or purchased using
their own money.
[
3.2 Soil Physicochemical Properties of Study Sites
The information on the soils physicochemical properties within the depth of 0 -10 cm (surface soil)
of all study sites such as home garden, rubber field, fruit orchard, oil palm field and pepper garden
were computed in the Table 2. Further detailed discussion of the report on soil physicochemical
properties will emphasize on the surface layer (0-10 cm) due to time limitation in completing
others soil physicochemical analysis.
3.2.1
Surface Soils Physicochemical Properties
As showed in Table 2, the surface soils (0-10cm) in the cash crop farming areas can be
characterized as strongly acidic in nature with pH(H 2O) level of less than 5. The acidic nature of
the sites in the cash crop farming areas including rubber field, fruit orchards, oil palm fields and
-1
pepper garden were caused by high level of exchangeable Al which ranged from 3.04 cmolckg to
-1
4.91 cmolckg . The soil organic matter of the soils in all sites excluding home garden was between the
ranges of 5.2 % to 6.2 % while the exchangeable bases such as K, Ca, Mg and Na of all sites were
-1
-1
low, ranging between 0.41 cmolckg to 1.00 cmolckg . The soil bulk density for the surface soil at all
-1
-1
sites except home garden ranging from 1.04 gmL to 1.19 gmL , giving rise to soils hardness from 14
mm to 19 mm. In each landuse type except home garden, the soil texture of study sites were acidic in
nature, relatively sandy with low clay which corresponds with the soils properties as reported by
Kendawang et al. (2005) on soils in secondary forests at Sabal area.
In general, the soils physicochemical properties of home garden were significantly different from
other cash crop farming areas such as rubber field, fruit orchard, oil palm field and pepper garden
(Table 2). Soils under home garden were relatively sandy with low clay contents. In spite, the
pH(H2O) of soils showed a high value of 7.08 at the surface layer, giving rise to high electrical
-1
conductivity value of 110.6 µScm . The contents of exchangeable bases, especially exchangeable
Ca were high while that of exchangeable Al was low. In addition, bulk density of home garden is
-1
significantly high with a value of 1.41 gmL , giving rise to high value of soil hardness. Based on the
field observation, the soils at all home garden sites were whitish-grey with sandstones. However, the
soils under other study sites were yellowish brown non-calcareous sedimentary rocks. The parent
materials of the home garden sites were different from the cash crops farming sites which were
categorized under non-calcareous sedimentary rocks.
In rubber field, the surface soils were acidic with low pH(H 2O) with higher contents of
exchangeable Al than soils in home garden. The value of pH(H2O) in rubber field corresponds with
the soil pH(H2O) of secondary forests in the study conducted by Tanaka et al. (2007), indicating
similarities between rubber field and secondary forest soil physicochemical properties. In addition,
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
negative correlation between pH(H2O) and exchangeable Al at the surface soil were observed (data
not shown). The exchangeable bases, especially exchangeable K at the rubber fields were very
low, indicating poor nutrients contents in the soils under rubber farmin g.
The soil physicochemical properties in fruit orchards were similar to those in rubber fields. The
pH(H2O) of fruit orchards was low with 4.96, giving rise to low electrical conductivity. Soil organic
matter of fruit orchard is 5.3 %, showing similarity to the soil organic matter contents in secondary
forest in the study conducted by Lattirasuva et al. (2010). The exchangeable bases of fruit orchard
were slightly higher than that of rubber field, especially exchangeable K. No substantial differences
were found between the soils properties in fruits orchard and rubber fields. The soils in fruits orchard
were acidic with high exchangeable Al contents.
The soils under pepper farm were characterized by a less acidic nature with higher contents of
exchangeable bases. Exchangeable K in pepper farm were high as compared with other study
sites. Clay contents of pepper garden were slightly higher compared with other landuse. However
Table 2. Soil physicochemical properties of surface soils (0-10 cm) of HG, Ru, Fr, OP and Pe
Sites
Soil
physicochemical
Home garden
Rubber
Fruit
Oil Palm
Pepper
properties
(n=6)
(n=5)
(n=6)
(n=6)
(n=5)
Surface soil , 0-10 cm depth
pH(H2O)
7.08±0.90b
pH(KCl)
5.88±1.36b
-1
EC (µScm )
110.6±46.7b
-1
Exch.Al (cmolckg )
0.18±0.06a
-1
Exch. K (cmolckg )
0.10±0.06n
-1
Exch. Na (cmolckg )
0.06±0.00n
-1
Exch. Mg (cmolckg )
0.22±0.08n
-1
Exch. Ca (cmolckg )
1.06±0.09b
a
-1
Sum bases (cmolckg )
1.44±0.17b
b
SOM (%)
3.3±0.9n
Clay (%)
6±4a
Silt (%)
29±23n
Sand (%)
65±26n
-1
Bulk density (gmL )
1.41±0.10b
4.82±0.21a
3.44±0.10a
39.1±8.3a
4.91±2.42b
0.07±0.02n
0.06±0.00n
0.09±0.07n
0.18±0.19a
0.41±0.25a
6.0±1.5n
15±7b
40±27n
45±25n
1.04±0.17a
4.96±0.20a
3.45±0.06a
39.0±9.3a
4.54±1.31b
0.10±0.04n
0.07±0.00n
0.15±0.13n
0.42±0.33a
0.73±0.47a
5.3±2.4n
18±5b
34±17n
49±19n
1.15±0.19a
4.97±0.26a
3.31±0.17a
42.1±13.6a
3.04±0.72b
0.08±0.03n
0.06±0.00n
0.23±0.13n
0.39±0.32a
0.76±0.44a
6.2±1.9n
12±3ab
37±23n
51±25n
1.19±0.07ab
4.89±0.25a
3.35±0.03a
49.8±18.9a
3.53±0.91b
0.13±0.01n
0.07±0.01n
0.22±0.07n
0.59±0.17a
1.00±0.25ab
5.2±0.6n
19±3b
43±24n
38±23n
1.19±0.09ab
c
Hardness (mm)
18±3ab
14±3ab
15±3a
15±2ab
19±2b
Parent Material
Whitish sandstone
Non-calcareous sedimentary rock
Means ± standard deviations: values in the same row followed by different letters are significantly
a
b
different at P < 0.05 (Tukey HSD test). Sum of Exchangeable Base (K, Na, Mg, Ca), SOM; Soil
c
organic matter, Determined using Yamanaka push type penetrometer.
, there is no significant correlation in clay contents between pepper gardens with other study sites.
Bulk density and soil hardness of pepper garden were relatively high, indicating considerable soil
compaction of the surface soils at the study sites.
In the oil palm field, soils physicochemical properties at the surface soils (0-10cm) showed similarities
with those found in pepper farm. Compared with rubber field, the soils were less acidic and richer with
nutrients. Soil organic matter at oil palm field was the highest among all other agriculture landuses.
However, there were no apparent correlations between the relationships of soil organic matter in oil
palm field with other study sites.
3.2.2 Soils Properties under Fertilizing Points of Pepper Farms and Oil Palm Fields
In pepper farms and oil palm fields, additional soil sampling point which is fertilizing point were
collected at the fertilizing circle of the pepper vines and oil palm stands. Both of these crops were
under intensive farming practices and need a higher maintenance as compared with other cash crops.
Table 3 compared the average values of selected soil physicochemical properties at fertilizing
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
point in pepper farm (Pe-F) and oil palm field (OP-F) as well as non-fertilizing point in pepper farm
(Pe-N) and oil palm field (OP-N) at the depth of 0-10 cm.
At fertilizing point, pH(H2O) of soils was lower than that of non-fertilizing point. In contrast, study
conducted by Tanaka et al. (2009) at Lubok Antu reported that the pH(H 2O) at the fertilizing point
should be higher than that of non-fertilizing point due to external bases added through fertilization.
Nevertheless, soil organic matter contents of fertilizing point (Pe -F) were higher than of nonfertilizing point (Pe-N). Presence of soil organic matter at the fertilizing point might c ause acidity
at the fertilizing point of the pepper farm. The contents of exchangeable bases, especially
exchangeable K were high at the fertilizing point of pepper farm, resulting in relatively high value
of electrical conductivity. However, the contents of exchangeable K in the fertilizing point of
pepper farm were low as compared to the study conducted by Tanaka et al. (2009). At the
fertilizing point (OP-F) and non-fertilizing point (OP-N) of oil palm field, the soils physicochemical
properties resembled as those in pepper farms.
Table 3. Comparisons of soil physicochemical properties of fertilizing point (FP) and non fertilizing
point (Non-FP) of OP and Pe at the depth of 0-10 cm
Sites
Soil Physicochemical
OP-N
OP-F
Pe-N
Pe-F
Analysis
(Non-FP)
(FP)
(Non-FP)
(FP)
pH(H2O)
4.97±0.26n
4.72±0.23n
4.89±0.25n
4.74±0.73n
pH(KCl)
3.31±0.17n
3.28±0.20n
3.35±0.03n
3.38±0.33n
-1
EC
µScm
42.14±13.56a
76.02±79.27ab
49.81±18.93a
244.54±198.21b
a
SOM
%
6.24±1.90n
6.34±2.73n
5.15±0.55n
5.88±1.31n
-1
Exch.Al
cmolckg
3.04±0.72n
3.51±0.88n
3.53±0.91n
3.99±2.25n
-1
Exch. K
cmolckg
0.08±0.03a
0.13±0.06a
0.13±0.01a
0.22±0.08b
-1
Exch. Na
cmolckg
0.06±0.00n
0.07±0.00n
0.07±0.01n
0.07±0.01n
-1
Exch. Mg
cmolckg
0.23±0.13n
0.15±0.10n
0.22±0.07n
0.31±0.14n
-1
Exch. Ca
cmolckg
0.39±0.32n
0.38±0.26n
0.59±0.17n
0.72±0.30n
b
-1
Sum
cmolckg
0.76±0.44n
0.73±0.34n
1.00±0.25n
1.32±0.40n
a
b
Soil organic matter of the soils, Sum of exchangeable bases (K, Na, Mg, Ca). Means ± standard
deviations: values in the same row followed by different letters are significantly different ar P < 0.05
(Tukey HSD test).
4. DISCUSSION
4.1 Soil Fertility under Vegetable Home Garden Cultivation
Soils under home garden were characterized by a less acidic with higher contents of nutrients,
corresponds with the soils properties reported by Tanaka et al. (2009) and Lattirasuvan et al.
(2010). The high nutrient contents of surface soils in the home gardens were not ascribable to
current fertilizers application activities. Based on the informat ion obtained from interview, home
garden received limited to no chemical fertilizers input. Therefore, it can be assumed that high
exchangeable Ca in the soils of home garden was due to the input from external sources of soils
under different parent material during the period of residential construction. The comparison
-1
between pH(H 2O) and exchangeable Ca (cmol ckg ) was showed in the figure below. The red
circle showed the home gardens site with high of exchangeable bases which resulted in the high
value of pH(H2O).
Another factor that might correspond to the high value of exchangeable Ca at the home gardens
was related with bulk density. High bulk density of the soils under home garden cultivation
probably caused by high intensity of human trampling regarding on activities such as planting,
weeding and harvesting the vegetables crops at the home garden sites. Lattirasuvan et al. (2010)
explained that high bulk density results in higher soils compaction which will lower the water
permeability, thus preventing the excess loss of nutrients leaching downwards despite of high
rainfall at the study area which is around 4000mm annually. USDA-NRCS (2008) reported that
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
high bulk density is an indicator of low soil porosity and soil compaction which will cause
restriction to root growth with shallow plant rooting. In addition, most of the home gardens studied
located at the flat area (slopes degree < 5°) which could prevent nutrients loss due to erosion and
runoff.
In addition to that, additional bases from ash effects due to burnt materials and leftover unburned
materials such as charcoal from household waste were one of the factors that increase the
exchangeable Ca in the soils. Based on field observation, the presence of charcoal and unburned
materials can be observed at all home garden sites during our field visitation, suggesting that the
sites were previously burnt. According to Nye & Greenland (1990), burning increases soil fertility
as it converted the burned household waste into nutrient – rich ash that deposited on the soil
surface and incorporated into the soil through rainfall and continuous cultivation. External input of
bases through burning provides additional nutrients to the vegetables cultivations in home garden
as an alternative to agrochemicals. W ith the slope less than 5° in the home garden, current
agricultural practices is considered sustainable with high nutrients input through natural
decomposition and regeneration with low input of agrochemicals.
4.2 Soil Fertility under Rubber and Fruit Cultivation by Local Farmers
Based on the soil analysis, the soils under rubber and fruit cultivation in Kruin Village, Sabal were
characterized by acidic nature and sandy in texture, which was mainly caused by the high
-1
-1
exchangeable Al at the study sites which is 4.91 cmolckg and 4.54 cmolckg . The high exchangeable
Al at both of the study sites were related to the parent materials which is highly weathered. von
Uexküll & Bosshart (1989) mentioned that high acidity due to aluminium occurrence has resulted
in lesser amount of available cations, which in turn resulted in low cation exchange capacity
(CEC) of the soil. In relation to this, the contents of exchangeable bases were very low in both
rubber fields and fruit orchards. Based on the interview, farmer s terminated fertilizers application
to the rubber stands 3 years after planting. As for fruit orchards, fertilizer applications were low or
limited. At most cases, the trees were left to bear fruits without or limited input of fertilizers.
Therefore, it can be assumed that low pH(H 2O) at the rubber fields and fruit orchards were caused
by low fertilizers input.
Study revealed that soil physicochemical properties under rubber and fruit cultivation at Kruin Village,
Sabal showed similarities. Table 4 below showed the comparison of selected soil physicochemical
properties between rubber fields and fruit orchards.
Table 4. Comparison of soil physicochemical properties under rubber and fruit cultivation
Sites (0-10cm)
Parent material
pH(H2O)
pH(KCl)
Bulk density
Sum of bases
Fruit
Non-calcareous
4.96
3.45
1.15
0.76
(8 - > 10 years)
sedimentary rocks
Rubber
Non-calcareous
4.82
3.44
1.04
0.34
(> 10 years)
sedimentary rocks
Similarities in soil physicochemical properties probably related to the similar farming practices under
rubber and fruit cultivation. Although the input of fertilizers were low, the farmers still able to sustain
the production of the crops. Therefore, it could be clarified that rubber and fruit cultivation with low
external input of agrochemicals can be regarded as sustainable farming in the upland farming system
of Iban culture. Moreover, rubber and fruits cultivation encouraged regeneration of forests from
previous shifting cultivation landuse. Based on the analysis, similarities of soils development under
fruits cultivation suggest that fruits trees might be suitable for the reforestation purposes at the study
areas in spite some farmers cultivated fruits for land security purposes. In addition, de Jong (2001)
pointed out that the rate of deforestation decrease after the rubber plantings were introduced into the
agriculture practice among the Dayaks farmers in Sarawak. Consequently, current low tapping
intensity was able to sustain the soil physicochemical properties, indicates that rubber plantings
encourage reforestation and sustainable agriculture as well as socio-economic developments of the
the local Iban farmers at the upland farming areas.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
4.3 Soil Fertility under Pepper and Oil Palm Cultivation of the Farmers at Kruin Village, Sabal
In the pepper farm and oil palm field, the soils were less acidic with higher exchangeable bases
compared to the rubber fields and fruit orchard. The bulk density in pepper farm and oil palm field
were also high, indicating regular farm works and human trampling effects which caused surface soil
compaction in the study sites. High exchangeable K, especially at the fertilizing circle indicates regular
fertilizers application at the pepper garden and oil palm field. Based on the interview, farmers applied
compound fertilizers N-P-K-MgO + Te with the ratio of 12:12:17:2 which are high in K 3 to 4 times
annually. Therefore, this has accounted for the high exchangeable K contents at the study sites,
particularly at the fertilizing circle.
Based on the soil analysis result on the present study, the soil physicochemical properties of the oil
palm field and pepper garden cultivated by local farmers showed similar properties. This is closely
related with the farming practices and management at both study sites. For pepper cultivation and
smallholder based of oil palm cultivation, methods of fertilizers application should be improved to
increase the efficiency use of chemical fertilizers and reduce the rate of soil erosion. Good agricultural
practices such as establishment of legume cover crops, intercropping, mixed farming and mulching
should be incorporated into the current farming practice.
5.0 CONCLUSION AND RECOMMENDATIONS
In nutshell, soils under most of the study sites were mainly acidic in nature , characterized by
sandy texture with relatively low nutrients contents. The chemicals level in the soils were low,
indicating low fertilizers input to the soils, contrary to the expectations that agrochemicals input at
the sloping and undulating uplands agriculture often result in leaching, nutrient runoff and erosion.
Study revealed that current farming practices in the upland agriculture were localized with little
agrochemicals input. Therefore, it does not resulted in negative impacts to the soils. With little
amount of fertilizers input, cash crops cultivated still, have the ability to sustain the production. In
addition to this, only a small portion of lands which are surrounded by secondary forest are
selected and cultivated intensively by farmers, thus, the small and acceptable rate of nutrients
runoff at the pepper garden and oil palm field does not reflect severe environment degradation.
Thus, it could be clarified that current farming practices in the upland area of Kruin Village, Sabal
is considered as sustainable as farmers are able to sustain the yield and productivity of the cash
crops cultivated with little impacts on soils.
Acknowledgement
This research was financially supported by the research grant by Borneo Rainforest Project
(Sarawak). We wish to express our gratitude to the Director and staff of the Forest Department,
Sarawak for their supportive assistance during the duration of this study. We would also like to extend
our thanks to community of Sabal Kruin (Rh Dingin, Rh Ijau, Rh Niah, Rh Baong and Rh Rendak)
village especially to Mr. Ikin Empati for their kind co-operation and hospitality during the field survey.
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
THE EFFECTS OF ROOTING MEDIA AND STOCK PLANT AGE ON ROOTING OF
Aglaia stellatopilosa
Johari, S.*, Tu, C.L., Yeo, T.C. and Manurung, R.
Sarawak Biodiversity Centre (SBC), KM20, Jalan Borneo Heights, Semengoh, Locked Bag No. 3032,
93990 Kuching, Sarawak (MALAYSIA)
*Email: jsuria@sbc.org.my
Abstract
Aglaia stellatopilosa Pannell (Meliaceae), also known as Segera (Iban), Rukang (Lun Bawang) and
Kela buno (Kelabit), a species endemic to Borneo, can be found in Sarawak, Sabah and Kalimantan.
Clonal propagation of this species is necessary to enable continuous supply of raw plant materials for
extraction of bioactive compounds and conservation of this species. This research is aimed at
developing a clonal propagation method for A. stellatopilosa through rooting of stem cutting.To
evaluate the rooting ability of cuttings, type of rooting medium and effects of stock plant age were
investigated. Significantly higher number of stem cuttings from mature tree rooted in sand:perlite (1:4
v/v) medium when compared to other media. Cuttings from young trees rooted well with at least 68%
rooting success rate whilst cuttings from mature trees rooted poorly with only a maximum of 17%
rooting rate. Cuttings obtained from young trees required less than 12 weeks to develop roots whilst a
period of 12 weeks is required to root cutting from mature trees. These results indicate that clonal
propagation of A. stellatopilosa is possible using rooted cuttings. Further work is required to improve
the rooting ability of the cuttings using juvenile shoots obtained by bending.
Keywords: Rooting ability, endemic species, bioactive compounds, conservation
1. INTRODUCTION
Aglaia is one of more than 100 species that belong to the family Meliaceae. Aglaia spp. commonly
found in Sabah and Sarawak, also occurs in Kalimantan but has not been recorded in Brunei
(Soepadmo et al. 2007). Aglaia spp. are dioecious trees or shrubs with small fragrant flowers. All
species of Aglaia are woody, ranging from a few metres to large trees up to 40m in height (Pannell,
1992). It is reported that A. stellatopilosa typically grows to a height of 8m tall and has a diameter of
16 cm at breast height. An Aglaia fruit bunch is similar to the tropical fruit Langsat from the genus
Lansium of the same family (Chuah et al. 2006).
Recently, Aglaia spp. have attracted considerable interests among scientists due to their importance
as source of bioactive products such as 1H-cyclopental [b] benzofuran derivatives (Kim et al., 2007
and Hwang et al., 2004). These compounds are active as insecticides and potentially as antitumor
agents. Aglaia spp. also produce other compounds such as rocaglamide derivatives, lignans,
flavonoid and bisamides which exhibit cytotoxic and antiviral properties. Hwang et al. (2004)
discovered that A. stellatopilosa contain a complex rocaglate that posses cytotoxic activity in human
cancer cell lines and have been shown to block protein biosynthesis and induce growth arrest in
certain tumor cell lines (Hwang et al. 2004). Apart from that, Chuah et al. (2006) reported the uses of
several Aglaia species in traditional medicine for treatment of tumours (bark); asthma, fever,
headache, jaundice, tonic and wounds (leaves); asthma, fever and jaundice (flowers); inflammation
(fruits).
Currently, there is demand for plant materials to provide sufficient quantity of targeted bioactive
compound for further studies. To our knowledge, there is very little information and research on in vitro
and vegetative propagation methods of A. stellatopilosa. For this purpose, in vitro and vegetative
propagation studies on A. stellatopilosa in the wild were done to provide adequate plant materials for
research. Therefore, this research was conducted to determine the appropriate rooting media and
types of stock plant for vegetative propagation of this species via stem cutting.
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2. MATERIALS AND METHODS
2.1 Samples collection
The samples were collected from Botanical Research Centre (BRC) and Sarawak Biodiversity Centre
(SBC) jungle. Cuttings from shoots of young trees (3-5 years old) and mature trees (20 years old and
more) were collected in April 2012. Stem cuttings 8 to 10 cm long with at least 5 nodes were made.
Leaves were trimmed so that only two pairs of leaflet from opposite side remained on the cuttings.
During the collection activity, the cut ends of the cuttings were wrapped in moist tissue paper and
aluminium foil.
2.2 Experimental designs
2.2.1 Effects of different types of rooting media
Stem cuttings from mature trees were used in this experiment. The cuttings were placed in four
different rooting media which are sand, perlite, sand:perlite (1:4 v/v) and commercial soil mix:perlite
(1:4 v/v) with eighteen cuttings per treatment. The cuttings were kept in polypropagator covered with
inverted V-shape acrylic top. Cuttings were watered daily in order to keep the rooting media moist until
the cuttings rooted. Rooted cuttings were then transferred into polythene bag filled with commercial
soil mix (primarily hardwood bark and vermiculite). All rooted cuttings were hardened under nursery
condition.
2.2.2 Effects of stock plant age
To test the effect of stock plant age, stem cuttings were collected from two types of stock plants;
young trees (3-5 years old) and mature trees (≥20 years old) with twenty five cuttings per treatment.
Perlite:sand (1:4 v/v) mix was used as the rooting medium. The cuttings were kept in polypropagator
covered with acrylic top and watered every morning. Rooted cuttings were transferred into polythene
bag filled with commercial soil mix and hardened under nursery condition.
2.3 Data collection and analysis
After 12 weeks, the appearance of root system was observed. The root system was gently washed
and the number of roots measuring at least 1mm long was determined for each cuttings. The number
and length of each root (primary and secondary) were counted and measured. The number of living
cuttings (rooted, callused, no response) and dead cuttings were recorded in both experiments. The
percentage of rooted stem cuttings from mature trees in different types of rooting media were
recorded, that is, rooted cuttings as a proportion of planted cuttings in each experiment unit.
Meanwhile, t-test (p < 0.05) was used to compare the number of roots per rooted cutting and longest
root length of primary root and secondary root from different stock plant age. The percentages of
rooted cuttings were calculated.
3. RESULTS AND DISCUSSION
3.1 Effects of different types of rooting media
The results show that cuttings taken from mature trees required 12 weeks to root. As shown in Figure
1, greater number of stem cuttings from mature tree rooted in sand:perlite (1:4 v/v) medium (17%)
compared to other media (0%). None of the cuttings propagated in sand rooted or even produced
callus. Although no rooting was observed, callus formation was present on cuttings in perlite (33%),
perlite:sand (1:4 v/v) (17%) and soil mix:perlite (1:4 v/v) (11%). The number of unrooted cuttings was
highest in sand (56%) compared to other types of media, that is perlite (6%), perlite:sand (1:4 v/v)
(11%) and soil mix:perlite (1:4 v/v) (11%). Highest cutting mortality was observed in soil mix:perlite
(1:4 v/v) (78%), followed by perlite (61%), sand:perlite (1:4 v/v) (56%) and sand (44%).Other than that,
the cuttings with no response on rooting media were higher in sand (56%) compared to other types of
media, that is perlite (6%), perlite:sand (1:4 v/v) (11%) and soil mix:perlite (1:4 v/v) (11%). Higher
number of stem cuttings dead were observed in soil mix:perlite (1:4 v/v) (78%), followed by perlite
(61%), sand:perlite (1:4 v/v) (56%) and sand (44%).
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Types of rooting medium seemed to affect the rooting ability of cuttings from mature trees. This may
be the reason for the higher number of cuttings that rooted in perlite:sand (1:4 v/v). Rooting
percentage increased in medium that is sterile, with good aeration or able to hold moisture (Longman,
1993). The effects of different media on the formation of roots and callus on the cuttings are shown in
Figure 2.
Figure 1. Effects of different types of rooting media on development of root, callus, without response
and dead cuttings obtained from mature trees
Note: The number at top of each bar denotes the number of cutting under each of the four categories
of response in each of the four rooting media.
a
b
Sand
Perlite
c
b
Sand:Perlite (1:4 v/v)
Soil mix:Perlite (1:4 v/v)
Figure 2. Effects of different types of rooting media on development of root (a; no response, b;
callused and c; rooted) on cuttings obtained from mature trees
3.2 Effects of stock plant age
The number of roots developed and the length of root per rooted cutting were significantly (p < 0.05)
influenced by the stock plant age (young trees and mature trees). Cuttings from young trees were
observed to require less than 12 weeks to develop roots whilst those from older or mature trees
required 12 weeks to root. More cuttings from young trees rooted (68%) compared to cuttings from
mature trees (4%) (Data not shown). The number of roots per rooted cutting and the root length of
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both primary and secondary roots were significantly (< 0.05) higher in cuttings obtained from young
trees than those obtained from mature trees (Table 1). The effects of stock plant age on root
development are shown in Figure 5. Cuttings taken from young trees rooted well and produced more
roots than cuttings taken from older or mature trees. Ky-Dembele et al. (2011) found that age of the
stock plant is the most critical factor affecting vegetative propagation. They are many literatures report
that cuttings taken from young trees much more easy to root than those from mature trees (KyDembele, 2011), although there are also some reports of successful propagation by cuttings from old
mature trees (Leakey, 2004). The ability of cutting to root has high correlation with carbohydrate level
in the stem (Baker, 1992), in which rooting is promoted by the production of specific sugars during the
period that the cuttings are in the propagator (Leakey, 2004).
During the plant sample collection, it was observed that the environmental factor such as temperature
and weather play a crucial role in ensuring the survival rate of cutting samples. Transportation of the
cutting samples from the collection site to SBC could also affect the outcome. Baker (1992), explained
that there are two main factors affecting rooting of cuttings which could be either internal or
environmental factor, or both. The ability of cutting to root has high correlation with carbohydrate level
in the stem (Baker, 1992), in which rooting is promoted by the production of specific sugars during the
period that the cuttings are in the propagator (Leakey, 2004). For plants that are difficult to root, stem
cuttings taken from young tree (in the juvenile growth phase) have been shown to root easily than
those taken from mature tree (Agbo and Obi, 2007). If due to some reasons where mature tree is
desired, the felling and subsequent coppicing of mature trees is generally regarded as the best way to
return to the juvenile state (Leakey, 2004).
Table 1. Mean number of roots per rooted cutting and length of longest primary and secondary roots
from mature and young stock plant
Stock plant
Primary root
Secondary root
No. of roots per rooted Longest root length No. of roots per rooted Longest root length
cutting
(mm)
cutting
(mm)
a
0.68 ± 0.68
a
0.16 ± 0.16
a
0.20 ± 0.20
a
b
6.28 ± 2.16
b
1.72 ± 0.69
b
1.89 ± 0.62
b
Mature tree
0.04± 0.04
Young tree
1.12 ± 0.22
All values are mean ± SE. Means with the same superscript in the same column are not significantly
different at p < 0.05 using t-test
Mature tree
Young tree
Figure 3. Effects of stock plant age on development of root
4. CONCLUSION
These experiments indicated that cuttings in sand:perlite (1:4 v/v) medium gave the highest rooting
percentage with good root development. Stem cuttings taken from young trees appeared to be the
more suitable as planting material compared to those obtained from mature trees. The cuttings
obtained from young trees rooted well whilst cuttings from mature tree rooted poorly. Further work is
required to improve the rooting ability of the cuttings by using juvenile samples induced by bending of
mature tree and young tree.
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Acknowledgement
We would like to extend our appreciation to the SBC for giving us the opportunity to conduct this study.
We would also like to thank our colleagues especially those directly involved in A. stellatopilosa
propagation work. We also gratefully acknowledge help from the Forest Department Sarawak and
Sarawak Forestry Corporation for their support in this project.
REFERENCES
Agbo, C.U., & Obi, I.U., (2007). Variability in propagation potentials of stem cuttings of different
physiological ages of Gangronema latifolia Benth. World Journal of Agricultural Science., 3(5):
576-581.
Chuah, C.H., Mok, J.S.L., Liew, S.L., Ong, G.H.C., Yong, H.C., & Goh, S.H. (2006). 101 plants to fight
cancer. Malaysia: Pepustakaan Negara Malaysia.
Hwang B.Y., Su B.N., Chai H., Mi Q., Kardono L.B., Afriastini J.J., Riswan S., Sastarsiero B.D., Jungle
R., Fairchild C.R., Vite G.D., Rose W.C., Farnsworth N.R., Cordell G.A., Pezzuto J.M.,
Swanson S.M., and Kinghorn A.D. (2004). Silvestrol and episilvestrol, potential anticancer
rocaglate derivatives from Aglaia silvestris. J Org Chem. 69(18):6156.
Kim, S., Hwang B.Y., Su B.N., Chai H., Mi Q., Kinghorn A.D., Jungle R., and Swanson S.M. (2007).
Silvestrol, a potential anticancer rocaglate derivative from Aglaia foveolata, induces apoptosis
in LNCaP cells through the mitochondrial/apoptosome pathway without activation of
executioner caspase-3 or -7. Anticancer Research. 27: 2175-2184.
Ky-Dembele, C., Tigabu, M., Balaya, J., Savadogo, P., Boussim, I.J., & Oden, P.C. (2011). Clonal
propagation of Khaya senegalensis: The effects of stem lenght, leaf area, Auxins, smoke
solution, and stock plant age. International Journal of Forestry Research.
Liu, Z., and Adams, J. (1996). Camptothecin yield and distribution within Camptotheca acuminata
trees cultivated in Lousiana. Can. J. Bot. 74: 360-365.
Leakey, R.R.B., (2004). Physiology of vegetative reproduction. In Encyclopedia of Forest Sciences.
Longman, K.A. (1993). Rooting cuttings of tropical trees. Commonwealth Science Council.
Pannell, C.M., (1992). A monograph of Aglaia. United Kingdom: HMSO.
Soepadmo, E., Saw, L.G., Chung, R.C.K., and Ruth Kiew. (Eds.). (2007). Tree flora of Sabah and
Sarawak (Vol 6). Kuala Lumpur: Ampang Press.
Zsuffa, L. (1992). Experiences in vegetative propagation of Populus and Salix and problems related to
clonal strategies. In F.W.G Baker (Ed.), Rapid propagation of fast growing woody species
(pp.86-97). United Kingdom.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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MICROPROPAGATION OF Aquilaria malaccensis LAMK. (KARAS) THROUGH SOMATIC
EMBRYOGENESIS
Zul Helmey, M. S.,* Sepiah, M. and Sani, H.B.
Plant Tissue Culture Laboratory, Department of Plant Science and Environmental Ecology, Faculty of
Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak.
*Email: zul_87fh@yahoo.com
Abstract
Aquilaria malaccensis δamk., locally known as ‘gaharu’ or ‘karas’, is of economic importance as
source of gaharu used for incense, traditional medicine and other products. Currently, the high value
of gaharu stimulates illegal harvesting and as a result, Aquilaria trees are often cut down
indiscriminately. Populations of this tree have declined and may lead to possible extinction in the near
future. The objective of this study is to develop an in vitro culture method for mass propagation of this
species via somatic embryogenesis. In this study, young leaves of A. malaccensis were used as the
explants and cultured on modified Murashige and Skoog (MS) medium with various plant growth
regulators (PGRs) at different concentrations. The highest percentage number of explant developed
embryogenic callus was obtained at concentration of 2.0 and 5.0 mg/L 2,4-D plus 0.5 mg/L BAP with
100% after four weeks. Modified MS medium added with 1 g/L activated charcoal, 0.1 mg/L Kinetin,
0.5 mg/L BAP and 2,4-D at 2.0 and 5.0 mg/L was the best medium for induction of somatic embryos
from embryogenic callus. Callus clumps formed somatic embryos within four weeks. For maturation,
modified MS medium supplemented with Kinetin at 2.0 and 4.0 mg/L, 0.01 mg/L Abscisic acid (ABA)
and 0.1 mg/L Gibberrelic acid (GA3) was the best promoted the maturation of somatic embryos.
Secondary (repetitive) somatic embryogenesis was also found in the culture. The mature embryos
grew into normal plantlets when cultured on medium with half-strength macro salts of modified MS
medium without plant growth regulators. Complete plantlets will be acclimatized and hardened using
soil, peat and sand in the ratio 1:1:1.
Keywords: Aquilaria malaccensis, embryogenic callus, somatic embryogenesis.
1. INTRODUCTION
Aquilaria species belongs to the family Thymelaeaceae, is of economic important source of non timber
forest product, gaharu or agarwood (Soehartono and Newton, 2000). The genus Aquilaria is
distributed in southern Asia from India to China and throughout most of Southeast Asia (Whitmore,
1972). In Malaysia, there are five species of Aquilaria, i.e., A. malaccensis, A. microcarpa, A.
beccariana, A. hirta and A. rostrata (Chang et al., 2002). Aquilaria malaccensis is the major producer
of gaharu in Malaysia. Gaharu also known as eaglewood or agarwood is a fragrant wood that has
been traded since biblical times for use in religious functions, medicinal and aromatic preparations
(Zich and Compton, 2001; Lim and Noorainie, 2010).
For hundreds of years, gaharu has been harvested from forests, and this has resulted in decline in the
number of Aquilaria trees. As a result, all species of Aquilaria including A. malaccensis have been
listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna
and Flora (CITES, 1994). In addition, the normal propagation of A. malaccensis by seed is difficult.
Because the seed low germination rate, low viability and delayed rooting of seedling (Rusli et al.,
2007). Thus, plant production through conventional method cannot be relied efficiently as the main
source of raw materials. In view of its potential commercial value as a plantation crop as well as to
conserve its germplasm and for production of gaharu, tissue culture via somatic embryogenesis can
be effectively used for plant regeneration and mass production of seedlings.
Tissue culture techniques as a means for conserving and multiplying medicinal plants have been
reported by Joshi and Dhar (2003), Fracaro and Echeverrigaray (2001) with the aim for large scale
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production of plant material. Somatic embryogenesis offers an alternative and efficient means for plant
multiplication (Gaj, 2004). This study aims to look into the micropropagation system of A. malaccensis
via somatic embryogenesis as a starting point for production of plantlets through in vitro cell culture
technique.
2. MATERIALS AND METHODS
The study started with an attempt to establish axenic (contamination free) culture. The axenic culture
could then be used in subsequent experiments on induction and development of somatic
embryogenesis. The healthy seedling of A. malaccensis used as stock plants were selected from the
UNIMAS Arboretum.
2.1 Induction of Embryogenic Callus
In this study, young leaves explants of A. malaccensis were surface sterilized in 15% for 10 minute of
Clorox, together with 0.1% Tween 20 (a wetting agent) with gentle agitation followed rinsed three to
five times (3-5 minutes each time) with sterilized distilled water. The explants were cultured in Petri
dish containing Murashige and Skoog (1962) medium without any hormone. For embryogenic callus
initiation, explants were cultured on modified MS medium containing 30 g/L sucrose and gelled with
0.3% (w/v) ‘Gelrite’ with supplement of 2,4-dichlorophenoxyacetic acid (2,4-D) plus 0.5 mg/L 6benzylaminopurine (BAP). The pH of the medium was adjusted to 5.8 prior to autoclaving. When
o
o
medium cooled down to about 50 C to 55 C, various concentration of 2,4-D ranging from 1.0 mg/L to
5.0 mg/L was added and dispensed to Petri dish. The cultures were incubated at room temperature
and in dark on the shelf of culture room condition until the embryogenic callus were formed. There four
replicates with four explants in each replicates. The callus cultures were separate and subculture to a
fresh medium every four weeks using the same conditions described above. Data on number of
explants that developed embryogenic callus and number of embryogenic callus in each Petri dish
were recorded weekly after sign of embryogenic callus formation.
2.2 Induction of Somatic Embryos
The embryogenic callus was cultured on modified MS solid medium supplemented with 30 g/L
sucrose, gelled with 0.3% (w/v) ‘Gelrite’ with supplemented 1.0 g/δ neutralize activated charcoal,
0.1mg/L Kinetin, 0.5 mg/L BAP and 2,4-D at 1.0, 2.0, 3.0, 4.0 and 5.0 mg/L. Four clumps of
embryogenic callus in each Petri dish with four replicates. The cultures were placed in the culture
room for 4 to 6 weeks under dark condition. At the end of the experiments, the frequency of embryo
formation was determined.
2.3 Maturation of Somatic Embryos
Somatic embryos at early developmental stages, mostly globular stage were selected and cultured on
a modified MS solid medium containing half-strength salt supplemented with 30 g/L sucrose, gelled
with 0.3% (w/v) ‘Gelrite’, 1.0 g/δ neutralized activated charcoal, 0.01 mg/δ Abscisic acid (ABA), 0.1
mg/L Gibberrelic acid (GA3) and different concentration of Kinetin at 1.0, 2.0 and 4.0 mg/L for four
weeks. Four clump of embryos in each Petri dish with four replicates. The cultures were placed in the
culture room for 4 to 6 weeks under light condition (16 hour photoperiod) at room temperature. The
number of each embryo stages and somatic embryos germinants was observed at the initial and the
end of the experiment.
2.4 Plantlet Conversion
Mature somatic embryos mostly at heart and torpedo stage were selected and cultured on a modified
MS solid medium with full- or half-strength macro salts and 20 or 30 g/L sucrose, without plant growth
regulators. Clumps of the embryos were cultured on a solid medium in each culture bottles for four
weeks. After four weeks of culture, numbers of somatic embryos produced shoot only, root only and
both shoot and root were determined.
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3. RESULTS AND DISCUSSION
3.1 Induction of Embryogenic Callus
For initiation of embryogenic callus formation, result show that 2,4-D combination of 0.5 mg/L BAP
were achieved number explants developed embryogenic callus from young leave explants for A.
malaccensis after four weeks (Table 1). White yellowish and soft consisted of many small nodular
structures of embryogenic callus were observed at all of the young leave explants (Figure 1A). The
highest percentage number of explant developed embryogenic callus was obtained at concentration of
2.0 and 5.0 mg/L 2,4-D plus 0.5 mg/L BAP with 100%. In an earlier study, Norazlina and Rusli (2006)
found that the highest frequency of embryogenic callus induction from young leaves of A. malaccensis
was cultured on MS medium supplemented with 2.0 mg/L 2,4-D plus 0.5 mg/L BAP. Similar pattern of
result was obtained in this study. Combination of 2,4-D and BAP at various concentrations led to a
faster growth of callus (Nurazah et al., 2009). Lowest number of explants produced embryogenic
callus were observed at 1.0 mg/L 2,4-D plus 0.5 mg/L BAP. All embryogenic callus culture was
transferred in new basal media for induction of somatic embryos.
Table 1. Percent of explants produced embryogenic callus after four weeks of culture initiation from
different concentration of 2,4-D plus 0,5 mg/L BAP
No. of explants produced
2,4-D (mg/L)
BAP (mg/L)
embryogenic callus (%)
1.0
0.5
65
2.0
0.5
100
3.0
0.5
70
4.0
0.5
95
5.0
0.5
100
*Four replicates for each treatment
3.2 Induction of Somatic Embryos
Embryogenic callus of A. malaccensis was white yellowish and soft consisted of many small nodular
structures transferred in MS medium supplemented with 1.0 g/L neutralize activated charcoal, 0.1
mg/L Kinetin, 0.5 mg/L BAP and 2,4-D at 1.0, 2.0, 3.0, 4.0 and 5.0 mg/L. Embryogenic callus of A.
malaccensis started to form somatic embryos after four weeks cultured. Some of white callus changed
to mostly white yellowish or greenish with solid structures called globular somatic embryos (Figure
1B). The concentration of 2,4-D with 2.0 and 5.0 mg/L 2,4-D plus 0.5 mg/L BAP and 1.0 mg/L Kinetin
affected higher formation of somatic embryos after four weeks cultured compared to other treatment
(Table 2). Embryogenic callus produce lower somatic embryos were observed at 1.0mg/L 2,4-D plus
0.5 mg/L BAP and 1.0 mg/L Kinetin. However, after six weeks cultured, most embryogenic callus
formed in clump with white yellowish or greenish solid structures at various development stages of
somatic embryos (Figure 1C). Somatic embryogenesis has already been reported in some woody
plants species i.e. Teak (Tectona grandis), sandal wood (Santalum album) and Eucalyptus globulus
(Kushalkar and Sharon, 1996; Bapat and Roa, 1999; Pinto et al., 2002). Induction of somatic embryos
with presence of activated charcoal can improve development of somatic embryos. This finding same
reported by Indra lyer et al. (2009), absence of activated charcoal in induction of somatic
embryogenesis of Myristica malabarica L. there was no embryogenic response but only callus
formation in zygotic embryos and their fragments. Activated charcoal adsorbs inhibitory substances
accumulating in the culture medium and is thus often used to reduce the oxidation of pehenolic
compound in tissue culture to improve cell growth and development (Ameena Abdulla and Khaled.,
2009)
3.3 Maturation of Somatic Embryos
A total of 80 clumps of somatic embryos were selected mostly at globular stage when the cultures
started. Four weeks later, the clump of somatic embryo increased to 100 to 150 embryos at different
developmental stages on half strength solid modified MS media with 0.01 mg/L ABA, 0.1 mg/L GA 3
and Kinetin at 1, 2, 4 mg/L. Kinetin at 2.0 and 4.0 mg/L was the best treatment for embryo maturation
and germination (Table 3). However, lower concentration of Kinetin decreased the number of somatic
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Table 2. Percent of embryogenic callus produced clump of somatic embryos after four weeks of
culture from media supplemented with different concentration 2,4-D plus 0.5 mg/L BAP and 0.1 mg/L
Kinetin
BAP
Kinetin
No. of embryogenic callus produces
2,4-D (mg/L)
(mg/L)
(mg/L)
clump of somatic embryos (%)
1.0
0.5
0.1
55
2.0
0.5
0.1
100
3.0
0.5
0.1
75
4.0
0.5
0.1
80
5.0
0.5
0.1
100
*Four replicates for each treatment
embryos germinated. During maturation process, somatic embryos of A. malaccensis changed in
shape, size, and color. At early developmental stage the shape of the embryo of A. malaccensis was
rounded or globular with greenish in color then turned green at later developmental stages (Figure
1D). Mostly clump of somatic embryos produced primary somatic embryos (Figure 1E). Increase
development of clump somatic embryos indicates that new somatic embryos were formed during the
culture. These new embryos are called secondary (repetitive) somatic embryos. Some embryos grew
further into later developmental stages, while other embryos demonstrated the tendency of repetitive
embryogenesis by budding off new globular embryos and contyledonary stage (Figure 1F). Therefore,
all stages of developing embryos with different sizes and colors were present at any one time over one
passage of culture. Formation of secondary embryos from primary embryos has also been reported in
other woody trees such as Albizzia lebbeck (Gharyal and Maheshwari, 1981) and Quercus robur
(Cuenca et al., 1999). Secondary embryogenesis has great potential for mass propagation and
repetitive embryogenesis can also be used for genetic transformation in forest trees (Merkle, 1995).
The embryo shape had been changed gradually from globular to heart-shape, torpedo, cotyledonary
and early germinant (Figure 2A).
Table 3. Percent of clumps of somatic embryos germinant after six weeks of culture from media
supplemented with different concentration of Kinetin plus 0.01mg/L ABA and 0.1 mg/L GA 3
Kinetin (mg/L)
ABA (mg/L)
1.0
0.01
2.0
0.01
4.0
0.01
*Four replicates for each treatment
GA3 (mg/L)
No. of somatic embryos germinant (%)
0.1
0.1
0.1
60
100
100
3.4 Plantlet Conversion
A total of 100 somatic embryos were select and used for plantlet conversion. Plantlet conversion was
achieved on the medium with half-strength macros of modified MS medium and sucrose at either 20 or
30 g/L (Table 4). Most plantlet had shoots and roots. Half-strength modified MS medium also gave
higher formation of root only than those full strength MS medium. Fotopoulos and Sotiropoulos (2005)
mention that the mineral concentration of the culture medium affect rooting characteristics and
proposed its reduction to half normal strength for rooting improvement. Similar results also were found
in somatic embryogenesis of tea where the use of modified half-strength macro salts increased the
conversion of somatic embryos into complete plantlets (Tahardi et al., 2000). However, for induction
shoot only, full-strength modified MS medium gave good shoot formation of plantlets.
Table 4. Effect of macro salts and sucrose on plantlet conversion of A. malaccensis on modified MS
medium without plant growth regulator.
Sucrose
Total of somatic
Both shoot
Treatment
Shoot only
Root only
(g/L)
embryos
and root
Full-strength
30
25
10
7
8
mMS
20
25
13
5
7
Half-strength
30
25
5
12
8
mMS
20
25
3
10
12
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* Five replicates for each treatment
A
B
C
D
E
F
Figure 1. (A) Embryogenic callus, (B) Early stage of somatic embryos, (C) A clump of somatic embryos
at various developmental stages, (D) Primary somatic embryos, (F) Secondary somatic embryos and
(G) Cotyledonary stage of somatic embryo. Bar: 0.5cm.
Root
A
B
C
Figure 2. (A) Somatic embryo development from globular to germinant, (B) Somatic embryos
germinant, (C) Complete plantlet derived from somatic embryos. Bar: 0.5cm
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According to Rajdan (1993), embryos might germinate and develop to plantlet on agar medium without
any growth regulators. Similar result also obtained in this study where somatic embryos cultured in full
and half modified MS medium with different concentration sucrose shown good germination of somatic
embryos and development of plantlet (Figure 2B). Different concentration of sucrose also effect on
development of somatic embryos. According to Anjaneyulu et al. (2004), cotyledon callus from
Terminalia chebula produced maximum number of plantlet develop from somatic embryos on MS
medium with high sucrose level. Similar result also obtained in this study. The effects of high sucrose
concentrations in the maturation medium may have resulted from high osmolarity, which has been
used to prevent precocious embryo germination, and enhance embryo maturation in some other plant
species (Pliego- Alfaro et al., 1996). All plantlets in the present study were transferred on same media
with same composition for the further growth. The regenerated plantlets will be acclimatized and
hardened using soil, peat and sand in the ratio 1:1:1.
4. CONCLUSION
As conclusion, 2,4-D at 2.0 and 5.0 mg/L 2,4-D plus 0.5 mg/L BAP was effective for induction of
embryogenic callus of A. malaccensis. The best medium for induction of somatic embryos from
embryogenic callus was modified MS medium supplemented with 1 g/L activated charcoal, 0.1 mg/L
kinetin, 0.5 mg/L BAP and 2,4-D at 2.0 and 5.0 mg/L. Kinetin at 2.0 and 4.0 mg/L promoted the
maturation of somatic embryos on medium containing 0.01 mg/L ABA and 0.1 mg/L GA 3. The mature
embryos grew into normal plantlets when cultured on medium with half-strength macro salts of
modified MS medium without plant growth regulators. Therefore, tissue culture via somatic
embryogenesis can be effectively used for plant regeneration and mass production of seedlings.
ACKNOWLEDGEMENT
The authors are grateful for the financial support provided by UNIMAS, under UNIMAS ZAMALAH
postgraduate scholarship. We are also grateful to the staff of Department of Plant Science and
Environmental Ecology, Faculty of Resource Science and Technology, UNIMAS, for their technical
assistance.
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IN VITRO CALLUS INDUCTION FROM LEAF AND PETIOLE EXPLANT OF GAHARU (Aquilaria
microcarpa BAILL.)
Rofiah, J.,* Sani. H.B. and Zul Helmey, M. S.
Department of Plant Science and Environmental Ecology
Faculty of Resource Science and Technology
Universiti Sarawak Malaysia, 94300 Kota Samarahan, Sarawak, Malaysia
*Email: jrofiah@gmail.com
Abstract
Aquilaria microcarpa Baill is a non – timber tree belongs to Thymeleaceae family. Gaharu is the
valuable product produce by the tree. It is economically important. It can serve many purposes such
as incense, fragrance and medicine. The study was carried out to establish an in vitro callus induction
of A. microcarpa Baill. Leaf and petiole from one–year–old potted seedling were surface sterilized with
10%, 15% and 20% of Clorox for 10, 15 and 20 minutes plus Tween20 followed by Benomyl for 1
hour. In the study, 10% of Clorox concentration with 20 minutes exposure produced more axenic
explant for leaf with 95% and petiole with 86.7%. Axenic leaf and petiole were placed into solidified
MS media supplemented with different concentrations of 2,4–dichlorophenoxy acetic acid (2,4–D)
alone at 0, 0.5, 1.0, 2.0 and 3.0 mg/L and also 2,4–D plus 0.5 mg/L of 6–benzylaminopurine (BAP) for
callus induction. After 4 weeks of culture, leaf significantly produce 100% of callus while petiole with
73.3% success in media supplemented with 2.0mg/l of 2,4–D plus 0.5mg/l of BAP. Fully developed
callus were subcultured after 4 weeks into fresh media to promote cell growth. The calli formed can be
induced to form new plantlets.
Keywords: Aquilaria microcarpa Baill.,, callus induction, leaf, petiole.
1. INTRODUCTION
Aquilaria is genus in Tymelaeceae family that produced resinous, fragrant and valuable heartwood
products as a result of defence mechanism of pathogen attack (Persoon & Beek, 2008). Gaharu has a
very high market value due to its fragrant resin. The resin is not limited to fragrant industry, in fact can
be used as medicine and some in religious purposes (Erdy, et al., 2011). However, the occurrence of
Aquilaria trees still not guaranteed to produce resin. In forests, only 10% of Aquilaria stands were
estimated by scientist that can produce resin (Gibson, 1977, cited in Donovan & Puri, 2004). Due to
the resin product, higher demand in market has caused excessive feeling of tress by local, thus
significantly reduced the tree population in natural forest.
Realising the depletion of agarwood trees distribution, many parties tried to plant the species trees at
commercial scale. But yet, poor supplies of seedlings usually slow down the effort. Rapidly losing
moisture content is one of the constraints that make gaharu seed to have poor germination (He, Qi, &
Hu, 2005). Besides, species of A.malaccensis are found to produce seeds after 7 to 9 years of growth,
while other species were identified can only produce seed once in their while life cycle (Chua, 2008).
As it is not reliable to plant seed that have sort of germination difficulties, in vitro propagation become
the potential alternative technique in producing the plantlets.
Through in vitro propagation, homogenous plant type of Aquilaria will be produced. Thus, similar
quality of resin desired will able to be gained. In direct organogenesis, manipulation of BAP with 0.5
ppm and TDZ with 0.25 ppm have produced optimum and best concentration in developing total bud,
size of bud and shoot sprout for A. malaccensis (Azwin, Siregar, & Supriyanto, 2006), While in indirect
organogenesis, callus tissue from sterile stem of A. sinensis was successfully induced in modified MS
media supplemented with 4 mg/L 2,4–D and 0.2 mg/L Kinetin (Qi, 1995). From experiment conducted,
small plantlets can be developed from calli by inducing shoot and root growth.
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2. MATERIALS AND METHODS
Several potted seedlings of Aquilaria microcarpa Baill. about the age of one years were brought from
External Lab and were maintained outside of Plant Tissue Culture (PTC) Laboratory, Level 2, Faculty
of Resource Science and Technology. The following process of surface sterilization, transfer and
incubation were conducted at Plant Tissue Culture (PTC) Laboratory. The explants used were leaf and
petiole.
2.1 Establishment of Axenic Explants
Leaf and petiole explants were left under running water for about 30 minutes to remove any foreign
materials. The explants were sectioned into convenient sizes to allow better contact with disinfectant
agent. Clorox® was used as disinfectant agent. There were 6 treatments conducted with 2
manipulated Clorox® concentrations (10 and 15%) and 3 different time exposures (10, 15 and 20
minutes). Few drops of Tween20 were added together with disinfectant solution. After time exposures,
disinfectant removed and rinsed 3 times with sterilized distilled water. The explants were soaked in
0.1% Benomyl for 1 hour. All treatments were having 5 replicates each and every replicates contained
4 explants for leaf and 3 explants for petiole. All explants were cultured on ½ MS media. Successfully
transferred plates were sealed with parafilm and were observed for 2 weeks. After 2 weeks, the axenic
explants were transferred on 1MS media supplemented with different concentration of 2,4–D for callus
induction. The numbers of axenic explants were recorded. Data were analysed using ANOVA and
Tukey HSD test.
2.2 Induction of Callus on Basal Media supplemented with 2,4–D alone and 2,4–D plus 0.5mg/L
BAP
Axenic leaf and petiole explants were transferred into 1MS media supplemented with manipulated
2,4–D concentrations. There were 5 different concentrations of 2,4–D tested consisted of 0.0 (control),
0.5, 1.0, 2.0 and 3.0 mg/L, same goes to 2,4–D plus 0.5 mg/L BAP. Therefore, there were comprised
10 treatments conducted. Each concentration consisted of 5 replicates with 4 leaf explants and 3
petiole explants in every plate. All plates were placed in dark condition for about 3 weeks with regular
inspection. Approximately after 21 days incubation in the dark, the frequent calli inductions were
calculated. For well developed callus within each treatment, they were subcultured first. Data of callus
formation was recorded and calculated after 3 weeks of incubation. ANOVA and Tukey HSD test were
used as data analysis.
3. RESULTS AND DISCUSSION
3.1 Establishment of Axenic Explants
Leaf explants used were taken from intermediate part of young and old leaves. Explants taken from
older trees sometimes giving serious problem of contamination (Bonga & Von Aderkas, 1992). After 2
weeks of culturing, some leaf explants turned browning, damaged and eventually died. Compared to
petiole, leaf explants showed low percentage of problems caused by contamination. However, most
explants were damaged after surface sterilization. For petiole, most of explants were contaminated
with fungi and bacteria even after 1 week of surface sterilized. Contamination reaction was rather
faster than leaf explants. Petiole and rachis part were also identified to easy contaminated after a
week cultured (Zul Helmey, 2010). From experiment conducted, it was found that 10% of Clorox®
concentration with 20 minutes of time exposure showed high percentage of producing axenic explants
with 95% for leaf explants and petiole with 86.7% success. After treated with 15% of Clorox®
concentration for 20 minutes, less axenic leaf explants were obtained. Meanwhile, petiole showed less
axenic after treated with 10% of Clorox® for 10 minutes. Cheng (2010) stated that higher
concentration of Clorox® with longer exposure time in surface sterilized particularly for leaf may cause
high possibilities damaging explants. Data analysis conducted using ANOVA showed that there was a
significant effect at 5% significant level between treatments of different concentration of Clorox® with
different exposure time in producing axenic leaf explants. According to mean comparison using Tukey
HSD test, treatment of 15% of Clorox® concentration with 20 minutes of time exposure showed
significantly different compare to other treatments. Treatment of 10% of Clorox® with 10 minutes
showed higher mean scores in producing axenic explants. However, there was no significant effect
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found in petiole explants even after mean comparison conducted (Table 1). The results indicated that
suitable sterilization protocol for petiole explants still not been developed.
Table 1 Percentage of axenic explants and its mean comparison number after treatments
Explant
Leaf
Petiole
Mean number of
Treatment (% / min)
Axenic (%)
Axenic (%)
axenic explants
T1
10 / 10
85.00
3.40 b*
60.00
T2
10 / 15
75.00
3.00 b
73.34
T3
10 / 20
95.00
3.80 b
86.67
T4
15 / 10
85.00
3.40 b
73.34
T5
15 / 15
75.00
3.00 b
66.67
T6
15 / 20
15.00
0.60 a
66.67
* Mean followed by different alphabetical letters is significantly different.
Mean number
axenic explants
1.80a
2.20a
2.60a
2.20a
2.00a
2.20a
of
3.2 Induction of Callus on Basal Media supplemented with 2,4–D alone and 2,4–D plus 0.5 mg/L
BAP
3.2.1 Induction of Callus on Basal Media supplemented with 2,4–D alone
Axenic leaf and petiole explants were transferred into full length of MS medium supplemented with
2,4–D hormones alone. Explants were observed developed into callus at the entire end within 3
weeks. However, some leaf and petiole explants were damaged and eventually died after week 3,
eventhough callus was growing at its ends. These calli were subcultures in fresh medium having
similar concentrations after 3 weeks to allow their growth. Latent contamination and browning of
explants that disrupting callus development frequently happened when petiole is used. Browning
problems caused the media surrounding explants appeared clear brown colour. Other explants were
immediately subcultured into another fresh medium as exudation from browning can caused growth
retardation. Medium appeared brown or black colour happened when plant tissues give off toxic
substances, hence subculturing is necessary to be done (Pierik, 1997).
Basal media supplemented with 2,4–D alone were manipulated into 5 different concentrations,
consists of 0.0(as control), 0.5, 1.0, 2.0 and 3.0 mg/L. Calli were observed started to develop when
cultured on media supplemented with 2,4–D. The situation indicated that there has been a response
between explant and hormone applied. Basal media with 2.0 mg/L 2,4–D explants showed 55% of leaf
explants have developed into callus, while only 40% petiole explants response to hormone applied
(Figure 1). Other concentrations indeed showed similar response for callus induction. Calli also turned
to develop under basal media of 3.0 mg/L 2,4–D but, eventually turned brown after a week transferred
into another fresh media. According to Cheng (2010), higher concentration of 2,4–D in media may
lead for disruption of callus growth. At one stage, high concentrations of synthetic auxins like 2,4–D
may possess herbicidal property that might inhibit callus formation (Siwach, Gill, & Kumari, 2011).
Data analysis was continued by using ANOVA and Tukey HSD test. Based from data obtained
through out experiment, the result indicated that leaf explants showed positive response with 2,4–D
concentration as there was a significant effect computed. Concentration of 2.0 mg/L 2,4–D showed
significantly effect in producing callus and produced higher mean scores rather that absence of 2,4–D
in media that do not stimulate any callus growth. Data gained proved that synthetic auxin
supplemented in media can induced callus growth regardless its concentrations applied as long as the
amount do not exceed lethal stages to explants. However, no significant differences found in data
computed for petiole explants (Table 2). Result showed that, too low and high concentration of 2,4–D
might stimulated less petiole explants forming callus. Yet, 2.0 mg/L 2,4–D produced more petiole
explants forming callus. It is also reported that using internode explants of Acacia mangium performed
low efficiency of callus induction (Xie & Hong, 2001).
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Table 2 Percentage of explants formed callus and its mean comparison number after treatments of
2,4–D alone.
Explants
Leaf
Petiole
Callus
Mean number of explants Callus
Treatments (mg/L)
(%)
formed callus
(%)
T1 0.0 (control)
0.00
0.00 a
0.00
T2 0.5
15.00
0.60 ab
20.00
T3 1.0
25.00
1.00 ab
26.67
T4 2.0
55.00
2.20 b
40.00
T5 3.0
50.00
2.00 b
33.33
* Mean followed by different alphabetical letters is significantly different.
a
Mean number of explants
formed callus
0.00 a
0.60 a
0.80 a
1.20 a
1.00 a
b
d
c
e
f
Figure 1. Callus induction of explants cultured in MS medium supplemented with 2.0 mg/L of 2,4–D
alone.
a
b
c
d
e
f
Callus inducted from leaf after 1 week cultured.
Callus developed from leaf after 3 weeks cultured.
Fully developed callus from leaf after 4 weeks cultured.
Swollen petioule after 1 week cultured.
Callus developed at entire cut end after 1 week cultured.
Fully developed callus from petioule after 5 weeks cultured.
*Bar=0.5cm
3.2.1
Induction of Callus on Basal Media supplemented with 2,4–D plus 0.5 mg/L BAP
Both axenic leaf and petiole explants obtained after surface sterilized were cultured in MS medium
supplemented with 2.0 mg/L of 2,4–D plus 0.5 mg/L of BAP. Within 2 weeks of cultured, explants were
observed having quick response to hormones applied and after 3 weeks, explants particularly leaf
showed ½ of its tissue growing callus while petiole at cut ends. After week 4, the successfully
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developed friable calli were transferred into another same concentration of fresh medium to allow
better performance of callus growth.
MS basal media supplemented with 2,4–D plus 0.5mg/L BAP were manipulated into 5 different of
auxin concentrations (0.0, 0.5, 1.0, 2.0 and 3.0 mg/L). From experiment, basal media with
supplementation of 2.0 mg/L 2,4–D plus 0.5 mg/L BAP hormones indicated the best growth
performances of callus. Similar result also found in Zul Helmey (2011), where as 2.0mg/L plus 0.5
mg/L produced high number of callus for A. beccariana. For leaf explants, 100% of explants were
successfully developed into callus while petiole significantly produced 73.34% explants produced
callus (Figure 2). Media supplemented with 0.5 mg/L 2,4–D plus 0.5 mg/L BAP likewise produced
good performance of callus at the first 3 weeks, but the growth stunted after 4 weeks and eventually
died. The situation was observed to happen due to response of same concentration of auxin and
cytokinin applied that caused uncertain growth of callus.
a
c
b
d
e
f
Figure 2 Callus induction of explants cultured in MS medium supplemented with 2.0 mg/L of 2,4–D
plus 0.5 mg/L BAP
*Bar=0.5cm
a
b
c
d
e
f
Callus developed from leaf after 1 week cultured.
Callus developed from leaf after 2 weeks cultured.
Fully developed callus from leaf after 4 weeks cultured.
Swollen petioule after 1 week cultured.
Callus developed at entire cut end after 2 week cultured.
Fully developed callus from petioule after 4 weeks cultured.
However, Pierik (1997) stated that, sudden stagnated of callus growth might be indicated the unfit
subculture medium. Another explants cultured in both 1.0 and 3.0 mg/L 2,4–D plus 0.5 mg/L BAP
showed better response in callus induction. Meanwhile for 0.0 mg/L 2,4–D, no reaction of callus
induced were observed even supplied with 0.5 mg/L BAP. Absent of 2,4–D inside media caused no
callus development as the hormone exert strong influenced throughout cell growth expansion, cell
division initiation and rise to organised tissue (Gaspar, et al., 1996)
Statistical data analysis continued using ANOVA, proceed with Tukey HSD test. After data analysis,
both explants showed significantly effect in response to hormone applied. According to result
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obtained, concentration of 0.0 mg/L 2,4–D plus 0.5 mg/L BAP showed significantly different between
another treatments. Treatment of 0.5, 1.0, 2.0 and 3.0 mg/L 2,4–D plus 0.5 mg/L BAP showed no
different between each other. However, 2.0 mg/L 2,4–D produced higher mean score in explants
formed callus rather than other treatment (Table 3).
Table 3 Percentage of explants formed callus and its mean comparison number after treatments of
2,4–D plus 0.5 mg/L BAP
Explants
Leaf
Petiole
Callus
Mean number of explants Callus
Treatments (mg/L)
(%)
formed callus
(%)
T1 0.0 + 0.5
0.00
0.00 a*
0.00
T2 0.5 + 0.5
80.00
3.20 b
40.00
T3 1.0 + 0.5
85.00
3.40 b
53.33
T4 2.0 + 0.5
100.00
4.00 b
73.34
T5 3.0 + 0.5
75.00
3.40 b
53.33
* Mean followed by different alphabetical letters is significantly different.
Mean number of explants
formed callus
0.00 a*
1.20 b
1.60 b
2.20 b
1.60 b
4. CONCLUSION
Plant propagation conducted through in vitro regeneration is a useful technique applied especially for
difficult–growing species. In establishing axenic explants of A. microcarpa, 10% of Clorox®
concentration with 20 minutes of time exposure showed high percentage of producing axenic explants
for both leaf and petiolule. Consideration during choosing suitable explants should be noted as it
influenced promising response. In surface sterilization applied to petiolule, mean score showed no
different between all treatments applied. Thus, the suitable sterilization protocol for petiolule explants
of A. microcarpa has not been developed yet.
MS media supplemented with 2.0 mg/L 2,4–D plus 0.5 mg/L BAP produced more explants forming
callus rather that 2.0 mg/L 2,4–D alone. Observation conducted within 4 weeks showed that MS media
supplemented with 2,4–D plus 0.5 mg/BAP response to callus growth more faster than 2,4–D alone.
Both explants were responded better when cultured into media of 2,4–D added with BAP. Instead of
2.0 mg/L concentration, callus also observed was inducted in another concentration as long as BAP is
added. Fraxinus micrantha, type of Himalayan temperate multipurpose trees species through in vitro
also found that induction using auxin alone is not sufficient enough and was found non–responsive,
thus requirement of cytokinins in optimum amount is needed as it is more promising for callus
induction, shoot proliferation and rooting (Bisht, et al., 2011).
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Persoon, G. A., & Beek, H. H. (2008). Growing ‘The Wood of The Gods’μ Agarwood Production in
Southeast Asia. Smallholder Tree Growing for Rural Development and Environmental Services,
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(Agarwood). In Y. P. S. Bajaj (Ed.), Medicinal and aromatic plants (Vol. 8, pp. 36). Germany:
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level on induction and long term maintenance of callus cultures of Ficus religiosa L. African
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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IMPORTANCE OF TOPOGRAPHY AND SOIL PHYSICAL PROPERTIES ON THE GROWTH OF
Shorea macrophylla UNDER REFORESTATION AT SAMPADI FOREST RESERVE
Nur Hanani Hanis, M.N.*, Wasli, M.E.*, Sani, H. and Nahrawi, H.
Department of Plant Science and Environemntal Ecology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
*Emails: wmeffendi@frst.unimas.my / hanani.hanis@gmail.com
Abstract
Shorea macrophylla are characterised as one of the Dipterocarp indigenous trees that exhibit a fast
growing ability. This species is said to be favourable on clay riparian alluvium and locally abundant on
damp soil on hillside by river and streams habitat. Several studies reported that S. macrophylla is less
favourable to ridge tops and upper slopes of a hill. Their growth patterns findings are very important,
as this species is commonly used in reforestation plantation, especially in Sarawak. In this study, the
relationship between soil physical properties at various topographical conditions were analyzed in the
respect of the growth of S. macrophylla planted in reforestation area of Sampadi Forest Reserve,
Sarawak. Study plots with the size of 50 m x 50 m were constructed at reforestation areas planted with
S. macrophylla at various age stands from 1996 (SM96), 1997 (SM97) and 1998 (SM98). In each
study plots, a topographical map was constructed by using the conventional method. By referring to
the constructed maps, soil samples were collected at 0 - 10 cm and 30 - 40 cm depths from several
random points within each contour zone for soil physical analysis. Soil physical analysis involved in
this study includes soil texture analysis, bulk density, porosity, water content, moisture content and
compaction. The findings showed that in relation between soil physical properties and topographical
characteristics, S. macrophylla shows strong adaptability across all range of elevation and other soil
physical properties. However, the presence of stagnant water and interruption of pioneer species in
between the planting lines greatly resulted in poor growth performance and survivability of S.
macrophylla.
Keywords: Shorea macrophylla, reforestation, topography, soil physical properties, Sarawak
1. INTRODUCTION
Reforestation are capable of restabilising more suitable living condition of flora and fauna, it support
more biological diversity and increase the resilience and adaptability of existing agricultural systems.
In the tropics, Shorea macrophylla could be seen as highly potential trees for reforestation purpose.
Azani et al. (2011) studied that, Shorea macrophylla is a fast growing species among Dipterocarp and
form wide spreading crowns even though it is planted under shades of higher trees. To-date in 2000,
over 11, 783 hectares of land under reforestation programme in Sarawak has been reforested with
Shorea macrophylla (Sarawak Forestry Department, 2009).
Shorea macrophylla is commonly found in lowland tropical rainforest and seldom occurs in 600m
altitude (Ashton, 1964). According to Rasip & Lokmal (1994), this species is confined on clay riparian
alluvium and locally abundant on damp soil on hillside by river and streams where is scattered and
rare. Shorea macrophylla also said to be less favourable to ridge tops and upperslopes. The growth
performance of Shorea macrophylla might associates in certain topography and soil texture.
Yamada et al. (1997) reported that the changes in elevation would consequently influence the soil
texture and water content of the soil. Yamakura et al. (1996) showed correlate changes in forest
architecture and topography in Bornean tropical rainforest at Lambir, Sarawak. From the findings, it
concludes that topography and soil physical properties play a significant role in the coexistence of
three different Scaphium species. Sabrina et al. (2005) also found that, survivability of trees in Lambir,
Sarawak were greatly influenced by soil texture. Growth and mortality of all tree species corresponds
to soil resource availability. The most clayish soil had the highest mortality rates follows by fine loams
and sandy loams. In different elevation, the habitat distributions of five species in the genus Shorea
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spp. were different in specific soil texture. Therefore, there is evidence that variation of topography
and soil physical properties affect the forest composition.
The relationship between topography and tree distribution in terms of spatial patterns of individuals
under natural environment also have been studied by many scientist (Itoh, 1995; Rogstad, 1990).
However, only few have reported on importance of topography and soil physical properties in growth
performance of tree planted in the artificial regeneration areas like reforestation area. Considering the
lack of studies, the objective of this study is firstly to establish a topographical map of each quadrate
plots. Second, is to determine the physical properties of different elevation in each quadrate plot.
Lastly, to analyze the relationship of topography and soil physical properties in growth performance of
Shorea macrophylla under reforestation environment to which plant survivability is predictable from
their soil condition and topographic variation.
2. MATERIALS AND METHODS
2.1 Site Description of Study Area
This study take place at Sampadi Forest Reserve (Sampadi FR), Sarawak, East Malaysia
0
0
(N01 30’16’’E10λ 55’03”) which is near to the Bau-Lundu road. The elevation of the study area is
ranging from 22 meter to 44 meter above sea level. The climate of West Sarawak is typical for humid
tropical lowland and characterised by heavy rainfall ranges from 3048 – 4572 millimetres annually with
0
a uniform relative humidity. The mean daily minimum and maximum temperature vary between 22 C
0
0
0
(72 F) and 31 C (88 F) (Andriesse, 1972). The soils in the study area were derived from
combination of sandstone, coarse-grained, humult ultisols and sandy residual parent material of Bako
series based on Sarawak soil classification system which corresponds to Ultisols soil orders according
to the USDA (United State of Department of Agriculture)-Soil Taxonomy Classification System.
This area was planted over a period of 5 years (1996-2000). In 1996, 1.6 hectares were planted
with Shorea macrophylla while in 1997 and 1998 1.1 and 1.2 ha respectively were planted. Another
1.3 ha were planted in August 1999.
2.2 Soil Collection and Analysis
Four study plots with 50 m x 50 m quadrate were constructed at various age stands of plantation from
year 1996, 1997 and 1998 (SM96, SM 97 and SM 98). In each study plots, topographical map were
constructed by using the conventional method. The microtopography measuring method was derived
from Almquist et al. (2002) by using clinometers, which predict the change in elevation from one tree
to another based on the conventional method. In this study site is the reforestation area, the distance
from each tree to another is uniformly at 5 m gap. Positive and negative values of reading from
clinometers indicate a convex (ridges) and concave (valley) land surface forms, respectively. The
values of initial elevation are recorded using GPS (Global Positioning System) device at the first row of
the planted trees and cumulated relative elevation is calculated for each row in the study plots. Any
significant microclimate features i.e., stagnant water, in the studied plot were noted in the
topographical map. Survivability and mortality of the trees were also recorded. As the area size of
each contour zones in the topographical map may differed within each study plots, composite soil
samples were collected from the depth of 0 - 10 cm (surface soil) and 30-40 cm (subsoil) from several
random points within each contour zone from the constructed map for soil physical analysis. Soil
physical analysis involved in this study includes soil texture analysis, bulk density, porosity, water
content, moisture content and compaction.
3. RESULTS & DISCUSSIONS
3.1 Soil Physical Properties of Sampadi Forest Reserve in SM96, SM97 and SM98
Figure 1 shows the topographical map of the study site SM96 in relation with the soil texture
classification. Soil textures on soil surface of SM96 were ranges from loam to clay loam. Table 1
shows the soil physical properties of the soil at surface soil (0 – 10 cm) and subsoil (30 – 40 cm)
collected at several random points within different elevations in SM96.
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Figure 1 SM 96 soil texture classification map
Table 1. Soil physical properties of SM96
Bulk
Water
Porosity
density
content
Elevation
-1
(g mL )
(%)
(%)
Moisture
content
Clay
(%)
--------- (%) ---------
Silt
Sand
Depth: 0-10 cm
25
1.07
0.60
45.1
7.5
20
25 55
26
1.12
0.58
36.0
2.6
19
29 53
27a
1.00
0.62
48.6
4.4
27
26 47
27b
0.98
0.63
42.0
4.7
30
28 42
28
1.11
0.58
43.4
3.6
23
33 44
Depth: 30-40 cm
25
1.65
0.38
25.2
1.1
18
27 55
26
1.53
0.43
25.0
2.2
25
36 39
27a
1.43
0.48
29.0
6.6
32
25 43
27b
1.46
0.45
29.0
3.8
32
32 36
28
1.46
0.45
28.3
2.6
26
32 42
1)
Hardness was measured using Hasegawa type penetrometer in 50 cm depth
Compaction
1)
Depth: 50 cm
26
31
43
43
24
The results showed that bulk density value of surface soil (0 – 10 cm) at the elevation of 27
-1
m, were less than 1.00 mL , and resulted in higher value in soil porosity. The lowest value in soil
water content was observed at the elevation of 26 m while elevation 27 m (a) showed high soil water
content. In addition, the lowest moisture content was observed at the elevation of 26 m while the
highest moisture content recorded was at the elevation of 25 m. The differences in terms of the
soil physical properties in SM96 could be described from the difference in soil texture in elevation of
27 m as compared to the other elevation. Clayey soils tend to have lower bulk densities and higher
porosities than sandy soil (Brady and Weil, 2008). As shown in Figure 2, comparison between the
values of the sand and elevation of SM96 showed significant correlation (r = 0.88). The result showed
that, significance difference shown between the changes of elevation (Figure 2). The significant
relationship between soil texture and site elevation is important in determine the natural distribution of
Dipterocarp species under tropic climate. As reported by Itoh et al. (2003), distribution of Dryoblanops
aromatica in natural distribution of Lambir Hills National Park was abundant at higher elevation in
sandy soils and on convex steep slopes. In contrast, Dryobalanops lanceolata preferred lower
elevation and less sandy soil. In high elevation Dryoblanops lanceolata could not become established
due to dry condition resulted from sandy soil. Besides that, distribution and growth performance of
trees of Scaphium borneense, Scaphium macropodum and Scapium longipetiolatum in Lambir Hills
National Park, was also closely related to the difference in their habitat preference to topography.
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Although the widespread of S. borneense was larger compared to the S. macropodum and S.
longipetiolatum, but in terms of the DBH (Diameter Breast Height) of trees it was less the smallest with
less than 41 cm. While the DBH of S. macropodum and S. longipetiolatum were exceeded 51 cm.
Therefore it is expected that the growth performance of the S. borneense was associate to its
topographical characteristics which was highly confined in convex slopes where the most part of the
soil were covered by sandy soil (Yamakura et al., 1996).
Figure 2 Correlation between elevation in SM96
(m) and sand content
** Significantly different at 0.01% Student t-test
Soil compaction recorded in number of strikes from 50 cm depth has showed that in higher elevation,
the soil compaction tended to be higher although the differences between the compared elevation was
only at 3 m above sea level. However at the elevation of 28 m, the soil compaction was observed to
be low in which almost similar to the soil compaction of the higher the soil compaction at the elevation
of 25 m. Waterlogged condition in most part of this contour may have contributed to the less
compaction in soil for this elevation (Figure 1). Glinski and Lipiec (2000) reported that soil compaction
will decrease abruptly with moisture content and accompanied with outflow of air to certain amount.
Table 2 shows the soil physical properties of the soil at surface soil (0 – 10 cm) and subsoil (30 – 40
cm) collected at several random points within different elevations in SM97. Figure 3 shows the
topographical map of the study site SM97 in relation with the soil texture classification. In soil the
depth of 0–10 cm the soil texture were consist of clay loam, loam, sandy clay loam and sandy loam. It
can be seen that at the elevation of 24 m (b) and elevation of 28 m the soil texture were the same
although both of the elevation were very distinct in 4 m difference metre above sea level. In the soil
depth of 30 – 40 cm, almost all of the soil textures in almost all of the elevation were changed to more
clayey. However, at 24 m (a) elevation, the loams textured were the same as in the surface soil. At the
elevation of 26 m, the texture was not only changing to more clayey but also more percentage of silt.
Table 2. Soil Physical Properties of SM97
Bulk
Water
Porosity
density
content
Elevation
-1
(g mL )
(%)
(%)
Moisture
content
Clay
(%)
--------- (%) --------
Silt
Depth: 0-10 cm
24a
0.91
0.66
59.8
5.0
22
31
24b
0.96
0.65
60.9
5.0
23
29
24c
0.94
0.65
61.0
4.4
25
25
25
0.98
0.64
53.3
5.4
23
27
26
0.90
0.67
66.6
5.7
35
26
27
0.98
0.65
59.7
5.9
33
32
28
1.18
0.57
44.6
3.6
20
28
Depth: 30-40 cm
24a
1.35
0.49
26.2
4.9
23
26
24b
1.25
0.53
40.8
3.4
29
30
24c
1.37
0.48
34.8
3.7
29
27
25
1.43
0.47
30.1
3.8
35
29
26
1.32
0.5
37.4
6.8
38
31
27
1.42
0.47
32.8
5.2
38
24
28
1.45
0.45
29.0
2.8
26
18
1)
Hardness was measured using Hasegawa type penetrometer in 50 cm depth
373
Sand
47
48
50
50
39
35
52
51
41
44
36
31
38
56
1)
Compaction
Depth: 50 cm
33
36
26
39
34
40
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Figure 3 SM 97 soil texture classification map
Table 3 shows the soil physical properties of the soil at surface soil (0 – 10 cm) and subsoil (30 – 40
cm) collected at several random points within different elevations in SM98. Figure 4 shows the
topographical map of the study site SM98 in relation with the soil texture classification. In soil the
depth of 0–10 cm the soil textures are varies from sandy loam and clay loam. It can be seen that all
the elevations in the depth of 30 – 40 cm in SM98 were comprised of the same soil texture which was
clay loam.
Table 3. Soil Physical Properties of SM98
Elevation
Bulk
density
-1
(g mL )
Porosity
Water
content
(%)
Moisture
content
(%)
Clay
Silt
Sand
(%)
--------- (%) --------Depth: 0-10 cm
22
0.87
0.67
57.4
7.1
35
22
43
23
0.92
0.65
53.0
5.4
32
18
50
24
0.93
0.65
58.1
5.9
34
25
42
25
1.13
0.58
42.0
3.3
18
22
60
Depth: 30-40 cm
22
1.33
0.50
22.3
3.4
29
31
40
23
1.37
0.48
33.3
4.9
33
28
39
24
1.21
0.55
43.0
4.1
39
28
33
25
1.47
0.44
29.7
2.7
37
29
34
1)
Hardness was measured using Hasegawa type penetrometer in 50 cm depth
Compaction
1)
Depth: 50 cm
36
33
38
44
Among of all elevation in SM98, only at the elevation of 25 m, the soil texture in depth of 0 – 10 cm
was different from the other elevation. This was due to the sand content that affecting the soil texture
-1
classification. Bulk density value across all elevations shows a uniform changes from 0.87 g mL to
-1
1.13 g mL . In relation to the increment of the bulk density value, the soil porosity shows a decrement
in value as well. The changes of these values were most likely caused by the percentage of clay and
sand. Therefore, the finding suggests that, statistically there were significant in correlation of elevation
with the values of the bulk density (r = 0.89) and the soil porosity (r = 0.99) in surface soil (Figure 5). It
shows that as the elevation gets higher, the higher the bulk density thus the lower the soil porosity in
study plot SM98.
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Figure 4 SM98 soil texture classification map
Figure 5 Correlation between elevation
in SM98 (m) and bulk density
-1
(g mL ) as well as soil
porosity (%)
** Significantly
Student t-test
different
at
0.01%
Soil water content of all elevation in the depth of 0 - 10 cm was ranges from 42.0 % to 58.0 %. In the
elevation of 22 m, 23 m and 24 m the water content were relatively the same. However, in the
elevation of 25 m the water content was clearly distinct with the value of 42.03 % which owes the
lowest water content among of all elevation. The highest moisture content was noted at the elevation
of 22 m with 7.1 % while the lowest moisture content was located at the elevation of 25 m with 3.3 %.
The moisture content of elevation 23 m and 24 m were relatively the same with the value that range in
between 5.4 % to 5.9 %. As shown in Figure 6, the elevations in SM98 and the moisture content
showed significant correlation (r = 0.89). Therefore, it suggests that the moisture content is lower as
the elevation is higher.
Figure 6: Correlation between Elevation
in SM98 (m) Moisture content (%)
** Significantly different at 0.01% Student
t-test
The lowest values of moisture content and water content were resulted from the highest content of
sand percentage which was 60 %. Here, the sandy loam texture of elevation 25 m affecting the values
of water content and moisture content greatly.
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3.2 Relationship of Microtopography and Soil Physical Properties on the Growth of Shorea
macrophylla
Table 4 shows the growth performance and survival rate of planted Shorea macrophylla in SM96,
SM97 and SM98 in relation to different elevation for each study sites. The results showed that the
survival rate of Shorea macrophylla in SM96 was 89.0 % followed by 81.0 % and 77.0 % in SM97 and
SM98, respectively. In SM96, the average DBH of Shorea macrophylla was higher as compared to the
trees in SM97 and SM98.
In order to clarify the importance of microtopography on the growth of planted trees in each study
sites, a comparison between the elevation and growth parameters as well as the soil properties were
conducted as shown in Table 5 and 6. The results suggested that both factors of microtopography
and soil physical properties of all study plots SM96, SM97 and SM98 may not be the vital edaphic
factor in determining the growth performance of Shorea macrophylla. Although some soil properties
and growth parameter showed significant correlation with the site’s elevation. The growth of Shorea
macrophylla has shown some distinct ability to adapt in across all elevations with variables of soil
physical. It was observed that there were lack of correlation in between soil physical properties and
the elevation in all study plots. In addition to that, no clear relationship was observed in terms mean
annual increment diameter (MAID) and mean annual increment height
Table 4. Growth Performance of Shorea macrophylla in respective elevation
Total
trees
Study
No.
of Survival
DBH
planted in each
Elevation
Plot
dead trees rate (%)
(cm )
elevation
SM96 25
7
2
71.4
5.9
26
51
6
88.2
8.1
27a
10
0
100.0
6.8
27b
22
3
86.4
7.9
28
10
0
100.0
8.6
Average
89.0
7.5
SM97 24a
8
6
25.0
3.9
24b
5
1
80.0
6.4
24c
14
4
71.4
4.1
25
36
2
94.4
8.6
26
27
4
85.2
7.6
27
8
2
75.0
3.8
28
2
0
100.0
7.8
Average
81.0
6.0
SM98 22
13
7
46.2
3.0
23
17
3
82.4
6.7
24
57
12
78.9
4.9
25
13
1
92.3
7.8
Average
77.0
5.6
Height
(m)
4.5
6.4
5.8
6.6
7.4
6.1
3.0
8.2
6.3
9.2
7.6
4.2
9.4
6.8
3.8
7.2
5.8
7.9
6.2
(MAIH) to elevations in all study plots. MAID and MAIH were calculated according to values of the
DBH and height of the planted trees in respective to the years of the planted trees. Moreover, soil
physical properties in each elevation were modified in the presence of natural phenomenon like solar
radiation, rainfall and flood that had occurred in the all study plot. From the field observation,
environmental variables such as light intensity and presence of water stagnant greatly influence in
growth performance of Shorea macrophylla compared to microtopography and soil physical properties
in SM96, SM97 and SM96.
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Table 5. Correlation between SM96, SM97 and SM98 study sites in all elevation zones with soil
physical properties
Pearson
Correlation
(2-tailed)
Elevation
Elevation
1.00
Bulk
density
0.65**
Porosity
Compaction
Moisture
content
-0.60*
0.12
-0.49
Water
content
-0.46
Clay
Silt
Sand
-0.25
0.62*
-0.15
Correlation is significant at the 0.05 level (*) and at the 0.01 level (**).
Table 6. Correlation between Elevation and growth parameter Survival rate, MAID and MAIH of all
elevations in SM96, SM97 and SM98
Survival
rate
(%)
Elevation, SM96
0.879*
0.736
0.861
Elevation, SM97
0.549
0.254
0.229
Elevation, SM98
0.871
0.774
0.791
Correlation is significant at the 0.05 level (*) by Pearson correlation
Elevation & growth parameter
MAID
-1
(cm year )
MAIH
-1
(m year )
Waterlogged condition together with the presence of water stagnant in the contour zone of 25 in
SM96, 24 m (a) in SM97 and 24 m in SM98 had resulted in poor growth performance of planted trees
in respective elevations. For example in the elevation of 25 m in SM96, although its survival rate was
71.4 %, the lowest DBH and height were recorded at this elevation with the value of 5.9 cm and 4.5 m.
Besides, the lowest survival rate in SM97 was located at the contour zone of 24 m (a) in which only 25
% of the planted trees available were able to survive. In relation to the poor survival rate, 3.9 cm in
DBH and 3.0 m in height were recorded. In addition, the lowest survival rate in SM98 was located on
the elevation of 22 m with the value 46.2%. Poor performance of growth in terms of its DBH and height
were also recorded with 3.0 cm in DBH of 3.8 m in height.
It was identified that, poor growth performance in the contour zones of 25 m in SM96, 24 m (a) in
SM97 and 24 m in SM98, were resulted from the water stagnant available. Poorly drained soil together
with limited aeration affects the root penetration. The influence of waterlogged condition on root
growth and functions is closely related to oxygen shortage. Water logged condition may decrease both
extraction of water by roots, especially from deeper soil layers and affecting root growth as a whole.
Oxygen plays fundamental role for plant root functioning, among other components, through its
availability by the roots system, its indirect effect changing the soil properties. Soil aeration status is
one of the most important factors influencing plants roots. It is expected that, the dead Shorea
macrophylla were subject to anoxia, many are exposed of oxygen deficiency caused by water
-1
submerged or compacted soil at the depth 30 – 40 cm with the highest of 1.65 mL in elevation of 25
m in SM96. Besides that, in relation to the soil texture in the elevation of 25 m in SM96 and 24 m (a) in
SM 97 along with presence of water stagnant, coarse textured soil signifies the restricting root growth
that is result from the rough of the sand particle which resists the particle displacement by slippage
(Cruse et al., 1980). Because of the lower surface area of the sandy materials, a smaller percent of silt
and clay is sufficient to cement sand grains than the same volume of finer particles (Ibanga et al.,
1980). The presence of these features greatly abrupt the soil condition physically
High mortalities were identified at the elevation of 22 m in SM96 and poor values growth performance
at the elevation of 27 m in SM97 with the values of 3.8 cm in DBH and 4.2 m height. High mortalities
and the poor growth performance of both elevations might be interrupted due to the presence of
pioneer species available, such as Dillenia pulchella, Acacia mangium and Hevea brasiliensis. The
large pioneer species may used up the nutrients available and the large crown creating high shades to
the surrounding. It is identified that, low light intensity resulted had caused the Shorea macrophylla
seedlings unable to be established on elevation of 22 m because of relatively low light intensity there.
Contrary to the previous study which was conducted in secondary forest, Shorea macrophylla was
well adapted in under shades of higher trees. Furthermore, pioneer species would produce suitable
conditions for the planted trees survival (Daisuke et al., 2009). However, light environment in
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degraded secondary forest may be diverse because light condition changes by forest structures such
as gap formation and time span after disturbance. Light is considered the most important
environmental factors affecting plant establishment and growth in a tropical forest. Processes
such as photosynthesis and phototropism depend on the availability of light sources for plants as they
seek out light sources for energy and survival. Besides that, growth performance is enhanced through
morphological and physiological acclimatization to light environment. Shade environment or low
light intensity would limit the photosynthetic capacity and biomass of a plant (Dan et al., 2002).Another
possibility that would lead to the mortality Shorea macrophylla was caused by herbivores attacks such
as beetles and worms (Daisuke et al., 2009).
Based on the findings, the best growth performance of planted trees were characterised by its high
survival rate of more than 80 %, high DBH value of more than 6.4 cm, high height value of more than
6.4 m. The high performance growth of Shorea macrophylla is due to the suitable condition of soil and
environment that existed. It is expected that, the most suitable growth of Shorea macrophylla is in
relatively sandy soil on the surface soil but yet clayey on the sub soil. Sandy soil on the surface soil,
would promote to the higher value of the bulk density and greater compaction.
Shorea macrophylla is adaptable to low and high elevations. Therefore, factors of mircrotopography
could be less critical in determining the growth performance of Shorea macrophylla. It is shown that, at
lowest elevation 23 m and highest elevation 28 m could achieve the best growth performance are
reported although both of the elevations were in 5 m difference. The bulk density value always
reflected to the ability of the roots to penetrate and plays a major role in nutrient uptakes as well as in
-1
-1
the stability aspect of the tree. Probably, the bulk density of 0.90 g mL in surface soil and 1.13 g mL
in subsoil were the best value of bulk density that promotes to the greater performance of the Shorea
macrophylla. Besides the value of bulk density, the value of the soil porosity was moderately low
compared to the other elevations. It is expected that the greater value of soil porosity would lead to the
greater water infiltration and thus enable the root growth. From the result, it showed that Shorea
macrophylla were fairly suitable to be grown in the lower value of moisture content and slightly higher
water content. In terms of soil texture preference, Shorea macrophylla were well adapted in various
types of soil texture that range in these study sites comprising of loam, clay loam, and silty clay loam.
Besides, the growth of Shorea macrophylla is also suitable in moderately compact soil with average
of 35 strikes.
4. CONCLUSIONS
Information on soil properties under secondary forest of various age stands is important in order to
know whether the site that have been implemented for reforestation activities were suitable for the
growth of the selected tree species. The findings suggest that, Shorea macrophylla is highly adapted
in all across range of soil physical characteristics and various elevations. However, in the presence of
water stagnant and interruption by pioneer species affect the growth of the planted Shorea
macrophylla fails to adapt in such situations. Stagnant water would greatly alter the ability of the roots
functioning in taking minerals and water for the development of the trees. Besides, in heavy shades
condition, growth of Shorea macrophylla is suppressed due to lower photosynthetic capacity and
biomass of the trees. Collectively, the growth performance of Shorea macrophylla can be considered
established in the 14 to 16 years old stand. It shows a high survival rate of more than 70%. The
survival rate in SM96, SM97 and SM98 were 89 %, 81 % and 77 % respectively. Shorea macrophylla
is also shown to be highly tolerant species in various harsh conditions such as flooding which occurred
annually in study sites and considerably in low fertile soils. Therefore, the planting of Shorea
macrophylla for reforestation purpose is highly recommended.
Acknowledgement
This research was financially supported by the research grant from Fundamental Research Grant
(FRGS: Grant No. FRGS/07(03)/760/2010(46)) from Ministry of Higher Education, Malaysia and
Grant-in-aid for research purpose by the Japan-Malaysia Association (JMA). We wish to express our
gratitude to the Director and staff of the Forest Department, Sarawak for their supportive assistance
during the duration of this study. We would also like to extend our thanks to Mr. Jonathan Lat for his
kind co-operation and assistance during the field survey.
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Almquist, B. E., S. B. Jack, & M. G. Messina. (2002). Variation of the treefall gap regime in a
bottomland hardwood forest: relationships with microtopography. Forest Ecology and
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Brady, N.C. and Weil, R.R. (2008). The nature and properties of soil (14th Ed.). Prentice Hall. Upper
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Cruse, R. M., Cassel, D. K., & Adverett, F. G. (1980). Effect of particle surface on densitification of
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Dan., B., Nick, B., Martin, S., Pedro, M. C., Yap S. W. (2002). Spatial structure of light and dipterocarp
seedling growth in a tropical secondary forest. Forest Ecology and Management.157: 65-75.
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Glinski. J., & Lipiec, J., (2000). Soil Physical Conditions and Plant Roots. United States: CRC Press
Ibanga. I. J., Bidwell, O. W., Powers, W.L., Feyernhern, A. M., & William, W. W.(1980). Soil
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Itoh, A., Takuo, Y., Tatsuhiro, O., Mamuro, K., Peter, A. P., James, L.F.,
Ashton, P. S. & Lee,
H. S. (2003). Importance of topography and soil texture in spatial distribution of two sympatric
diterocarp trees in a Bornean rainforest. Ecological Research 18: 307- 320
Jomo, K. S., T. T. Chang & K. J. Khoo. (2004). Deforesting Malaysia: The political Economy and
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Social Ecology of Agriculture Expansion and Commercial Logging (1 Ed.). New York: Zed
Books Limited.
Mohammad Azani, A., Nik Muhammad Majid, & Magtro, S. (2011). Rehabilitation of Tropical
Rainforest Based on Indigenous Species for Degraded Areas in Sarawak, Malaysia in
Proceeding of Rehabilitation of Degraded Tropical Forest Ecosystem, 2-4 November 1999,
Bogor, Indonesia, Centre for International Forestry research, Bogor, Indonesia. pp. 226.
Rasip, A.G., & Lokmal, N. (1994). six year growth and survival rate of Shorea macrophylla planted
under pine plantation. In Wickneswary et al. (Eds.). Proceeding International Workshop, BioReforestation, Kangar, Malaysia. pp79-82.
Rogstad, H. S. (1990). The biosystematics and evolution of the Polyathia hypoleuca species complex
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347-408.
Sabrina, E. R., Davies S. J., King D. A. & Tan, S. (2005). Soil-related performance variation and
distribution of tree species in a Bornean rainforest. Journal of Ecology (95): 879 – 889.
Yamada, T., Yakamura T., Kanzaki, M., & Itoh, A. (1997). Topography-dependent spatial
pattern and habitat segregation of sympatric Scaphium sp. in a tropical rainforest at Lambir,
Sarawak. TROPICS (7): 57-66.
Yamakura, T., Kanzaki, M., Itoh, A., Ohkubo, T., Ogino, K., Chai, E. O. K., Lee, H. S., Ashton, P. S.
(1996). Forest structure of Lambir rainforest in Sarawak with spatial referenced to the
dependency of its physiognomy dimension on topography. TROPICS (6): 1-18.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
GROWTH PERFORMANCE OF PLANTED Shorea macrophylla UNDER LINE PLANTING
TECHNIQUE
*
*
M. Perumal , M. E. Wasli , H. Sani, A. Said and H. Nahrawi
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
*Emails: mugunthanperumal89@gmail.com / wmeffendi@frst.unimas.my
Abstract
A study on the growth performance and survival rate of planted Shorea macrophylla of different age
stands was conducted at Sampadi Forest Reserve, Sarawak. The objectives of this study were to
assess the growth performance and survival rate of planted S. macrophylla under reforestation
activities that were implemented by Forest Department, Sarawak along with several agencies of Japan
as to obtain preliminary data for further improvement of future reforestation activities in Sarawak. In
this study, S. macrophylla planted under line planting method was selected. Study plots were
established in the reforestation areas (planted with S. macrophylla in the year 1996; SM96, 1997;
SM97, 1998; SM98 and 1999; SM99). Diameter at breast height (DBH), total height and survival rate
were measured. The findings indicated that the growth performance in terms of survivability and mean
annual increment of diameter (MAID) in SM96 were the highest and showed better growth as
compared to SM97, SM98 and SM99. On the other hand, the mean annual increment of height (MAIH)
in SM97 revealed the greatest height among other plots. The survival rate of S. macrophylla in SM96,
SM97, SM98 and SM99 were 89%, 81%, 77% and 51% respectively. Substantial growth performance
and high survival rate of S. macrophylla implied that microclimate condition such as competition
between planted and existing pioneer species of the study area may have affected the planted S.
macrophylla. Notwithstanding, it is recommended that proper silvicultural technique is crucial to
manage the existing pioneer tree species towards the success of this reforestation programme.
Keywords: reforestation, Shorea macrophylla, growth performance
1. INTRODUCTION
Tropical rainforests are recognized as the richest ecosystems in the world in terms of structure and
species diversity (Whitmore, 1998). On a large regional and global scale, tropical rainforests have an
outstanding role and major influence in ameliorating and maintaining global climate change by
reducing the accumulation of greenhouse gases (Shukla et al., 1990). Though occupying only 7 % of
the earth’s land surface, over half of the planet’s life forms are found in tropical rainforests (Whitmore,
1998). Tropical rainforests not only sustain biodiversity but provide homes to indigenous peoples,
pharmacopeias of natural products, and provide crucial ecosystem services, such as flood
amelioration and soil conservation.
Nonetheless, despite the multi-functional roles and richness, tropical rainforests are fragile habitats
and are being destroyed rapidly. Conversion of forested areas to non-forest lands such as to pasture
and agriculture have resulted in the permanent reduction of indigenous species including timber
species such as Dipterocarp spp. from Dipterocarpaceae family (Montagnini et al., 1997). Commercial
logging activities, shifting cultivation, urbanization industry, natural disturbances like landslide and
other forms of encroachment are all principal causes of deforestation in tropical regions (Geist and
Lambin, 2002; ITTO, 2002). Due to the consequence of deforestation, excessive forest harvesting and
shifting cultivation, tropical lands become rapidly eroded and infertile, and degraded farmlands are
commonly abandoned.
Reforestation plays a vital role in maintaining our tropical rainforests and may offer one means of
mitigating these processes of degradation while sustaining resident human communities. The
importance of reforestation in the tropics includes productions of timber and other goods and services
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as well as aiding the recovery of biodiversity by re-establishing forest cover (Parrotta et al., 1997;
Lamb et al., 2005; Benayas et al., 2009). However, lack of information regarding tree species
performance has been identified as major limitation on the success and adoption of diversified
reforestation strategies in restoring the tropical rainforests. In Peninsular Malaysia, stated that
information on soil properties under rehabilitation of degraded forest land and growth performance
including survival rate of planted dipterocarp species such as S. macrophylla and non-dipterocarp
species is still limited (Arifin et al., 2008).
Since S. macrophylla has been identified as one of the valuable timber tree species which benefits
future reforestation and rehabilitation efforts in Sarawak, it is very crucial to conduct the study on the
growth performance of this particular species. This preliminary study is to clarify fundamental
information on the suitability of S. macrophylla of Dipterocarpaceae family planted using line planting
technique for future reforestation and rehabilitation activities in Sarawak. Hence, the objectives of this
study are to evaluate and assess the growth performance and survival rate of planted S. macrophylla
at various age stands under reforestation activities that were implemented by Forest Department,
Sarawak along with several agencies of Japan in Sampadi Forest Reserve. Besides that, the objective
of this study is to obtain preliminary data which will be used by Forest Department, Sarawak for further
improvement in the planning strategies of their future reforestation activities.
2. MATERIALS AND METHODS
This study was conducted at Japan-Sarawak Friendship Forest reforestation area located in Sampadi
Forest Reserve (N01°34’13’’E10λ°53’12’’) δundu, Sarawak and is located about 72 km southwest of
Kuching. The Sampadi Forest Reserve covers approximately 5,163 hectares and has a humid tropical
climate, associated with peaks of seasonal changes of rainfall and temperature. The topography at the
study site was of low undulating with an average elevation of 87 m above sea level. Sampadi Forest
Reserve has a tropical seasonal climate (no dry season) with all months receiving on average more
than 100 mm with a subtropical wet forest biozone. Average annual precipitation was 4,100 mm
(Vincent and Davies, 2003). The soils in the study area were derived from combination of sandstone,
coarse-grained, humult ultisols and sandy residual parent material of Bako series based on Sarawak
soil classification system which corresponds to Ultisols soil orders according to the USDA-Soil
Taxonomy Classification System (Soil Survey Staff, 2006).
Since the year 1996, Forest Department of Sarawak, along with various international agencies had
initiated a reforestation programme by planting various types of Dipterocarp species (mainly of
Dipterocarpaceae family) in the study area such as S. macrophylla. The reforestation sites were
established under secondary forests with all tree seedlings were planted under line-planting technique
with lines cut 5 m apart and trees planted at 5 m interval along the lines. Preparation and maintenance
of planting lines were conducted manually and large pioneer species were left uncut when preparing
the planting lines.
In this study, assessment on the growth performance of S. macrophylla that were planted in Sampadi
Forest Reserve at various age stands from the year 1996, 1997, 1998 and 1999, were conducted.
Approximately 400 S. macrophylla seedlings were planted for each age stand under line planting
2
2
technique. Study plots with the size of 50 x 50 m (four subplots with 25 x 25 m in each subplot) were
constructed within the compartment planted with S. macrophylla at four different ages of tree stand
(planted in year 1996, 1997, 1998 and 1999). In these plots, measurement on the height, diameter at
breast height (DBH), survival rate and light intensity of planted trees were conducted. Nonetheless,
abbreviation were coded to represent the studied plots; SM96 (S. macrophylla planted in year 1996),
SM97 (S. macrophylla planted in year 1997), SM98 (S. macrophylla planted in year 1998) and SM99
(S. macrophylla planted in year 1999). At present the selected S. macrophylla were 16 years old, 15
years old, 14 years old and 13 years old respectively.
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3. RESULTS AND DISCUSSION
3.1 Survival rate of planted Shorea macrophylla in SM96, SM97, SM98 and SM99
The assessment of sufficient number of species survival such as S. macrophylla when planted under
various age stands on a degraded forest land under rehabilitation or plantation programs is required
towards better recommendation of species selection in the future. The survival rate for planted S.
macrophylla at various age stands in SM96, SM97, SM98 and SM99 are shown in Figure 1.
11 %
19 %
89 %
23 %
49 %
81 %
51 %
77 %
Survival rate (%)
Mortality rate (%)
Figure 1. The survival rate of planted Shorea macrophylla in plot aged 16 years (SM96), plot aged 15
years (SM97), plot aged 14 years (SM98) and plot aged 13 years (SM99)
The results showed that the survival rate for planted S. macrophylla in SM96 and SM97 were 89 %
and 81 % respectively whereas the survival rate for SM98 and SM99 were 77 % and 51 %
respectively. The highest survival rate of planted S. macrophylla was from the plot aged 16 years
(SM96) with 89 % and the lowest survival rate was from the plot aged 13 years (SM99), with the
percentage of survival rate, 51 %. Relatively, when all the study plots were compared, it clearly
showed that planted trees in SM99 has the lowest survival rate.
There are several factors that contribute to these findings such as the microtopography, intervention
on man silviculture and the thinning regime of pioneer species such as Acacia mangium, Macaranga
spp., Dillenia suffructicosa, and Ficus spp.. Environmental factors such as drought season, diseases,
planting technique and weed competition influence the growth performance and survival of planted
species. Besides, two major factors that usually influence the seedlings survival are light intensity and
the amount of available moisture especially during the initial stage of stand establishment (Evans,
1992).
3.2 Growth performance of planted Shorea macrophylla in SM96, SM97, SM98 and SM99
3.2.1Tree height and Diameter at breast height of planted Shorea macrophylla
The number of occurrence and total height of planted S. macrophylla at various age stands in
Sampadi Forest Reserve were shown in Figure 2. Based from the histogram obtained, the frequency
of encountered trees with the height class of 5.01 m to 10.00 m showed the highest with total
frequency of 174 trees among all the four study plots surveyed; SM96, SM97, SM98 and SM99. This
followed by the height class of 10.01 m to15.00 m with a total of 68 trees, ≤ 5.00 m class with 48 trees
and 15.01 m to 20.00 m with 8 trees. There were no trees measured with height more than 20.00 m in
all the study plots. Generally, there were no much differences in the diameter at breast height (DBH)
class for S. macrophylla compared with the tree height class. The number of occurrence and diameter
at breast height (DBH) of planted S. macrophylla at various age stands in Sampadi Forest Reserve
were shown in Figure 3. Based from the histogram obtained, the frequency of encountered trees with
the diameter class of 5.1 cm to 10.0 cm showed the highest with total frequency of 175 trees among
all the four study plots surveyed; SM96, SM97, SM98 and SM99. This followed by the diameter class
of 10.1 cm to 15.0 cm with a total of 5λ trees, ≤ 5.0 cm class with 55 trees and 15.1 cm to 20.0 cm
class with 9 trees. There were no trees measured with DBH more than 20.0 cm in all the study plots.
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Figure 3. Diameter at breast height (cm)
distribution of planted Shorea macrophylla in
SM96, SM97, SM98 and SM99
Figure 2. Total height (m) distribution of
planted Shorea macrophylla in SM96, SM97,
SM98 and SM99
b
b
b
a
c
c
b
a
Figure 5. The mean average diameter at breast height (cm) of planted Shorea macrophylla in SM96,
Figure 4. The mean average total height (m) of planted Shorea macrophylla in SM96, SM97, SM98
and SM99; Different letter indicate significant differences at 5 % using Tukey test SM97, SM98 and
SM99; Different letter indicate significant differences at 5 % using Tukey test
Nonetheless, based from the Figure 4, the average total height of S. macrophylla in SM97 showed the
highest total height with 7.45 m followed by SM96 with 6.32 m and SM98 with 6.07 m. The average
total height of planted S. macrophylla in the year 1999 recorded the lowest among all study plots with
3.84 m. About two stems of S. macrophylla planted in SM98 were at height of more than 16.00 m. On
the other hand, the average diameter at breast height decreases and is directly proportional to the
planting year of S. macrophylla. SM96 recorded the highest average reading in diameter at breast
height with 7.8 cm whereas the lowest average diameter was from SM99 with only 3.3 cm. The
average diameter at breast height for SM97 and SM98 were 7.1 cm and 5.3 cm respectively.
Approximately four stems of S. macrophylla planted in SM97 were in diameter above 16.0 cm.
From the field observation, the growth in terms of height and diameter of planted trees in SM99 was
gradually low as compared with other study plots. There are several environmental factors resulting in
this Shorea species being the shortest in height of SM99 plot. Most of the planted S. macrophylla were
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damaged at some part of the top shoots by falling branches, twigs, and large leaves of uncut pioneer
species such as the Acacia mangium and Dillenia suffructicosa and probably resulting S. macrophylla
being the shortest in height in the Sampadi Forest Reserve. Moreover, most of the S. macrophylla
seedlings were attacked by termites and herbivores such as ants, worms and beetles leading to
slightly unpromising growth as shown in Figure 4 and Figure 5.
In addition, in plot SM99, unapplied silvicultural treatment significantly affected the mortality rates in
which, had a large impact on the growth performance of S. macrophylla. Competition of undergrowth
vegetation could be assumed as another reason why most of the growth of S. macrophylla in SM99
was stunted. On the other hand, the growth performance in terms of height and diameter in SM96 and
SM97 were slightly better than SM99. Better height and diameter growth of the species could be due
to adapt well to the locality or species-site matching and ability to tolerate water stress and other
unfavorable site conditions. The variations in growth of planted species are due to specific reaction of
the species to environmental conditions such as climate, soil and topography (Azani et al., 2003).
Furthermore, growth in terms of height and diameter increment is also influenced by the availability of
space between trees (Mohd Zaki et al., 2003). However, this would be most likely to promote weed or
climbers to grow and invade the site especially in the plot SM99.
3.2.2 Mean annual increment in height (MAIH) and diameter at breast height (MAID) of Shorea
macrophylla in SM96, SM97, SM98 and SM99
The results for mean annual increments in height and diameter at breast height among species after
16 years, 15 years, 14 years and 13 years of planting are shown in Figure 7 and Figure 8. In similarity
to survival rate, generally there were significant differences (p < 0.05) of the mean annual increments
in height and diameter among species being planted in the study plots. The average value of the
-1
mean annual increment of height (MAIH) of S. macrophylla planted in SM97 (0.47 m year ) recorded
the highest MAIH of planted trees among the plots surveyed, followed by SM98 with MAIH 0.40 m
-1
-1
-1
year , SM96 (0.37 m year ) and the lowest SM99 (0.27 m year ). On the other hand, the average
-1
value of the mean annual increment of diameter (MAID) of planted trees in SM96 (0.46 cm year )
-1
-1
attained significantly higher MAID than SM97 (0.44 cm year ), SM98 (0.35 cm year ) and SM99 (0.23
-1
cm year ) respectively.
0.80
b
b
0.70
a
c
bc
b
0.60
MAID (cm year-1)
ab
a
0.50
0.40
0.30
0.20
0.10
0.00
SM1996
SM1997
SM1998
SM1999
Figure 7 & 8. The mean annual increment in height (MAIH) and diameter (MAID) of planted Shorea
macrophylla in plot aged 16 years (SM96), plot aged 15 years (SM97), plot aged 14 years (SM98) and
plot aged 13 years (SM99); Different letter indicate significant differences at 5 % using Tukey test
Basically in terms of mean increments of height and diameter, the growth performance of planted S.
macrophylla is relatively slow. Although these mean annual increments (MAI) are quite slow after 16
years of planting, S. macrophylla still can adapt well and survive under harsh conditions such as
annual flooding. There were no similar characteristics in terms of growth was found and observed
compared when early plantation establishment was done. However, there are several factors such as
microtopography, soil condition and microclimate influence the growth performance of the planted S.
macrophylla in the study plots.
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3.2.3 Suitability and growth performance of planted Shorea macrophylla in comparison with previous
studies on reforestation in Malaysia
In order to compare and find out the suitability of S. macrophylla as one of the appropriate species for
reforestation and rehabilitation purpose, findings in this study with previous studies in Malaysia on
experimental reforestation efforts to rehabilitate degraded land by using indigenous Dipterocarp tree
species under various form of enrichment planting technique were compared as shown in Table 1.
Even though the planted tree species in previous studies varied from this study, it should be noted that
the tree species used in these studies are of the same family which is Dipterocarpaceae.
Table 1. The growth rates of planted species in this study and other previous study on reforestation in
Malaysia
Stand
Survival
MAIH
MAID
Planting
Studied species
Source
-1
-1
age (yrs)
rate (%)
(m yr ) (cm yr ) technique
Shorea macrophylla
16
89
0.37
0.46
LP
Present study
Shorea macrophylla
15
81
0.47
0.44
LP
Present study
Shorea macrophylla
14
77
0.40
0.35
LP
Present study
Shorea macrophylla
13
51
0.27
0.23
LP
Present study
Shorea leprosula
Hamzah et al. 2009
6
22
1.08
1.10
OP
Shorea parvifolia
Hamzah et al. 2009
6
30
1.00
1.01
OP
Shorea leprosula
Arifin et al. 2008
12
57
1.24
1.44
MSFP
Shorea parvifolia
Arifin et al. 2008
12
40
1.01
1.20
MSFP
Shorea pauciflora
Abdu et al. 2008
11
34
0.67
0.74
LP
Shorea macroptera
Abdu et al. 2008
11
47
0.61
0.67
LP
Shorea pauciflora
Abdu et al. 2008
11
36
0.77
1.08
GP
Shorea macroptera
Abdu et al. 2008
11
54
0.74
0.86
GP
Planting techniques: LP, line planting; OP, open planting; MSFP, Multi-storied Forest Planting; ICPS,
Island Corridor Planting Scheme
In the present study, regardless of the difference in stand age after planting, planting techniques and
types of planted tree species, the survival rate of planted S. macrophylla was higher than the result
reported by previous studies. In contrast, the survival rate of S. macrophylla was higher than the result
reported on S. leprosula and S. parvifolia at 6 years and 12 years after planting on a degraded forest
land in Pasoh Forest Reserve, Negeri Sembilan and in Perak, Peninsular Malaysia respectively (Arifin
et al., 2008; Hamzah et al., 2009). Nonetheless, in terms of mean annual increment in height (MAIH)
and diameter (MAID) of S. macrophylla planted in SM96, SM97, SM98 and SM99 were lesser as
compared to the results reported by Arifin et al. (2008) and Hamzah et al. (2009). However it should
be noted that under the same technique of enrichment planting, at the age stand of 16 years, 15
years, 14 years and 13 years of planting, the survival rate of S. macrophylla were higher compared to
Shorea pauciflora (34%) and Shorea macroptera (47%) planted under line planting technique by Abdu
et al. (2008). On the contrary, the mean annual increment in height (MAIH) and diameter (MAID) of S.
macrophylla were lower compared to planted S. pauciflora and S. macroptera under both line and gap
planting techniques.
The effect of extreme heat, strong winds and over exposure of light to the planted seedlings under
open planting could be among the major reason behind the low survival rate of planted S. leprosula
and S. parvifolia. Full sunlight received by the seedlings has caused scorching of leaves hence
resulting in low survival rate (Suhaili et al., 1998). As can be seen from this study, although the
survival rate of the planted S. macrophylla in Sampadi Forest Reserve were substantially higher, it is
expected that in the future, the survival rate of S. macrophylla will decline when the planted trees
reached its maximum maturity due to competition for space, available nutrients in soil and light
requirement for the planted trees. In the case of planted dipterocarp species like Shorea sp., several
researchers have also reported similar trend of low survival rate when involving planting dipterocarp
species on degraded secondary forest. For example, Azman et al. (1990) reported that S. leprosula
and S. parvifolia survival rate of 30 % and 20 %, respectively at 15 years after planting. Another study,
Adjers et al. (1996) reported that the survival rate of planted ten dipterocarp species varied widely
from 5.9 % to 77.8 % three years after planting in degraded secondary forest subjected to shifting
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cultivation in Kalimantan, Indonesia. Dipterocarp species shows relatively slow growth rate but it is
adapting well to sufficient amount of sunlight when planted in land with poor soil properties (Appanah
and Weinland, 1993; Adjers et al., 1996; Vincent and Davies, 2003).
Environmental factors such as weather condition in terms of sunlight intensity, pest attack (termites),
animal distribution, planting technique, weed competition and poor soil condition are the factors
possibly could lead to variation in growth performances and survival rate of Dipterocarp species. Study
has been reported that S. macrophylla is susceptible to termite attack. The sap-sucking insect,
Helopeltis clarifer has been recorded to kill seedlings in nursery. Moreover, growing space also may
contribute to the growth performance (Mohd Zaki et al., 2003).
One of the advantages adapting enrichment planting such as line planting technique in this study is
that, it enables the local vegetation such as some kind of undisturbed pioneer species which has
grown naturally before establishment of planting lines such as Acacia mangium, Dillenia suffructicosa
and Macaranga spp. to provide optimal shade condition for the planted dipterocarp seedlings which is
well known as shade tolerant when they are sapling as to reduce overexposure to sunlight.
Nevertheless, silvicultural maintenance activities can be carried out easily as the spaces between the
planted seedlings are uniform. Notwithstanding, good silvicultural practice in the reforestation area in
Sampadi Forest Reserve should be considered as tree species like Acacia mangium, Dillenia
suffructicosa and Macaranga spp. in SM96, SM97, SM98 and SM99 would generate a competition for
the S. macrophylla. As described by Abdu et al. and Arifin et al. (2008), the survival rate of planted
Shorea sp. was significantly higher under narrow than wider opening of both line and gap planting
techniques in Peninsular Malaysia. Generally, dipterocarp seedlings have been found to survive better
in shaded sites as compared to open sites (Adjers et al., 1996; Romell et al., 2007).
4. CONCLUSION
Based from the findings obtained and compared, S. macrophylla is considered as suitable as a
planting material for reforestation in the study area. The findings of this study shows positive progress
in terms of survival rate and growth performance of planted Dipterocarp species especially S.
macrophylla. This finding indicates that S. macrophylla is suitable for reforestation activities to be
carried out in Sampadi Forest Reserve and the positive outcome could be achieved from such
reforestation effort. Nonetheless, the high survival rate of planted S. macrophylla in study plots of
SM96 and SM97 with 89% and 81% respectively implies that S. macrophylla is a suitable species to
be planted under line planting technique for reforestation activities in this study area. Moreover, well
adaptability and survivability of planted S. macrophylla which can grow under severe conditions such
as flood can be one of the possible reasons as planting species for reforestation purpose. Besides
that, the probable reason behind the substantial growth rate in terms of height and diameter
associated with high survival rate of S. macrophylla could be due to the adaptability of planted trees to
grow under certain soil condition or competition for light between planted trees and naturally grown
vegetation which was undisturbed with the planting line. Thus, in order to find out other factors which
could affect the growth performance and survival rate of S. macrophylla under line planting technique,
further detailed studies are required.
Acknowledgement
This research was financially supported by the research grant from Fundamental Research Grant
(FRGS: Grant No. FRGS/07(03)/760/2010(46)) from Ministry of Higher Education, Malaysia and
Grant-in-aid for research purpose by the Japan-Malaysia Association (JMA). We wish to express our
gratitude to the Director and staff of the Forest Department, Sarawak for their supportive assistance
during the duration of this study. We would also like to extend our thanks to Mr. Jonathan Lat for his
kind co-operation and assistance during the field survey.
references
Abdu, A., Tanaka, S., Jusop, S., Majid, N.M., Ibrahim, Z., Sakurai, K., & Wasli, M.E. (2008).
Assessment on soil fertility status and growth performance of planted dipterocarp species in
Perak, Peninsular Malaysia. Journal of Applied Science, 8, 3795-3805.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Adjers, G., Kuusipalo, J., Hadengganan, S., Nuryanto, K., & Vesa, L. (1996). Performance of ten
dipterocarp species in restocking logged-over forest areas subjected to shifting cultivation.
Journal of Tropical Forest Science, 9, 151-160.
Appanah, S. & Weinland, G. (1993). Planting quality timber trees in Peninsular Malaysia- A review.
Forest Research Institute of Malaysia, Kuala Lumpur, Malaysia.
Azani, M.A., Muhamad, N.M.N., Hanum, I.F., Zaki, H.M., Kamil, Y.M., & Yusoff, B.M. (2003). Species
trial and soil characteristics of rehabilitated logged-over forest at Pasoh, Negeri Sembilan,
Malaysia. In: Rehabilitation of Degraded Tropical Forests, Kobayashi, S., Y. Matsumoto and E.
Ueda (Eds.). Shigeo Kobayashi, Malaysia, ISBN: 4-9901797-0-6.
Azman, H., Razali, W.M.W., Shahrulazman, I., & Rahman, A.K. (1990). Growth performance of
indigenous species under enrichment planting in logged-over forests. In: Malaysian Forestry and
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Benayas, J.M.R., Newton, A.C., Diaz, A. & Bullock, J.M. (2009). Enhancement of biodiversity and
ecosystem services by ecological restoration: a meta-analysis. Science, 325, 1121-1124.
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Evans, J. (1992). Plantation forestry in the tropics. (2 ed.). Oxford: Oxford University Press.
Geist, H.J. & Lambin, E.F. (2002). Proximate causes and underlying driving forces of tropical
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Hamzah, M.Z., Arifin, A., Zaidey, A.K., Azirim, A.N., Zahari, I., Hazandy, A.H., Affendy, H., Wasli, M.E.,
Shamsuddin, J., & Muhamad, M.N. (2009). Characterizing Soil Nutrient Status and Growth
Performance of Planted Dipterocarp and Non-Dipterocarp Species on Degraded forest land in
Peninsular Malaysia. Journal of Applied Science, 9 (24), 4125-4223.
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Lamb, D., Erskine, P.D. & Parrotta, J.A. (2005). Restoration of degraded tropical forest landscapes.
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Mohd Zaki, H., Azani, M.A., Faridah, H.I., Kamil, Y.M., & Muhamad, M.N. (2003). Recovery
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Montagnini, F., Eibl, B.M., Grance, L., Maiocco, D., & Nozzi, D. (1997). Enrichment planting in
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CONCEPTUAL FRAMEWORK FOR THE ASSESSMENT OF ECOLOGY AND CARBON
SEQUESTRATION AT SECONDARY FORESTS IN SARAWAK
Karyati
1,2*
1
1
, Isa B. Ipor , Ismail Jusoh and Mohd Effendi Wasli
1
1
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300,
Kota Samarahan, Sarawak, Malaysia.
2
Faculty of Forestry, University of Mulawarman, Kampus Gunung Kelua, Samarinda, East Kalimantan,
Indonesia, 75119.
*Email: karyati.hanapi@yahoo.com
Abstract
A conceptual framework is a fundamental structure to guiding principles of research within a discipline.
Various frameworks on assessment of secondary forests had been reported in the tropic region.
However, there is limited information available on the conceptual framework of ecology and carbon
sequestration assessment under secondary forest at Sarawak. This paper was conducted to
determine the conceptual framework for the assessment of ecology and carbon sequestration at
various stage secondary forests in Sabal, Sri Aman. The conceptual framework was determined
based on research plans to bring out in the process of research. Field surveys were conducted at 3,
5, 10, and 20 years old secondary forests (hereafter called Temuda I, Temuda II, Belukar I, and
Belukar II respectively) at the study sites. The assessment is mainly to determine diversity of tree
species, soil properties, growth increment, above ground biomass, and carbon sequestration of
secondary forest at the different stages of fallows. Conceptual framework for assessment of the study
can help a researcher determine methodology or way to answer the study questions.
Keywords: Conceptual framework, ecology, carbon sequestration, and secondary forest.
1. INTRODUCTION
Conceptual framework is a type of intermediate theory that attempt to connect to all aspect of inquiry,
such as problem definition, purpose, literature review, methodology, data collection and analysis
(Anonim, 2012). Few studies had been done on the framework of tropical forest (Chokkalingam, et al.,
2001; De Jong et al., 2001; Ponce-Hernandez, 1999). There are little information exists on the
conceptual framework for ecology and carbon sequestration of secondary forests with various stage
stand, because most of previous framework were mainly pointed out on dynamics and diversity of
tropical secondary forest particularly. However, species diversity, soil properties, growth increment,
above ground biomass, and carbon sequestration all can play major roles in determining ecology and
carbon sequestration of secondary forests. The proposed conceptual framework will be use to
determine the comprehensive view on ecology and carbon sequestration under secondary forest at
Sarawak, Malaysia.
2. MATERIALS AND METHODS
To contribute an understanding of ecology and carbon sequestration on different age secondary
forests, the study was carried out in a chronosequence of different stage of fallows ranging in age from
3, 5, 10, and 20 years in Sabal, Sri Aman, Sarawak, Malaysia. The conceptual framework was
developed based on steps of research questions or problems. It was constructed according to the
purposes of the study. This framework provides description to give comprehensive understanding
about the study assessed.
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3. RESULTS AND DISCUSSION
3.1 Conceptual Frameworks for Assessment on Ecology and Carbon Sequestration of
Secondary Forests
Conceptual framework was developed for the planned research (see Figure 1). The steps of
constructed framework were arranged based on objectives of the study. The conceptual framework
provides several useful functions (De Jong et al., 2001). The framework reflected the main
assessment process that done in the field and laboratory works. The Framework was essential to
lead research activities. This paper identified the factors related to ecology and carbon sequestration
under secondary forests. The framework provided some information as a basis to identify clearly at
different stages. Therefore, it provides general information about analysis of the study on ecology and
carbon sequestration of secondary forests. Because of this feature, the conceptual framework was a
useful tool to identify viable secondary forest management and policy options. In principle, it is
necessarily required as important information to manage and conserve secondary forests.
3.2 Implications of the Study
Results from past work and field experiences showed that the forests are complex with pronounced
stratification with the ecosystems held together in a dynamic state. Species composition is influenced
by processes of competition and interactions between plants and their biotic and physical
environments (Odera, 2002). Secondary forests provide a wide range of benefits at local, national,
and global levels. One of these benefits is carbon sequestration. U.S. Environmental Protection
Agency (2010) define that terrestrial carbon sequestration is a process where carbon dioxide (CO 2)
from the atmosphere is absorbed by trees, plants, and crops through photosynthesis and stored as
carbon in biomass (tree trunks, branches, foliage, and roots) and soils. Carbon sequestration rates
vary by tree species, soil type, regional climate, topography, and management practice. Fisher and
Binkley (2000) state that the current distributions of tree species (and understory species) show strong
relationships with temperature and water supplies. So the changes in these factors could alter
species distributions and species differ substantially in their effects on soil biogeochemistry. Forest
management practices have major effects on soil carbon pools, and management changes over the
decades even when climate does not.
This paper addresses on issues of ecology and carbon sequestration, because ecology and carbon
sequestration of secondary forests remain poorly understood, and few in-depth data are available. A
theoretical framework is proposed to address this gap in the literature and to provide a basis for
understanding ecology and carbon sequestration of secondary forests. The conceptual framework
focused on the design of studies on species diversity, diameter increment, soil properties, above
ground biomass, and carbon sequestration. The overall contribution of the framework is proposed to
determine the scope of study on ecology and carbon sequestration of secondary forests. A secondary
purpose of the framework is to illustrate how to assess field and laboratory works of this study.
CONCLUSION
The conceptual framework was determined as basic information about the study was
assessed. The conceptual framework provides a brief and holistic overview of study on ecology and
carbon sequestration under secondary forests. This framework can be used as a model of operations
research on the related study. An understanding of the relationships among species diversity, soil
properties, growth increment, above ground biomass, and carbon sequestration is essentials.
Information about the sequence of change in community biomass and environment of the secondary
forests are important to evaluate and conserve them during the course of succession.
389
Floristic
Structure,
Composition ,
and Diversity
Ecology
Soil
Properties
Morphological
properties
Physicochemical
properties
Selected
Trees
390
Forest
Secondary
forest (SF)
Different
horizon
0-10 cm
20-30 cm
N
H'
C
e
R
ISS
IDS
Colour, field texture, consistence,
structure, rock fragment, mottling, root
Texture, BD, hardness, pH, EC, TC, TN,
OM, Porosity, Ca, Mg, K, Na, CEC, P.
Acacia mangium
Cratoxylum arborescens
Cratoxylum glaucum
Endospermum diadenum
Euodia glabra
Macaranga gigantea
Macaranga triloba
Vernonia arborea
Vitex pubescens
Rainfall
Temperature
Humidity
Light intensity
Temuda I
(3 yr old SF)
Temuda II
Growth
Climate elements
Diameter
(5 yr old SF)
Increment
increment
Belukar I
DBH increment
(10 yr old SF)
Belukar ------------------------------------------------------------------------------------------------------------II
(20 yr old SF)
Leaf
Destructive
Tree
Allometric
Branch
Sapling
sampling
AGB
Biomass
equation
Stem
Seedling
technique
Carbon
sequestration
Carbon
sequestration
Carbon stock =
AGB × Carbon concentration
ECOLOGY &
CARBON
SEQUESTRATION
OF SF
Annual carbon sequestration =
Carbon stock / age
Abbreviations : DBH = diameter at breast height ; H = height ; f = frequency ; SA = sample area ; N = number of individuals ; G = number of gen era ; IV = important value ; SDR = summed
dominant ratio ; N = number of individuals ; H' = Shannon-Wienner diversity index ; C = Simpson dominance index ; e = Pielou evenness index ; R = Margalef richness index ; ISS = Sorensen’s
index of similarity ; IDS = Sorensen’s index of dissimilarity ; BD = bulk density ; EC = electrical conductivity ; TC = total carbon ; TN = total nitrogen ; OM = organic matter ; Ca = calcium ; Mg
= magnesium ; K = potassium ; Na = ; CEC = cation exchange capacity ; P = phosphorus ; AGB = above ground biomass.
Figure 1. Conceptual framework (Source : Karyati et al., 2011).
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
DBH & H
f, SA, N, G
Species Abundance
Curve
Basal area
Floristic composition
Family composition
Species composition
SDR & IV
Species Diversity
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
References
Anonim. (2011). http://en.wikipedia.org/wiki/Framework. Retrieved December 08, 2011.
Chokkalingam, U., Smith, J. & De Jong, W. (2001). A Conceptual Framework for the Assessment of
Tropical Secondary Forest Dynamics and Sustainable Development Potential in Asia. Journal
of Tropical Forest Science, 13(4): 577-600.
De Jong, W., Chokkalingan, U., Smith, J. & Sabogal, C. (2001). Tropical Secondary Forests in Asia :
Introduction and Synthesis. Journal of Tropical Forest Science, 13(4): 563-576.
rd
Fisher, R.F. & Binkley, D. (2000). Ecology and Management of Forest Soils. 3 Edition. Canada:
John Wiley & Sons. p. 433.
Karyati, Ipor, I.B., Jusoh, I. & Wasli, ε.E. (2011). “Frameworks of Study on Ecology and Carbon
st
Sequestration of Secondary Forests at Sarawak”. Presented in 1 Postgraduate Symposium on
nd
st
Resource Science and Technology (20 -21 December 2011) at Universiti Malaysia Sarawak
(Unimas), Sarawak, Malaysia.
Odera, J.A. (2002). The State of Secondary Forests in Anglophone Sub-Saharan African Countries:
Challenges and Opportunities for Sustainable Management in Africa. Workshop on Tropical
Secondary Forest Management in Africa.
http://www.fao.org/DOCREP/006/J0628E/J0628E13.htm
Ponce-Hernandez, R. (1999). Assessing the Carbon Stock and Carbon Sequestration Potential of
Current and Potential Land Use Systems and the Economic Rationality of Land Use
Conversions. In World Soil Resources Report 86, pp. 77-92, FAO , Rome.
U.S. Environmental Protection Agency. (2010). Carbon Sequestration in Agriculture and Forestry.
http://www.epa.gov/sequestration/international.html
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
AN ACCOUNT OF SELECTED Shorea spp. OF LAMBIR HILL NATIONAL PARK, SARAWAK
1*
1
1
1
1
Linda, L. & Tawan, C.S. , Ipor, I.B. ., Meekiong, K. . , Jusoh, I. . & Affendi, S.
2
1
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, University Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
2
Forest Department Sarawak, Wisma Sumber Alam, Jalan Stadium, Petra Jaya, 93660, Kuching
Sarawak
*Email : lindaliman1172@gmail.com
Abstract
δambir Hill National Park is located at the latitude 4˚ N, longitude 114˚ E in εiri Division, Sarawak. It
was gazetted on the 26 June 1975 covering an area of about 6952 hectares. This includes the
spectacular central portion of the Lambir hills featured by a rugged east-west trending sandstone
escarpment with height of about 465 m above sea level. Lambir Hill National Parks is recognised as
very important regional and global conservation for its mega tropical plant diversity. There are
basically four main types of forest namely the rich natural mixed dipterocarp forest which covers half
of the area while the remaining consists of kerangas, logged-over, and the secondary forests.
Previous studies indicated that this national park recorded 1173 different species of flora. With the
high number of tree diversity, Lambir Hill National Parks is rated as having one of the highest flora
diversity of the old tropical rainforest. As an effort to obtain more information of the tree flora,
taxonomic study of seven selected species of Shorea found within Lambir Hill National is carried out.
Detail morphological characteristics, ecological information of the seven species are described.
Key word: Lambir Hill National Park, Flora diversity, Shorea species
1. INTRODUCTION
δambir Hill National Park is located at the latitude 4˚ N, longitude 114˚ E in εiri Division, Sarawak. It
was gazetted on the 26 June 1975 covering an area of about 6952 hectares. This includes the
spectacular central portion of the Lambir hills featured by a rugged east-west trending sandstone
escarpment with height of about 465 m above sea level (Watson, 1985). Lambir Hill National Parks is
recognised as very important regional and global conservation for its mega tropical plant diversity.
There are basically four main types of forest namely the rich natural mixed dipterocarp forest which
covers half of the area while the remaining consists of kerangas, logged-over, and the secondary
forests. Previous studies indicated that this national park recorded 1173 tree species in 286 genera
and 81 families in only 52 ha (Lee et al., 2002) . Most of the emergents in this forest are Meranti,
(Shorea sp.), Keruing (Dipterocarpus sp.) and Kapur (Dryobalanops sp.) As an effort to obtain more
information of the tree flora, taxonomic study of seven selected species of Shorea found within Lambir
Hill National is carried out.
Shorea Roxb. ex Gaertn.f. (DIPTEROCARPACEAE) is a genus of tree native to rain forest of South
East Asia. The genus Shorea is named after Sir John Shore, Governor-General for British East India
Company (1793-1798). The genus Shorea is well distributed in Borneo, 139 species are known, of
which 91 are endemic, and 130 occur in Sabah and Sarawak (Ashton, 2004).The species of Shorea
can be distinguished from other genera in Dipterocarpaceae based on their morphological characters
such as leaf, wood, fruit, nut and flower and sometimes resin chemistry. The studies aimed to provide
taxonomy identification and description of tree species that could serve supplementary project such as
Hyperspectral Airborne Imaging System pursued by the team from Sarawak Forestry Department.
2. MATERIALS AND METHODS
This study is based mainly on observations of natural population during fieldwork in Lambir Hills
National Park, Sarawak (2011- 2012). Using canopy crane plot, with 80 m in height and arm of 75 m
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long, data collection was carried out in systematic manner, which include photos of tree crowns. All
specimens of selected species was collected and preserved in large plastic bag containing 70%
alcohol to keep it from fungal infection. Each specimen was dried and mounted on a sheet of stiff
paper or card 42 cm x 29 cm. Then, the specimens were brought to Herbarium, Forest Research
Centre (FRC) Kuching for species identification.
3. RESULTS & DISCUSSION
3.1 Description
1. Shorea acuta P.S. Ashton (Figure 1a, b & c)
Small to emergent tree, 30-50 m tall, 0.8-1. 2 m dbh, buttresses stout. Bark pale reddish brown,
cracked, flaky, fissured. Inner bark reddish brown, fibrous. Presence of dammar, brown to dark brown.
Twigs terete, slightly compressed, with stipule scars. Crown: uneven. Leaves simple, alternate,
lamina broad elliptic-obovate 10-28 cm x 5.0-14 cm; apex broad acumen up to1.5 cm long. Base
obtuse/cuneate, without domatia. Margin entire. Leaf texture coriaceous, glossy upper surface and
glabrous/glabrescent below. Leaf colour green, sometimes pale brown above. Midrib prominent below,
slightly sunken above. Secondary veins pinnate, 10-13 pairs, prominent on below. Tertiary veins
scalariform, slender. Petiole 1.5 cm long, stout. Stipule 10 x 5 mm oblong, obtuse, and caduceus.
Flower: buds ellipsoid, obtuse; sepal subequal, ovate; petals linear, dark crimson. Fruit: pinkish
green; wing reddish/ pinkish, unequal, 3 longer lobes spatulate to 12 x 3 cm, 2 short lobes unequal,
linear to 6x 0.8 cm. Nuts: ovoid, 3 x 2 cm. Distribution : Endemic to Borneo. In Sarawak, recorded
from Limbang, Marudi and Miri districts. (specimen ref: S.46478, S.46482, S.46591). Present in
Lambir Hills and Mulu NPs.
2. Shorea beccariana Burck (Figure 2a, b & c)
Tree up to 60 m tall, 1-1.5 m dbh, buttress 1.5 m tall. Bark smooth, with hoop marked, flacky
especially in large trees. Inner bark pinkish brown. Slash wood pinkish. Presence of dammar, brown to
dark brown . Crown often uneven, spreading, golden – suffused from below. Leaves simple, alternate
spiral, lamina elliptic-ovate 10-20.4 cm x 5.0-8 cm; apex broad acumen up to 0.8 cm long. Base
obtuse sometimes cuneate. Margin entire. Leaf texture coriaceous, glabrous, sometimes glaucous at
abaxial surface. Leaf colour golden brown, lepidote, to dull mauve grey below. Indumentums
glaucous, and glabrous at abaxial surface. Midrib shallow and sunken at adaxial surface, prominent at
abaxial surface. Secondary veins pinnate, 11-14 pairs, slender. Tertiary veins scalariform, prominent
at abaxial surface. Petiole 2.0-4.0 cm long, rufous brown puberulent. Stipule 10 x 5 mm oblong,
obtuse, small and caduceus. Flower: buds small, sepal greenish yellow , petal greenish brown or pink.
Fruits: Green; calyx glabrescent. Wings reddish, lobes unequal, 3 longer lobes to 20 x 3 cm, shorter
lobes, linear 10 x1 cm. Nuts: ovoid 4x3 cm. Distribution: Endemic to Borneo. In Sarawak, recorded
from Bintulu, Kapit, Kuching, Limbang and Miri district. (specimen ref: S46476, S32410). Present in
Lambir, Bako and Mulu NPs.
3. Shorea crassa P.S. Ashton (Figure 3a, b & c)
Tree, 40 m tall, 1 m dbh, buttresses 1.5 m tall. Bark purplish brown, crack and flaky. Twigs terete/
compressed with pale ascending stipule scars. Leaves simple, alternate, lamin, 8-14 cm x 5-7 cm;
shape elliptic to ovate, apex acuminate; 0.8 cm long at tip; base cuneate to obtuse; margin entire. Leaf
texture coriaceous, pale brown with lepidote at abaxial surface; Midrib prominent at abaxial surface,
sunken at abaxial surface. Secondary veins 7-11 pairs, prominent at abaxial surface, evenly spaced.
Tertiary veins scalariform, sinuate. Petiole 3.5-4 cm long. Stipule ovate, acute, 6 x 5 cmm. Flower:
buds small,calyx greenish yellow / cream, petals red at the base, yellow above, stamen and style
yellow. Fragrant. Fruits: green; calyx reddish, unequal lobes, 3 longer lobes to 8. 5 x 2.5 cm, 2
shorter lobes, to 5 x 0.8 cm. Nuts: ellipsoid, to 2 x 2.2 cm. Distribution : Endemic to Borneo. In
Sarawak, recorded from Bintulu, Kapit, Kuching, Lundu and Miri districts (specimen ref: eg. S.46484,
S.44064). Present in Mulu N.P.
4. Shorea curtisii Dyer ex. King (Figure 4a, b & c)
Tree, 50 - 60 m tall, 1.5 to 2 m dbh; buttresses 1.5 – 2 m tall, stout. Crown: Hemispherical cauliflower
shaped. Bark : pinkish brown, fissured; inner barks thick, pinkish brown. Twigs : slender, terete.
Leaves : simple, alternate; lamina 5-9 x 2- 3.8 cm, lanceolate- ovate. Apex acumen to 0.7 – 1.0 cm;
base cuneate. Leaves texture coriaceous, colour pale pink to grey lepidote below. Midrib slender,
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terete, sunken on adaxiaL, prominent on abaxial, obscure. Secondary veins 9-11 pairs, slender,
arched, prominent below. Tertiary veins, scalariform , slender. Flowers: in small buds; petal reddish.
Fruits: lightly green. Calyx lobes unequal, 3 longer lobes to 6 x 0.8 cm, 2 shorter lobes, linear, 3.5 x
0.3 cm. Nut ovoid, 1 x 1.3 cm. Distribution: SE Peninsular Thailand, Sumatra (Singkep & Lingga),
Peninsular Malaysia and Borneo. In Sarawak, recorded from Bintulu, Mukah and Miri districts
(specimen ref: eg. S46483, S23690). Frequent in Lambir Hills and Mulu NPs.
5. Shorea fallax Meijer (Figure 5a, b & c)
Tree, 40 m tall, 1 m dbh, buttresss 1.5 m tall, stout. Bark smooth sometimes flaky, with hoop marked;
grey to brown. Inner bark dark brown, slash wood dark brown. Twigs ribbed.Crown dense, irregular,
more or less hemispherical. Leaves simple, alternate spiral, 5-15 cm x 3-7 cm, shape, oblong to
ovate; apex acuminate to 0.8 cm tip; base obtuse, margin entire, to slightly wavy. Leaf colour, purplish
brown at adaxial surface, brown at abaxial surface when dry; flushing leaves bright green flushing
leaves. Mid rib flatten to shallowly furrowed at adaxial surface, prominent at abaxial surface.
Secondary veins 14-17 pairs, prominent at abxial surface. Tertiary veins sclariform, distinct at abaxial
surface. Petiole 1.8-2.5 cm long. Stipule broad hastate, caduous, 18 x 8 mm. Flower: bracts cream,
petals yellow, stamen and pistil light cream. Fruits: green, subsessile. Calyx lobes unequal, 3 longer
lobes to 5 x 0.5 cm, 2 shorter lobes, linear 2 x 0.1 cm. Nut ovoid, 3 x 1 cm, apiculate. Distribution:
Endemic to Borneo. In Sarawak, recorded from Belaga, Bintulu, Kapit, Lawas, Lundu, Marudi, Miri
and Tatau districts (specimen ref: eg. S46429, S31702). Present in Lambir Hills and Mulu NPs.
6. Shorea kunstleri King (Figure 6a, b & c)
Tree, up to 50 m tall, dbh 1.5-2.0 m , buttresses 1.5 m tall, spreading. Bark grey brown, fissured,
flaky. Presence of yellow to brownish resin (dammar). Crown cauliflower shape, diffuse, sometimes
spreading. Leaf simple, alternate, lamina 8-14 cm x 4-7 cm, broad ovate, apex acuminate, with 1 cm
long tip, base obtuse.; margin entire. Leaf texture coriaceous, smooth; green to dark green when fresh
sometimes with domatia. Midrib shallowly fissured, furrow above, channeled. Secondary veins
slender, 6-8 pairs, slightly raised at adaxial surface, indistinct at abaxial surface; Tertiary veins
slender, scalariform. Petiole geniculate, slender , about 2-3.8 cm long. Stipule oblong- lanceolate,
10x3 mm long, greyish. Flower: in small buds, petal yellow, pink at base. Fruits young fruit, green;
calyx lobes unequal, 3 longer lobes to 8 x 2 cm, 2 shorter lobes, linear, to 4 x 0.3 cm. Nut ovoid, 2 x 1
cm. Distribution: North Sumatra, Peninsular Malaysia and Borneo. In Sarawak, recorded from
Belaga, Bintulu, Kuching, Lawas, Miri and Samarahan districts (specimen ref: eg. S.46525, S.32451).
Common in Lambir Hills NP, also present in Kubah NP.
7. Shorea ochracea Symington (Figure 7a, b & c)
Tree 60 m tall, 1.5 m dbh. buttresses up to 1.5 m tall, stout. Bark dark brown with mottled pale brown,
fissured, flaky, inner bark yellowish brown. Dammar pale creamy to yellowish. Twigs terete , stout,
with stipules scars. Crown irregular hemispherical, dense. Leaves simple, alternate spiral; lamina 1522 cm x 8-12 cm; broad elliptic to oblong; apex acuminate, 0.4 cm long at tip; base cordate. Leaf
texture thickly coriaceous, bright yellow lepidote below. Mid rib obscure sunken above, prominent at
abaxial surface. Secondary veins 25-30 pairs, closely arranged, arched, prominent at abaxial surface.
Tertiary veins scalariform. Petiole stout 1.4-2.0 cm, stipule elliptic, obtuse 20 x 18 mm. Flower buds
pale green covered with fine golden hairs, strong fragrance. Fruits: chocolate brown, young fruit hairy.
Calyx reddish, lobes unequal, 3 longer lobes to 10x 2.5 cm , 2 shorter lobes, to 5 x 0.5 cm.
Distribution: In Sarawak, recorded from Belaga, Bintulu, Kapit, Limbang and Miri districts (specimen
ref: eg. S.46447, S. 29695, S.15117, S.32359). Present in Lambir Hills and Mulu NPs.
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Figure 1 Shorea acuta P.S. Ashton
A: showing the fresh twigs
B: Image of the crown
C: Image of the tree bark
B: Image of the crown
C: Image of the tree bark
Figure 2 Shorea beccariana Burck
A: showing the fresh twigs
Figure 3 Shorea crassa P.S. Ashton
A: showing the fresh twigs
B: Image of the crown
C: Image of the tree bark
Figure 4 Shorea curtisii Dyer ex. King
A: showing the fresh twigs
B: Image of the crown
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Figure 5 Shorea fallax Meijer
A: showing the fresh twigs
B: Image of the crown
C: Image of the tree bark
B: Image of the crown
C: Image of the tree bark
Figure 6 Shorea kunstleri King
A: showing the fresh twigs
Figure 7 Shorea ochracea Symington
A: showing the fresh twigs
B: Image of the crown
C: Image of the tree bark
3.2 Ecology and Habitat
Shorea is found growing in various kind of vegetation from the vast coastal peatswamp forest to
montane forest, but most of species are found in Mixed Dipterocarp Forest such as Lambir. The
Lambir Hills are dominated by soft friable sandstone derived from Neogene sediments ( Liechti et al.
1960). Sandstone derived soils are humult ultisols , or sandy haplic acrisols, with a surface horizon of
loosely matted and densely rooted raw humus, low nutrient status and low water retention capacity (
Ashton & Hall 1992, Davies et al. 1998), where its long term mean of annual rainfall is estimated to be
c. 3000 mm with all month averaging > 100 mm, with periodic short term drought (Watson, 1985).
Uses: Shorea is undoubtly renowned timber for its excellent resistance to damp conditions which
makes it highly competent at combating insect attacks and decay. Its formidable strength, and
extremely durable puts it in good stead as the leading hardwood in the light construction industry.
Besides being well traded timber product, Shorea trees also provide a rich catalogue of natural oils.
The broken down resin can be manufactured in to several household products such as candles, soaps
and other novelties. While their nuts, some of it can be eaten, other than being altered and molded in
to a chemical substitute for cocoa butter, or for making lipstick (Ashton, 2004).
3.3 Conservation status
Shorea is a large genus, and vulnerable to extinct in the future. The population of Shorea in the world
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is obviously effected by logging activities for timber trade. Some of the species such as Shorea
beccariana Burck, Shorea crassa P.S. Ashton, Shorea fallax Meijer, and Shorea ochracea Symington
have not yet recorded in Catalogue of Life, and its conservation status in the International Union for
Conservation of Nature (IUCN) is not been assessed. While, species like Shorea acuta P.S.Ashton
and Shorea kunstleri King are critically endangered and need to be update (IUCN, 2012). There are
strict regulations regarding the harvesting of Shorea; to be granted permission to cut down the tree, it
has to be larger than 20 inches in diameter otherwise the harvesting is deemed illegal. The demand
for timber trade for Shorea still remains high as it is one of the important timber export in Indonesia,
Malaysia and Vietnam.
4. CONCLUSION
Lambir Hills National Parks is not only potential for tourism, its existence also important to protect and
conserve tree flora like Shorea from over-exploited or extinction. Moreover, Lambir Hills is rated as
having one of the highest flora diversity of the old tropical rainforest, which makes the park an ideal
location for ecological research. Therefore, it is important that a functional park steering committee,
which includes local member community co-operate with each other to enhance ownership toward the
park. This can be achieved through environmental awareness and education campaigns and
activities.It is hoped that this studies can help in providing fundamental data that serve as baseline for
conservation management and supplementary project such as Hyperspectral Airborne Imaging
System pursued by the team from Sarawak Forestry Department.
References
Ashton, P.S. (1988). Dipterocarp biology as a window to the understanding of tropical forest
structure.Annual Review of Ecology and Systematic! 19: 347-370.
Ashton, P. S.(1995). What can be learned from a 50-ha plot which cannot be learned any other way?
In: H. S. Lee, P. S. Ashton, and K. Ogino (eds),Long Term Ecological Research of Tropical Rain
Forest in Sarawak. Studies of Global Environmental Change with Special Reference to Asia and
Pacific Region. Ehime University, Japan. Pp. 207-220.
Ashton, P.S. & P. Hall (1992). Comparisons of structure among mixed dipterocarp forests of NorthWestern borneo. Journal of Tropical Ecology,80(3): 459-481.
Ashton, P.S. (2004). DIPTEROCARPACEAE. In: E. Soepadmo, L.G. Saw and R.C.K. Chung (eds.),
Tree Flora of Sabah and Sarawak. Volume Five. A joint publication of Sabah Forest
Department, Forest Research Institute Malaysia and Sarawak Forest Department, Malaysia. Pp.
63- 388.
Baillie I.C. and P.S. Ashton, M.N. Court, J.A.R. Anderson, E.A. Fitzpatrick and J. Tinsley. (1987). Site
characteristics and the distribution of trees species in mixed dipterocarp forest on tertiary
sediments in Central Sarawak, Malaysia.Journal of Tropical Ecology,3: 201-220.
Baillie I.C. and P.S. Ashton. (1983). Some soil aspects of the nutrient cycle in mixed dipterocarp
forests in Sarawak. TropicalRain Forest: Ecology and Management (eds. S.L. Sutton, T.C.
Whitmore and A.C. Chadwick).Pp 347-356, Blackwell Scientific Publications. Oxford.
Davies, S.J. & Becker, P. (1996). Floristic composition and stand structure of mixed dipterocarp and
heath forests in Brunei Darussalam. Journalof Tropical Forest Science 8: 542-569.
Davies, S. J., Palmiotto, P., Ashton, P. S., Lee, H. S. &Lafrankie, J. V. (1998). Comparative ecology of
11 sympatric species of Macarangain Borneo: tree distribution in relation to horizontal and
vertical resource heterogeneity. Journal of Ecology 86: 662-673.
IUCN (2012). IUCN Red List of Threatened Species. Version 2012.1. <www.iucnredlist.org>.
Downloaded on 10 September 2012.
Lee, H. S., Davies, S. J., LaFrankie, J. V., Tan, S., Yamakura, T., Itoh, A., Okhubo, T., and
Ashton, P. S. (2002). Floristic and structural diversity of mixed dipterocarp forest in Lambir
Hills National Park, Sarawak, Malaysia. Journal of Tropical Forest Science, 14(3): 379-400.
Lee, H. S., Ashton, P. S., Yamakura, T., Tan, S., Davies, S. J., Itoh, A., Chai, E. O. K., Okhubo, T.,
and LaFrankie, J. V. (2005). The 52-hectare Forest Research Plot at Lambir Hills, Sarawak,
Malaysia: Tree distribution maps, diameter tables and species documenta-tion. Forest
Department Sarawak, The Arnold Arboretum-CTF Asia Program, Smithsonian Tropical
Research Institute, Kuching, Sarawak, Malaysia.
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Liechti, P., Roe, F.W. & Haile, N.S. (1960). The Geology of Sarawak and The Western Part of North
Borneo, Geological Survey Department, British Territories in Borneo, Bulletin 3, vol. 1(Text),
Kuching, Sarawak, Malaysia.
Watson, H.
(1985). Lambir Hills National Park: Resource Inventory With Management
Recommendations. Kuching, Sarawak, Malaysia: National Park and Wildlife Office, Forest
Department. Pp. 13-169. (Unpublished report).
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ESSENTIAL OIL OF Neolamarckia cadamba (Roxb.) Bosser
Tawan, C.S.*, Syarifah Hanisah, S.M, Mohd Razib, A. & Ipor, I.B
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, Unioversity Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
*Emaill: cheksum@frst.unimas.my
Abstract
Neolarmarckia cadamba (Roxb.) Bosser belongs to the family Rubiaceae It has a wide range of
distribution throughout the world including India, Nepal, Thailand, Indo – China, Malaysia Archipelago
and Papua New Guinea. In Malaysia, it occurs widely in the secondary forest and in disturbed areas
and along the roadsides of the lowland and hill areas. N. cadamba is considered a light demanding
and a fast growing species. Due to its fast growing ability and good timber characteristics, this
species is chosen as one of the species for agro –forest plantation. About 18,851 ha or a total of 8% of
the total planted forest in Sarawak has been planted with this species. It is for the production of sawn
timber, veneer, chips and pulp. The petioles of this species produce aromatic smell when crushed.
Thus, an attempt to extract and analyse the essential oil from the fresh petioles was conducted. Fresh
samples were collected from the trees growing in the secondary forest within Kota Samarahan
Division, Sarawak. The extraction was based on the hydro distillation technique and essential oil
obtained was purified using anhydrous sodium sulphate. The essential oil obtained was then analysed
using Gas Chromatograph – Mas Spectrophotometer (Shimadzu GC-MS QP-5000). From this
analysis, it showed that the petioles of N. cadamba contained methyl salicyclate and caryophyllene
which have not be reported elsewhere. Both compounds are well known for medicinal purpose and
have been found in other plant species. This finding may be very useful for further analysis of the
essential oil from N. cadamba to diversify its potential utilisation apart from timber production in the
future.
Key words : Neolamarckia cadamba, essential oil, agro-plantation, Sarawak.
1. INTRODUCTION
Neolamarckia cadamba (Roxb.) Bosser is an indigenous species belongs to the family Rubiaceae
which is widely used as timber. It has been identified as one of the fast growing timber and trial plots
have been established in Sarawak. According to Jugi et al., 2010, N. cadamba has a high quality of
wood, a fast-growing species and produce plenty of seeds.
This plant species is locally known as Kelampayan. It can attain a height of 45 m and diameter at
breast height up to 100 cm or more with large pagoda-shaped crown. It produces globose head of
flowers and the fruits are small capsules, packed closely together to form a fleshy yellow to orange
globose infructescence containing of about 8000 seeds. The small capsules split into four parts
releasing the seed at maturity. There are approximately 20,000 seeds per gram. N. cadamba wood
are used for sawn timber, veneer, chips, pulp and composites (Monsalud & Lopez 1967; Peh 1970;
Phillips et a1. 1979; Logan et al., 1986.
N.cadamba also possesses aromatic petioles which act as an indicator for the presence of essential
oil. However, update there is no reports have been published on the type of essential oil presence in
this plant. Previous researchers have mentioned many potential of N. cadamba on medicinal value
extracted from leaves, but hardly being commercialised.
This study was conducted to identify the types of natural compound found in the petioles of N.
cadamba which may be of potential use. N. cadamba is the indigenous plant species that have cover
8 % of planted forest area in Sarawak, which is aimed to support the growing demand of timber
industry as the timber production from the natural forests in Sarawak are greatly depleting. Sarawak
has established Licensed Planted Forest by choosing the fast-growing species with valuable
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characteristics for industrial wood, fuel, amenity, conservation ada protection (Sabang, 2010).
Investigation of the essential oil from N. cadamba will thus help to diversify the usage not only for
timber but of chemical constituent that could be valuable in pharmaceutical or fragrance industry in the
future.
2. MATERIALS AND METHODS
2.1 Samples collections
The sample of Neolamarckia cadamba leaves were collected from the tree growing in the secondary
forest of Kota Samarahan. N. cadamba usually found in group, which the location can easily
recognised. The samples were taken by cutting down branches and stem of N. cadamba. The fresh
leaves were placed in the plastic bags and brought immediately to the laboratory for extraction.
2.2 Essential Oil Extraction (Hydro-distillation)
The N. cadamba petioles were detached from the lamina and were cut into smaller pieces. Oil extracts
from petioles N. cadamba were made using the hydro-distillation with distill water by using Clevengertype apparatus, done at Sarawak Biodiversity Centre with permission. The extraction of 300 g samples
N. cadamba was done in 4 L of distilled water. A stirrer was placed together with the samples into the
bottom flat flask. The petioles were left to boil with 350 rpm for about 6-8 hours. Then, n-pentane was
applied to dissolve all the oil in the extraction machine. The oil was placed in the darker vial.
Anhydrous sodium sulphate, Na2SO4 was mixed with cotton in a pipette together to absorb the
excess water from collected essential oil. The essential oil was dropped into the pipette and few drops
of Dicloromethane (DCM) were added to allow the essential oil flow through the pipette.
2.3 Analysis of Extraction Oil
The essential oil extracted was diluted with ratio 1 δ of essential oil to the 200 δ of DCε. Three
replicate of essential oil for N. cadamba petioles were prepared. The preliminary analysis of essential
oil from N. cadamba was analysed by using gas chromatography analysis, Gas ChromatographyMass Spectrometry (GC-MS) following the method by Derwich et al. (2010). The chemical
compositions of essential oils were determined by GC-MS using a CP-SIL 5 HP fused silica column.
The identification of different chemical constituents was done by mass spectrometry with the specified
GC-MS condition as in Table 1. The column used was Varian capillary column (CP-Sil 5 HP; 60 m
length, 0.32 mm of diameter & Film thickness 0.25 m). The column temperature was programmed
from 40 °C (5 min), to 5 °C /min-50 °C, to 3 °C /min-280 °C (15 min). The temperature of the injector
was fixed to 250 °C and the one of the detector to 320 °C. Electrons impact: 70 ev. The debit of gas
vector (Helium) was fixed to 1.5 mδ/min. The volume of injected specimen was of 1 δ of diluted
essential oil in 200 δ DCε solution. The constituents of essential oils were identified in comparison
with their ‘spectres’ of mass with those gathered in a library of (NIST-MS) type.
3. RESULTS AND DISCUSSION
The essential oil identified from the petioles of N. cadamba were Methyl salicylate and Caryophyllene,
which has not been reported before. The range value based on the composition area was as shown in
Table 1.
Table 1 Analysis of the essential oil extracted from the petioles of N. cadamba.
Compound
Retention
Time (min.)
Area composition (%)
R1
R2
R3
*RANGE value
Methyl salicylate
20.0
68.2
54.5
32.8
35.4
Caryophyllene
27.5
17.4
17.2
36.4
19.2
*Range value = highest value of percentage of percentage composition minus lowest value of the
percentage area composition. R– replicate.
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Previous studies indicated that Methyl salicylate compound has potential in pharmaceutical for the
treatment of rheumatic disease and act as anti inflammatory agent (Jan et al. 2003). It produces a
similar smell that of Wintergreen oil - Gaultheria procumbens (Dweck, 2009) . The compound
Caryophyllene, which produces woody spicy fragrance believe to exhibit an anti-cancer property
(Opdyke, 1973.) It is an anti inflammatory property and has been added in the manufactured of skin
care product. It has also been recorded that Caryphyllene was identified the major compound in
Salvia rosifolia of Turkey. (Ozek et al. 2010).The presence of these compounds would be an
additional added value to this species as it is selected as one of the fast growing species for forest
plantation. Other than for the production of timber, the natural essential oil presence in the petioles
would be another future potential resource to be extracted for commercialisation. The analysis of
these compounds should be carried out further and evaluated.
4. CONCLUSION
The petioles of Neolamarckia cadamba contained two types of essential oil namely Methyl salicylate
and Caryphyllene. The presence of these essential oil would be important secondary products to be
utilised once the resources are plentiful obtainable from the plantation of this species in the future.
Acknoeledgement
The authors wish to express our gratitude to the Dean of Faculty of Resource Science and
Technology UNIMAS, Chief Operating Officer, Sarawak Biodiversity Centre for giving permission
using the facility. We would like also to thank En. Benedict Samling, Pn. Noor Pahtiwi , En. Sekudan
Tedong, En. Hidir Marzuki, En. Salim Arip. En. Muhd Najib Fardos, En. Mohd Rizan Abdullah,
En.Mohd Shafiq Sahat, En. M. Nurfazillah M. Ramzie and Pn Fatimah Daud and staff of Sarawak
Biodiversity Centre for the Support rendered in this research.
REFERENCES
Derwich, E., Z. Benziane and A. Boukir. (2010). Chemical composition of leaf essential oil of Juniperus
phoenicea and evaluation of its antibacterial activity. International Journal and Agriculture
Biology,12:199-204.
Dweck, A. C. (2009). Toxicology of essential oil reviewed. Retrived 3rd May 2011 from
http://www.zenitech.com/documents/Toxicity_of_essential_oils_p1.pdf.
Environmental
and
social sustainability report for Indonesia. (2009). Growing a sustainable future. PT. Purinusa
Ekapersada : Indonesia.
Ismail, J., Jusoh, M. Z. and Sabri, H. M. (1995). Anatomical variation in planted Kelempayan
(Neolamarckia cadamba, Rubiaceae). Journal IAWA Journal , 16(3): 277-287.
Jan, K., Janusz, S. C., and Wlodzimierz R. (2003). Validation of a method for simultaneous
determination of menthol and methyl salicylate in pharmaceuticals by capillary gas
chromatography with cool on-column injection. Acta Poloniae Pharmaceutica-Drug Research,
60(5):343-349.
Jugi, E. (2010). Planting and Maintenance of Kelempayan (Neolamarckia cadamba) in the field.
Proceedings of a planted forest technical workshop, May 25. Sarawak.
Logan, A.F. Balokis, Y.K., and Tan, Y.K. and Phillips, F.H. 1986. Pulping properties of regrowth
Anthocephalus chinensis and Macaranga hosei from Sarawak Forests. Tropical Science, 26:4558
Monsalud, M.R. and Lopez, F.R. 1967. Kaatoan Bangkal – a wonder tree. The Philippines
Lumberman, 13:60-64.
Opdyke, D. L. J. (1973). Monographs on fragrance raw materials. Caryophyllene. Food and Cosmetics
Toxicology, 11: 1059–1060.
Ozek, G., Demirci, F., Ozek, T., Tabanca, N., Wedge, D. E., Khan, S. I., Can Baser, K. H., Duran, A.
and Hamzaoglu, E. (2010). Gas chromatographic–mass spectrometric analysis of volatiles
obtained by four different techniques from Salvia rosifolia
Sabang, J. (2010). Planted Forest Sites and Tree Species. Sarawak Forestry Corporation Sdn. Bhd.
Proceedings of a planted forest technical workshop, May 25. Sarawak. 34.
Peh, T.B. 1970. Present status of pulping and paper making research with special reference to the
possible utilisation of tropical hardwoods. Malayan Forester, 33: 324-327.
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THE SPECIES DIVERSITY OF Mapania aublet (CYPERACEAE) FROM MALAYSIA
Zinnirah, S.* and Meekiong, K.
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, Sarawak, Malaysia.
*Email: szinnirah@frst.unimas.my
Abstract
Twenty (20) species of Mapania were collected from Peninsula Malaysia and Borneo, of which three,
M. multiflora, M. sapuaniana and M. sp. 1 are new records for Borneo. From published literature,
Malaysia recorded a total of 32 species. A field key is provided.
1. INTRODUCTION
Cyperaceae is the third largest family in the monocotyledons and seventh largest family in the
angiosperms with 106 genera and 5387 species (Govaerts et al., 2007). They form a huge,
morphologically diverse, geographically widespread, and ecologically and economically important
family (Naczi, 2005). Nevertheless, there are some species which are narrowly distributed, specific to
certain habitats and of conservation concern (Naczi and Ford, 2008). The difficulty of assessing the
specific level of endemism was mention by Goetghebeur (1998), due to lacking of recent revisions and
reliable continental checklists as many species are known from one single specimen or a single
locality only.
Mapania first described by Aublet (1775) and allies form one of the two branches at the base of the
Cyperaceae phylogenetic tree. This group of mostly forest-dwelling sedges is widely distributed
throughout the tropics. Many Mapania occur in rainforest at low altitudes, a habitat that is threated
globally. Many are also endemic, narrowly distributed and of conservation concern. Several Mapania
species are known to be used in basket and mat-making, while others are known to local people for
medicinal purposes such as a fever remedy. The greatest diversity in the genus occurs in Borneo with
25 species (of 84 estimated worldwide), of which 12 are endemic (Simpson, 1992). Knowledge of the
genus is limited due to lack of detailed studies.
As there is an increase of habitat destruction to rainforest nowadays, the threat to Mapania species is
alarming. Through several fieldtrips conducted in 2008, it was found that most forests that existed
within the last 5-10 years have been cleared for oil palm and rubber plantations, as well as logging.
Suitable habitat which was known to be able to hold four to six species previously (Simpson, 1992)
was discovered with only one to two species or none at all. This reality signifies that the disturbance
outcome is a decline in the number of species. Mapania species are in danger of going extinct before
they can be described and classified.
In this paper, we attempt an updated list to this genus, including new putative species, as well as
presenting modified key for Malaysian taxa.
2. MATERIALS AND METHODS
Mapania samples were collected from Peninsula and East Malaysia through several fieldtrips
conducted in December 2007 to February 2008 and from November 2008 to February 2009. Of the
84 species recognised, 42 are distributed in Southeast Malaysia, in which 32 species in Malaysia. A
list of Mapania species collected was given in Table 1.
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3. RESULTS
Collections were made in Peninsula Malaysia and Sarawak. Among the interesting specimens
collected were a number of new species; M. multiflora, an interesting linear-oblong leaved species
with paniculate inflorescence; M. sapuaniana, a broad leaf pseudopetiolate species that has maroon
purple coloration on the pseudopetiole and inflorescence; M. sp. 1, a species that resembles M.
longiflora, but with a distinct fruit shape. The descriptions of the new species have been submitted for
publication.
Table 2 The Mapania species found during the fieldtrips in West and East Malaysia.
No. Taxa
Locality
M. bancana
1
Pantu, Sri Aman, Sarawak.
M. cuspidata var. cuspidata
2
Jln Endau Rompin, Johor
Bukit Larut, Perak
Lambir, Miri Sarawak
3
M. cuspidata var. petiolata
4
M. debilis
5
M. enodis
6
7
M. holttumii
M. kurzii
8
M. latifolia
M. longiflora
9
10
11
12
13
14
15
16
17
M. lorea
M. meditensis
M. monostachya
M. obscuriflora
M. palustris
M. richardsii
M. sessilis
M. tenuiscapa
M. wallichii
New taxa
M. multiflora sp. nov
18
M. sapuaniana sp nov
19
M. sp. 1
20
Sg. Strass, Pakan, Sarawak
Lambir, Miri, Sarawak
Bkt. Sembiling, Limbang, Sarawak
Hutan Lipur Gunung Arong, Johor
Gunung Gading NP, Lundu, Sarawak
Tekam Forest Reserve, Pahang.
Hutan Lipur Ulu Sedim, Kedah
Sebangkoi, Sarikei, Sarawak
Jln Trusan Lawas, Sarawak
Sampadi, Lundu, Sarawak.
Hutan Lipur Gunung Arong, Johor.
Tapang Rumput, Sri Aman.
Bukit Larut, Perak.
Tekam Forest Reserve, Pahang.
Penang Hill, Penang
Bukit Larut, Perak.
Tekam Forest Reserve, Pahang
Gombak Forest Reserve, Selangor.
Lanjak Entimau, Kapit, Sarawak.
Lambir, Miri, Sarawak.
Batu Berkarang, Limbang, Sarawak.
Tapang, Rumput, Sri Aman, Sarawak.
Bukit Sembiling, Limbang, Sarawak.
Lambir, Miri, Sarawak.
Lanjak Entimau, Kapit, Sarawak.
Lanjak Entimau, Kapit, Sarawak.
Hutan Lipur Bukit Hijau, Kedah.
Lanjak Entimau, Kapit, Sarawak.
Jalan Endau-Rompin, Johor.
Lambir, Miri, Sarawak.
Lambir, Miri, Sarawak
Sampadi, Lundu, Sarawak
Kg. Pueh, Sematan, Sarawak.
Jalan Endau-Rompin, Johor.
Hutan Lipur Gunung Arong, Johor.
Batu Berkarang, Limbang, Sarawak.
Lanjak Entimau, Kapit, Sarawak.
Tapang Rumput, Sri Aman, Sarawak.
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Key to Malaysian species
1. Inflorescence paniculate
2
Inflorescence capitates or with solitary spike
4
2. Exocarp thin in cross-section, detached from endocarp and
disintegrating
M. multiflora
Exocarp thick in cross section, persistent
3
3. Spicoid bracts 2.1-2.5 mm long, floral bracts 1.6-2.5 mm long
M. bancana
Spicoid bracts 3.0-4.2 mm long, floral bracts 2.7-3.7 mm long
M. sumatrana
4. Culms central, involucral bracts foliaceous/ glumaceous
M. latifolia
Culms lateral; involucral bracts usually glumaceous
5
5. Leaves without a distinct pseudopetiole, the leaf blade gradually narrowed
into sheath
6
Leaves with a distinct pseudopetiole between leaf blade and sheath
11
6. Leaf blade up to 2 cm wide, usually very dark green often with metallic blue sheen,
sheath narrowly lanceolate, 0.5-0.8 (-1.2) cm wide, usually hardly distinguishable
from pseudopetiole
M. caudata
Leaf blade usually over 2 cm wide, yellowish green/mid to dark green
7
7. Sheath oblong lanceolate, leaf blade base apex gradually narrowed
into sheath (Borneo)
M. sapuaniana
Sheath broadly lanceolate to ovate, leaf blade base apex abruptly narrowed
into sheath
8
8. Inflorescence subsessile, hidden by leaf sheath, spicoid and floral bracts
18-44 mm long
M. meditensis
At least some inflorescences with culms, not hidden by leaf sheaths, spicoid bracts up to
12 mm long, floral bracts up to 11.5 mm long
9
9. Young emerging leaf salmon pink to reddish on underside in fresh materials,
plant glabrous, inflorescence usually 1 spike, 1.5 cm or more long
M. cuspidata
Young emerging leaf light green on underside in fresh material; plant hispid
on either culm or sheath or both, inflorescence with (1-) 3-12 spikes, if 1 spike
then spike less than 1 cm long
10
10. Leaves 4.2-11.5 cm wide, sheath glabrous, spikes 1.8-3.0 cm long, spicoid
bracts 9.5-11 mm long
M. holttumii
Leaves 2.7-3.9 cm wide, sheath usually hispid, spikes 0.7-1.0 cm long, spicoid bracts
5.0-5.3 mm long
M. hispida
11. Leaf apex abruptly narrowed, broadly obtuse to rounded
12
Leaf apex gradually to very gradually narrowed, acute
13
12. Inflorescence usually with more than 1 spike, spicoid bracts 6-8 mm long, fruit
2.4-2.8 x 1.4-1.6 mm (Borneo)
M. richardsii
Inflorescence with 1 spike only, spicoid bracts 9-13 mm long, fruit
3.9-5.3 x 1.9-2.6 mm (Borneo)
M. borneensis
13. Spike sessile or culm up to 0.6 cm long, the spike 1.3 to 1.6 cm long
14
Spike usually on an elongated culm over 1 cm long, if subsessile then
spikes 2.5 cm or more long
15
14. Spicoid bracts 10 – 13.5 mm long, lowest 2 floral bracts 12-17 mm
long (Malay Peninsula)
M. micropandanus
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Spicoid bracts 7-10 mm long, lowest 2 floral bracts 7.5-9.5 mm long
16
15. Spike 1.4-1.5 cm long, spicoid bracts 9-10 mm long, fruit 5-6 mm long
M. maschalina
Spike 0.8-1 cm long, spicoid bracts 7 mm long, fruit 3.2-3.5 mm long
M. obscuriflora
16. Fruits lageniform (flask-shaped)
M. enodis (in part)
Fruit not lageniform
17
17. Leaves crowded and strongly 3-ranked at base
18
Leaves not as above
19
18. Fruit ellipsoid to obovoid, apex shortly apiculate, 4-5.5 mm x
2.3-2.7 mm (Borneo)
M. graminea
Fruit obovoid, apex apiculate, elongated
M. sp. 1
19. Inflorescence comprising 3-6 closely compressed spikes, almost completely
enclosed by involucral bracts, even at maturity; spicoid bracts membranous,
18-21 mm long, lowest 2 floral bracts 20-21 mm long (Borneo)
M. longiflora
Inflorescence comprising (1-)2 –many spikes, partially or not enclosed by
involucral bracts, spicoid bracts subcoriaceous to coriaceous, up to
18 mm long; lowest 2 floral bracts up to 18 mm long
20
20. Leaf blade usually up to 1 cm wide, inflorescence 1.5 cm wide
21
Leaf blade 1.1 cm or more wide, inflorescence 1.5 cm or more wide
22
21. Involucral bracts 0.2-0.4 cm long, much shorter than the spike; spikes distinct,
spicoid bracts 3.5-4.5 mm long, fruits 3.3-4 mm long (Borneo)
M. angustifolia
Involucral bracts 0.5-1 cm long, at least half as long as spike, spikes rather indistinct,
spicoid bracts 5.2-6.5 mm long, fruits 2.9-3 mm long (Borneo)
M. debilis
22. Stigma branches 2, staminate flowers 2 per spicoid (Borneo)
M. foxworthyi
Stigma branches 3, staminate flowers 3 per spicoid
23
23. Spicoid bracts ovate, dark brown or dark reddish brown, keel on lowest
2 floral bracts narrowly winged (Malay Peninsula)
M. kurzii
Spicoid bracts oblong to lanceolate, light to mid-brown; keel on lowest 2
floral bracts wingless
24
24. Culms more than 1.5 m wide, spikes usually more than 4 (widespread), if less
than 4 then involucral bracts 0.7 cm or more wide (N Malay Peninsula)
M. palustris
Culms less than 1.5 cm wide
25
25. Culms up to 0.5 mm wide; leaf-blade up to 0.7 cm wide
M. monostachya
Culms 0.6 mm wide or over, leaf-blade usually over 0.6 cm wide
26
26. Spicoid bracts and lowest 2 floral bracts 10-29 mm long
27
Spicoid bracts and lowest 2 floral bracts usually up to 9.7 mm long, if more
than fruit lageniform
31
27. Spicoid bracts membranous, 27-29 mm long, lowest 2 floral bracts 26-28 mm long
(Malay Peninsula & Borneo)
M. sessilis
Spicoid bracts subcoriaceous to coriaceous, up to 18 mm long, lowest 2 floral bracts
up to 18 mm long
28
28. Leaf blade 2.4-4.5 cm wide, involucral bracts 0.8-1.2 cm wide
M. squamata
Leaf blade 0.6-2.3 cm wide, involucral bracts 0.4-0.8 cm wide
29
29. Leaves not strongly 3-ranked, 1.3-2.2 cm wide, spicoid bracts 10-14.5 mm
long, lowest 2 floral bracts ± mm wide
M. wallichii
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Leaves strongly 3-ranked, 0.6-1.4 cm wide, spicoid bracts 10-14.5 mm long;
lowest 2 floral bracts up to 1 mm wide
30
30. Culms mostly scabrid, 2.9-4 mm wide, leaf sheath somewhat shiny, mid to dark
chocolate brown; involucral bracts ± 0.8 cm wide (Borneo)
M. spadicea
Culms mostly smooth, 1-2.3 mm wide, leaf sheath dull greenish to mid brown;
involucral bracts 0.31-0.5 cm wide (Borneo)
M. graminea
31. Fruit lageniform (flask-shaped), (5.5) 6-9 mm long, apex long apiculate
M. enodis
Fruit ellipsoid to obovoid, 3-6 mm long, apex shortly apiculate
32
32. Leaf blade thickly coriaceous, 1-nerved, flat to inverse w-shaped in cross section;
leaf margins densely serrulate, feeling distinctly rough to touch, spike
1.5-1.9 cm long
M. lorea
Leaf blade subcoriaceous, 3-nerved, flat in cross section; leaf margins indistinctly
minutely serrulate, feeling ± smooth to touch, spike 0.9-1.5 cm long
M. tenuiscapa
Acknowledgements
The first author would like to thank the Government of Malaysia and Universiti Malaysia Sarawak for
providing full scholarship to permit this study being carried out.
References
Aublet, F. (1775). Histoire des plantes de la Guiane Francaise rangées suivant la méthode sexuelle
avec plusieurs mémoires sur différents sujets intéressants relatifs ὰ la culture et au commerce
de la guiane Franҫaise et une notice de I Ile de France Ouvrage, Paris.
Goetghebeur, P. (1998). Cyperaceae. In Kubitzki, K. (Ed.) The families and genera of vascular plants:
Alismatanae and Commelinanae (except Graminae). Germany, Springer-Verlag.
Govaerts, R., Simpson, D.D., Bruhl, J.J., Egorova, T., Goetghebeur, P. & Wilson, K.L. (2007). World
Checklist of Cyperaceae. Sedges., Kew Publishing.
Naczi, R.F.C. (2005). Insights on Using Morphologic Data for Phylogenetic Analysis in Sedges
th
(Cyperaceae). 17 International Botanical Congress. Vienna, Austria, Springer.
Naczi, R.F.C. & Ford, B.A. (2008). Sedges: Uses, Diversity and Systematics of the Cyperaceae,
U.S.A, Missouri Botanical Garden Press.
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CHARACTERISTICS OF PADDY FARMERS HOUSEHOLDS IN EAST KALIMANTAN, INDONESIA
1,2*
Karmini
and Abu Hassan Md Isa
2
1
2
Faculty of Agriculture, University of Mulawarman, East Kalimantan, Indonesia
Faculty of Economics and Business, Universiti Malaysia Sarawak, Sarawak, Malaysia
*Email: karminiunmul@yahoo.com
Abstract
Paddy farmers households are target in agricultural development because of their important position
as actors of paddy farming, producers and consumers of rice and also labors in other economic
sectors. The information about their characteristics is needed as a foundation to arrange and develop
effective strategies for the development of paddy farming in rural areas. This study discovered some
of the characteristics of paddy farmers households include ethnicity, gender, status in household,
marital status and number of family members.
Keywords: paddy farmer, household, characteristic, East Kalimantan.
1. INTRODUCTION
Paddy is one of the most important commodities in agricultural sector. Rice is a staple food for most
Indonesian. Paddy farming is becoming the potential source of income for many farm households in
East Kalimantan. The development of paddy farming in rural areas should be had priority in
agricultural development. Agriculture development is needed not only to increase farmer’s income but
also to achieve food security, improve competitiveness and increase value added of agricultural
products.
Paddy farmers households have important position in agricultural development as actors of paddy
farming, producers and consumers of rice. Paddy households also have ability to influence the
development of other economic sectors because the members of paddy households are involved in
non paddy farming jobs. Paddy farmer households are target in agricultural development. However,
there is limited information existence on the characteristics of paddy farmers households in East
Kalimantan, Indonesia. This information is needed as a foundation to propose the effective strategies
for the development of paddy farming in rural areas.
The objective of this study is to identify the characteristics of paddy farmers households in East
Kalimantan, Indonesia.
2. METHODOLOGY
This study was conducted in East Kalimantan Province, Indonesia. A two stage cluster sampling was
used to determine the 3 districts (Kutai Kartanegara, Penajam Paser Utara and Bontang) and the 9
subdistricts for the study areas and the number of respondents. Total of 380 households of paddy
farmers were selected in the sampling frame. Respondents reside in Tenggarong Seberang (128
households), Loa Janan (17 households), Muara Muntai (4 households), Babulu (128 households),
Penajam (84 households), Waru (16 households), South Bontang (2 households), North Bontang (1
household) and West Bontang (0 household). Descriptive statistics were used to identify the
characteristics of paddy farmers households in East Kalimantan, Indonesia.
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3. RESULTS AND DISCUSSION
Paddy is grown by very different types of farmers (Uphoff et al., 2008). Most of wetland paddy farmers
in East Kalimantan are Javanese community (93.16%). There are as many as 119 respondents of
Javanese which cultivate only wetland paddy and 235 respondents have other activities besides
paddy farming. They came from Java to East Kalimantan through transmigration program. Therefore,
the wetland fields for the most part are located in transmigration resettlement. As shown in Table 1.,
the number of Sundanese, Kutainese, Bugisnese and Banjarnese respondents are 7, 6, 6 and 2
households. It meant that paddy farming could be done by anyone without ethnic background
restriction because everyone can learn method to paddy cultivation. Paddy is main staple for all
ethnics in research location and all farmers commonly have conventional knowledge to paddy
cultivation from elder generation in their families. Collective farmers do some activities such as
planting and harvesting that lead easier process to adopt new technology.
Table 1. Characteristics of respondent.
No.
Characteristic
1
Ethnicity
(household)
2
Gender
(person)
3
Status in
household
(person)
4
Marital status
(person)
5
Number of
family
members
(household )
Javanese
Sundanese
Kutainese
Bugisnese
Banjarnese
Others
Total
Female
Male
Total
Household head
Spouse
Children
Children in law
Grand children
Other household members,
family
Other household members,
non family
Total
Single
Married
Divorced
Widow
Total
1-2
3-4
5-6
7-8
9-10
Total
Source of household
income
Paddy &
Paddy
non paddy
farming
farming
119
235
5
2
0
6
1
5
1
1
2
3
128
252
201
487
218
525
419
1,012
131
255
118
243
146
437
3
28
16
32
5
14
1
2
420
152
251
8
7
418
34
73
1,011
419
575
4
9
1,013
25
143
20
1
0
128
77
6
1
252
Total
Percen
-tage
354
7
6
6
2
5
380
688
743
1,431
386
361
583
31
48
93.16
1.84
1.58
1.58
0.53
1.32
100.00
48.08
51.92
100.00
26.97
25.23
40.74
2.17
3.35
19
1.33
3
1,431
576
827
16
12
1,431
59
0.21
100.00
40.25
57.79
1.12
0.84
100.00
15.53
216
97
7
1
380
56.84
25.53
1.84
0.26
100.00
There is small difference in nursery activity of wetland paddy farming system among ethnics in
research location. Some Kutainese respondents sow seed directly in wetland to minimize planting
cost because agricultural labor is scarce in their area. Most of others community choose to cultivate
the young plants in seedbeds. This study found no difference farming system or method for land
cultivation, fertilizing, weeding, transplanting, controlling pests and diseases, harvesting and post
harvesting among ethnics. However, this research found that the frequency of fertilizing, weeding,
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transplanting, controlling pests and diseases differs among respondents because of capital and labor
limitation.
The parity in gender and the identification of male and female roles are considered to be a main key to
reach effective and productive rice farming (Lambrou, 2004). The data showed that paddy household
members are mainly male at 51.92% and the rest are female. There are male, which as many as 218
of 419 persons in households have only paddy farm income. Others 525 of 1,012 persons in
households have paddy farm and non paddy farm income. It showed that paddy household has big
potency of male labor who could be employed in paddy farming and non paddy farming activities.
Although gender has important role in family and production process or family business, there is a
distinction between the roles of male and female in paddy farming. Male labors are commonly
engaging in land cultivation, weeding, processing, management and crop protection. On the other
hand, females get to involve in activities, which related to the planting and harvesting.
In similar to Zambrana et al. (2007) that mentioned that gender influences the knowledge and use of
input and also Marenya and Barrett (2007) that found that gender of the household head has a
significant positive effect on the likelihood of adoption. Male labors in paddy farming in East
Kalimantan adopt more new technology than female labors such as use new hybrid seed and try new
brand of fertilizer and pesticide. This study found that male and female have same access to resource
and service. Female labors have same opportunity to attend extension to improve their knowledge and
to increase rice productivity. There are 201 female persons in paddy households that only have paddy
farm income and others 487 in paddy households that have both paddy and non paddy farm income.
Paddy farmer households are largely dominated by children at 40.74%, then followed by household
head at 380 persons and his spouse at 361 persons. Other household members are children in law,
grand children, family and non family. Their total number are small approximately 7.06%, in paddy
households that only have paddy farm income and both paddy and non paddy farm income. It meant
that the most of paddy farmer households have the small number of labor force and family labors but
have big number of family dependent. The small number of family labor means small potency to
obtain variety income from many sources. The big number of family dependent leads more household
expenditure for consumption, education and others.
The status in paddy farmer household will distinguish their roles in decision making on paddy farming.
Related to gender, Barham and Chitemi (2009) mentioned that the leadership by gender associates to
the performance improvement. The gender structure affect the marketing performance which become
an enabling factor for male-dominated. Household head has a role as decision maker in most of
paddy household aspects. They organize labor and raw materials in paddy farming and look for
potential buyer. Although research by Danes et al. (2007) discovered that female personnel
management has nine times greater to gross revenue rather than male owners. It implies that the
increasing of a female role in business would positively impact the revenue. It also indicates that
females have more potencies to develop business when given opportunity to work in formal and
informal sector. However, this study found that male and female have specific role in paddy farming
and it is very difficult to replace its role. All paddy households only employ male labor to cultivate land,
they did not use female labor. Paddy household prefers to employ a bigger number of female labors
in paddy planting than male labor although male labor can do it.
This research found that approximately 57.79% the members of paddy households are married and
other 40.25% are not married yet. There are as many as 1.12% the members of paddy households
are divorced and 0.84% are widow. This study discovered that the number of married persons is
bigger than single, divorced and widow in paddy household that do only paddy farming and both
paddy and non paddy farming. It meant that paddy farming is done most by farmer family because
they have wetland field and responsibility to their spouse and children. Family has bigger potency to
have bigger income from many sources rather than individual because of more many family labors.
According to Danes et al. (2007), the marital status is related to the difference of household income.
The statistic indicated that significant influence between marital status and the wealth of household.
Most of the respondents (216 households) have family members between 3 and 4 persons. It meant
that the majority of paddy households have small household size. They are commonly only household
head, spouse and their children. There are 105 respondents, in paddy households that only do paddy
farming and both paddy farming and non paddy farming, who have big household size or more than 5
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persons. Household size determines the consumption of foods, clothes, house, education, recreation
and etc. Paddy households that have the smaller household size will have more attention to the
wealth of household because of more time to take care children and they could save more income
from reducing household expenditure. The larger household size has the more potencies of labor if
the household has many members in productive age. It is believed that the household size will affect
the family income.
4. CONCLUSION
Paddy farmer households in East Kalimantan, Indonesia, have some socio characteristics. Most of
the members of paddy households in the study areas are Javanese community. Male is the dominant
in the paddy farmer households. Household head has role as the manager of paddy farming. Majority
of paddy farmer households have the members between 3 and 4 persons, most of them are married.
References
Barham, J. & Chitemi. (2009). Collective Action Initiatives to Improve Marketing Performance: Lessons
from Farmer Groups in Tanzania. Food Policy, 34(1):53-59.
Danes, S.M., Stafford, K. & Loy, J.T. (2007). Family Business Performance: The Effect of Gender and
Management. Business Research, 60:1058-1069.
Lambrou, Y. (2004). Gender and Rice. Rome: Food and Agriculture Organization of the United
Nations. www.rise2004.org. pp.1-2.
Marenya, P.P. & Barrett, C.B. (2007). Household-level Determinants of Adoption of Improved Natural
Resources Management Practices among Smallholder Farmers in Western Kenya. Food
Policy, 32:515–536.
Uphoff, N., Kassam, A. & Stoop, W. (2008). Short Communication. A Critical Assessment of a Desk
Study Comparing Crop Production Systemsμ
The Example of the ‘System of Rice
Intensification’ versus ‘Best εanagement Practice’. Field Crops Research, 108:109–114.
Zambrana, N.Y.P., Byg, A., Svenning, J.S., Moraes, M., Grandez, C. & Balslev, H. (2007). Diversity
of Palm Uses in The Western Amazon. Biodivers Conserv, 16(10):2771-2787.
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BIOACTIVITIES OF Jathropa curcas Linn LATEX
Mohamad Syakir Mohd Sarib*, Ismail Jusoh and Nur Diyana Ishak
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak (MALAYSIA)
*Email: same_owner@yahoo.com
Abstract
Jatropha curcas L. is a perennial tree under Euphorbiaceae family and well known for alternative
source of biodiesel obtained from its seed. The study examined the latex of J. curcas crude that might
be toxic to human and plant pathogen fungi namely Fusarium oxyporum, F. solani and Aspergillus
niger; wood decay fungi which were Trametes versicolor and Gleophylum trabeum. The specific
objectives of this study were firstly to determine the toxicity of crude powdered J.curcas latex,
secondly to determine the chemical composition of crude powdered J. curcas latex and thirdly to
determine the chemical constituents of crude powdered J.curcas latex. Three different concentrations
of J. curcas latex (100 mg/ml, 50mg/ml and 25 mg/ml) were prepared for in vitro bioassay test using
agar well diffusion method. The wells were filled with concentration prepared and the growths of
microorganisms were observed after 72 hours incubation period. The crude latex of J.curcas was
subjected to composition and constituent analysis using Gas Chromatography – Mass Spectroscopy
(GC/MS). In toxicity test, all tested fungi showed significant inhibition to all fungus tested except A.
niger. The most abundance compound determined from were dotriacontane (24.00%) and 1,2benzenedicarboxylic acid (40.96%), Further study should be carried out to tap the potential of J.
curcas latex as active ingredient in the pharmaceutical and pesticide production.
Keywords: Jatropha curcas; pure powdered latex; toxicity; chemical composition; chemical constituent.
1. INTRODUCTION
Jatropha curcas Linn. belongs to the family Euphorbiaceae, known as ‘Jarak Pagar’ in εalay and
widely known as physic nut. It is a drought resistant and perennial tropical plant that can be grown in
low to high rainfall areas either in the farms as a commercial crop or on the boundaries as a hedge to
protect fields from grazing animals and to prevent erosion (Irvine, 1961). It will grow under a wide
range of rainfall regimes from 250 to over 1200 mm per annum (Katwal & Soni, 2003). The extract of
its leaves has antifungal properties (Garcia & Lawas, 1990). Fruit of J. curcas is highly toxic and may
lead to death if consumed. According to Goonasekera et al. (1995), the fruit may cause pregnancyterminating in rat which benefit the pest management industry. However, the toxicity can be removed
and utilized to many other purposes. The water extract of J. curcas branches showed inhibition on the
HIV induced cytopathic effect with low cytotoxicity (Matsuse et al., 1999).
1.2 Jatropha curcas Latex
The latex of J. curcas obtained from its branches reported to contains an alkaloid known as jatrophine,
which is believed to be having anti-cancerous properties (Henning, 2003). The healing effect of
curcain a proteolytic enzyme from the latex on wound has been demonstrated (Nath & Dutta, 1991).
The latex combined with the powdered leaves is applied to sluggish wounds while when formulated as
enema it is used for the treatment of gonorrhoea (Irvine, 1961). The latex also used in healing of
wounds, refractory ulcers, and septic gums and as a styptic in cuts and bruises. A proteolytic enzyme
(curcain) has been reported to have wound healing activity in mice (Nath & Dutta, 1997). The latex is
used to treat fungal infections in the mouth, bee and wasp stings and digestive problems of children in
Mexico (Schmook & Serralta-Peraza, 1997). The chemical composition of J. curcas latex was reported
to contain curcacycline A that has anti tumour properties (Van den Berg et al., 1995) and curcain, a
protease (Nath & Dutta, 1991). Previous study on the latex using IR spectrum of ethyl acetate extract
reported the presence of aromatic phenolic compounds and phenolic compounds which generally
behave as acids that have high antimicrobial activity (Gisvold, 1977). In phytochemical screening
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study done by Suhaili et al. (2011) on the crude latex extract revealed the presence of saponins and
tannins as had previously been reported on other parts of the plant. Saponins and tannins, in
particular, have been reported to possess antimicrobial activity (Zakaria et al., 2010)
2. METHODOLOGY
2.1 Latex Collection
The fresh latex of J. curcas was collected from Jatropha orchard owned by Carbon Capital Coperation
in Sadong Jaya, Kota Samarahan. latex were collected and stored into clean sterilized bottle warped
with aluminum foil to prevent phytochemical reaction. Sample collected were then directly brought into
the laboratory and stored in 4°C.
2.2 Drying and Powdering of Latex
Latex was spread on sterilized glass Petri dish and kept in fume cupboard in dark condition for 24
hours. Dried latex were subsequently scrapped off carefully using sterilized glass slide and mashed to
obtain finer poder. The powder was then sterilized using UV light inside laminar flow for 10 minutes.
2.3 Fungi Preparation
Trametes versicolor, Aspergillus niger, Fusarium oxysporum, F. solani, and Gloeophylum trabeum
cultured on Malt Extract Agar (MEA) was used for culturing all the fungi. 15ml of MEA solution was
poured into sterile disposable Petri dish and left cools until it solidified. Fungi were then inoculated
from stock culture onto agar plate and were re-inoculated after one week to obtain pure culture of
fungus.
2.4 Toxicity test
The agar well diffusion method adapted from Suhaili et al. (2011) and Oyi et al. (2007) was employed
in this study with several modifications. Three well were made using cork borer (10 mm diameter) in
25 ml agar. Three different concentrations of J.curcas latex diluted using metanol (25 mg/ml, 50
mg/ml, 25 mg/ml) filled into well, each 30µl and left for 20 minutes. Plug of test fungi (± 5mm X 5mm)
from cultured stock were next placed on the centre of solidified agar. Then it was incubated at 30 ºC
th
and the data were recorded on 5 day after incubation. A plate with single well was prepared and filled
with 30µl methanol for each fungi tested as a control. Antifungal activities were calculated by
measuring the zone of inhibition or clear zone. Inhibition zones above 20 mm classified as “strong”, 1λ
mm to 15 mm as “moderate” and below 14mm as “weak” activities.
2.5 Fractionation and Chemical Analysis
Column chromatography use to extract chemical present in pure powdered Jatropha curcas latex
using 19 solvent system with different ratio of n-hexane, dichloromethane, ethyl acetate and metanol.
Fraction of 100% solvent n-hexane and methanol were then subjected to Gas Chromatography
equiped with Mass Spectrometer. Identification of chemical components was made based on
comparison with GC retention times of standard references materials. It was also assisted by
comparing the mass spectra obtained during analysis with those mass spectra stored in the National
Institute of Standard and Technology (NIST) standard library incorporated in the GC/MS data system.
3. RESULTS AND DISCUSSION
3.1 Toxicity Test
The toxicity tests of crude powdered latex of J. curcas were carried out by using 100mg/ml, 50 mg/ml
and 25 mg/ml concentrations diluted in methanol. From the test, it was observed that J.curcas latex
has toxicity towards T. versicolor, G. trabeum, F. oxysporum, F. solani in all concentrations of crude
powdered J.curcas latex applied. Fusarium solani was recorded to have the least inhibition for all
concentrations of powdered J.curcas latex. Figure1 shows the example for the formation of inhibition
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zone against F. solani with different concentrations of crude J. curcas latex observed from toxicity test.
However, A. niger showed no significant effect of J.curcas latex on the growth of this fungus in any
concentration of J. curcas latex (Figure 2).
100 mg/ml concentration showed strong inhibition with inhibition diameter more than 20 mm.
Gleophylum trabeum was observed to strongly inhibited with 27 mm inhibition zone. F. Oxysporum
and T. versicolor recorded 25.7 mm zone of inhibition. F. solani formed inhibition zone of 20 mm. For
50 mg/ml concentration, inhibition zone of T. versicolor was 22 mm, indicated strong inhibition while
the lowest inhibition diameter was F. solani with 16 mm indicated moderate activity. Both F.
oxysporum and G. trabeum showed inhibition zone of 21 mm. For 25 mg/ml concentration, inhibition
zone was strong on both F. oxysporum and T. versicolor with 20 mm while less inhibition was on F.
solani with 14 mm which indicated moderate activity. G. trabeum was observed to inhibit by 19 mm
zone of inhibition which indicated moderate inhibition.
Figure 1 Formation of inhibition zone against
Fusarium solani.
Figure 2 No inhibition zone showed against
Aspergillus niger.
Table 1 shows the summarize zone of inhibitions of J.curcas latex and the reading was taken as mean
of three replicates. Fig. 3 visualized the inhibition classification of different types of fungi and its
concentration and shows different fungi has different inhibition strength for each concentration. Pure
latex of J.curcas has toxicity affect on several human and plant pathogen fungi. Study done by
Schmook and Serralta-Peraza (1997) proved that J. curcas latex can be used to treat fungal infection
Henning (2003) analyzed the presence of alkaloid in J. curcas called jatrophine and it’s believed to
have anti dermatomucosal disease. Ambuse & Bhale (2012) reported J. curcas latex showed toxicity
toward the growth of Fusarium proliferatum and F. pytium. It also been reported that the crude extracts
of ethyl acetate and methanol from stem bark has no significant affect towards A. niger (Gupta et al.,
2010).
Table 1 Inhibition zones of fungal species in agar well diffusion plate assay of crude powdered J.
curcas latex after 5 days incubation
Conc. of latex (mg/ml)
Fungi / Zone of inhibition (mm)
FS
FO
TV
GT
100
20 ± 2.3
25.7 ± 0.3
25.7 ± 0.8
27 ± 1.5
-
50
16.3 ± 1.2
21.7 ± 0.3
22 ± 1.2
21.7 ± 0.9
-
25
14 ± 1.2
20 ± 0.6
20 ± 2.3
19 ± 1.7
-
-
-
-
-
-
Methanol
AN
FS = Fusarium solani; FO = Fusarium oxysporum; TV = Trametes versicolor; GT = Gleophylum
trabeum; AN = Aspergillus niger; - = No inhibition
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Figure 3 Inhibition classification of three concentrations of J. curcas latex against tested fungi
3.2 Chemical Constituents and Compound Analysis
The GC/MS analysis revealed the most abundance compounds in hexane and methanol fraction were
dotriacontane and 1,2-benzenedicarboxylic acid.
3.2.1 n-hexane Fraction
Major chemical constituents of hexane fraction of J.curcas latex was dotriacontane with percentage of
composition 24.00% (Fig 4). Other significant compounds were pentatriacontane (20.33%),
hexatriacontane (19.79%), tetracosane (19.08%), tetratricontane (10.33%) and nonacosane (5.56%)
(Table 2).
The chemical constituent in hexane fraction contain nonacosane was reported to responsible in the
inhibition of several types of bacterial formation (Yayli et al, 2006). Nonacosane is known as a plant
compound that responsible in formation many of waxy layer of long chain paraffin (Hankin &
Kolattukudy, 1968).
Figure 4 Gas chromatogram traced by GC/MS for n-hexane fraction of powdered J.curcas latex
Tetratriacontane was also reported in the extract of Dictyopteris membracea significants for
antimicrobial activities (Ozdemir et al, 2006). Dotriacontane was reported to showed toxicity affect on
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the microbe tested. (Amin & Sleem, 2007). This compound was also found in extract of Sideritis
scardica and it showed antimicrobial properties (Vanja et al., 2012). Pentatriacontane was also
reported to has no toxicity against Candida albicans, Aspergillus niger and Mucor. (Naik and Jadge,
2010). Hexatriacontane was classified as flavanoid group and has very high medicinal value (Vaishali
et al., 2009). Yassa et al. (2009) reported Hexatriacontane can be used as active preventing agent for
many diseases and also has antioxidant affects. Tetracosane was also reported in flower extract of
Allium atroviolaceum and showed positive antibacterial activity (Dehpour et al., 2011). It also found in
the essential oil of Geranium columbinum that has toxicity affects (Radulovic et al., 2011).
Table 2 Major chemical constituents identified in n- hexane fraction of pure latex of J.curcas
3.2.2 Methanol Fraction
Methanol fraction is assumed to consist of intermediate polar compounds such as fatty acids and most
abundance chemical identified was mainly classified in alcohol group. Figure 5 shows presence of 1,2benzenedicarboxylic acid (40.96%), ß-Sitosterol (35.06%), 2-Hexyl-1-decanol (9.30%) and 4,6Cholestadien-3.beta.-ol (4.72%) (Table 3).
2-hexyl-decanol was reported to has antibacterial activities (Najiah et al,. 2008) and also found in Thai
honey bee which has antibiotic affect for many human diseases (Suwannapong et al., 2011). 1,2benzenedicarboxylic acid is a plasticizer compound reported to have high toxicity and antimicrobial
effect against Listeria (Hsouna, 2011) and reported as the active phytochemical compound (Chen et
al., 2011). 4,6-Cholestadien-3.beta.-ol was found in extract of Azadiracta indica which had showed
antibacterial activity (Moorthy & Boominathan, 2011).
Figure 5 Gas chromatogram traced by GC/MS for methanol fraction of powdered J.curcas latex
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ß-Sitosterol was also been found in the root and kernel meal of J.curcas in phenolics and flavonoids
analyses by HPLC analysis (Oskoueian et al., 2011). The chemical was also found in the leaves of J.
curcas. ß-Sitosterol has no antifungal effect (Hegazy et al, 2010), but it had significant effect on
bacteria (Gohari et al., 2009)
Table 3 Major chemical constituents identified in methanol fraction of pure latex of J.curcas
4. CONCLUSION
The toxicity test showed J. curcas latex inhibited Trametes versicolor, Gleophyllum trabeum, Fusarium
oxysporum, Fusarium solani, but negative to Aspergillus niger indicate the powdered latex has limited
inhibition affect different type of organisms. The chemical constituents of powdered J. curcas latex
shows significantly inhibited fungal growth. Extensive research need to be done with various
concentrations of Jatropha curcas latex and organisms. Extraction of pure compounds to obtain
significant active ingredient and to find which inert ingredient has potential to amplify the chemical
activation, will lead into the commercialization of J. curcas, and hence benefit industries related with
Jatropha.
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Vanja, T., Dragica, B., Ivana, A., Sofija, D., & Ksenija, A. (2012). Chemical and antimicrobial
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ANTIFUNGAL ACTIVITIES OF METHANOL EXTRACTS FROM Eusideroxylon zwageri
HEARTWOOD
1*
1
3
Sim, S. P. , Ismail, J. , & Zaini, A
1
Department of Plant Science and Environmental Ecology
Department of Chemistry, Faculty of Resource Science and Technology, University of Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
2
*Email: spsim9@yahoo.com.my
Abstract
Durability and strength of wood are important in construction industry. Natural durability of
Eusideroxylon zwageri is known to be very high. One of the reasons for high wood durability is the
presence of extractives. The objectives of this study were firstly to determine the amount of methanol
crude extracts from E. zwageri, secondly to identify the chemical constituents of methanol extracts and
thirdly to assess antifungal activity of methanol extracts. Extraction by using methanol were carried
out. Gas chromatography–mass spectrometry techniques were used to identify and characterize the
chemical constituents and compositions of methanol crude extract fractions from E. zwageri. The
antifungal activities were determined methanol extracts using agar dilution method. Methanol crude
extracts from E. zwageri was 8.37%. Methanol crude extract from E. zwageri was toxic to Trametes
versicolor, Gloeophyllum trabeum and Chaetomium globosum. Hexanedeconic acid, methyl
hexadecanoate, methyl octadeconate and -cadinol might be responsible to the antifungal activities in
E. zwageri.
Keywords: Eusideroxylon zwageri, chemical constituents, antifungal
1. INTRODUCTION
Antifungal compounds in wood can be extracted using different solvents. Methanol is a polar solvent
and it usually used to extract polar chemical compounds from wood. Polar compounds in wood include
alcohols, ketones, carboxylic acids, phenols, carbohydrates and fatty acids. Extractive compounds
have significant impact on properties of wood which are durability and strength, odour and taste,
inflammability, toxicity, density, economic value and factory uses (Negi, 1997). Since Eusideroxylon
zwageri Teijsm. & Binnend is very durable timbers due to their extraneous substances, thus, it is
important to identify the compounds responsible for the decay resistance in E. zwageri heartwood.
Currently chemical wood preservatives are used to treat non-durable timbers.
Chemical-based preservatives lead to a number of environmental concerns, thus the potential of
natural wood preservatives as effective replacement has gained interest of many researches. The
discovery of these environmental-friendly compounds may replace the role of toxic chemical, which
currently used as wood preservatives. The advantages of using natural wood preservatives to treat
wood are, as natural product they are usually harmless to human and environment. The potential
development of wood extractives as natural wood extractives not only important to provide alternative
treatment for wood preservation industry but also may be useful for therapeutic and cosmetic
industries. Wood extractive consists of different kinds of chemical compounds. It is non-cell wall
component which are small molecules. Wood with high amount of extractive is more resistant to decay
(Zabel & Morrell, 1992; Eaton & Hale, 1993; Schultz et al., 1995; Schultz & Nicholas, 2000; Ismail &
Ipor, 2003). Currently there is limited information on the chemical compounds from E. zwageri.
The objectives of this study were firstly to determine the amount of methanol crude extracts from E.
zwageri. Secondly, to identify the chemical constituents of methanol extracts from E. zwageri. Thirdly,
to assess antifungal properties methanol extracts.
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2. METHODOLOGY
2.1 Preparation of wood samples
Wood samples of Eusideroxylon zwageri (belian) was obtained from Kakus, Bintulu, Sarawak. Wood
samples were grinded by using a grinder to produce wood meal. E. zwageri wood meals were
extracted using methanol. A total of 1300 g of wood meal was immersed in two liter methanol in
separator funnel in room temperature. After three days, the crude extract solvent was drained and
collected into a round bottle flask. The solvent was evaporated into dryness using vacuum rotary
o
evaporator at 35 C to obtain pure crude methanol extract. Crude extracts was weighed. Three
replicates of extraction were done. The amount of crude extract expressed as percentages according
the equation below:
Crude extract (%) = (Sample 1/Sample 0) x 100
Sample 1 = weight of crude extract
Sample 0 = weight of wood sample
2.2 Fractionation of crude methanol extract
Methanol crude extract was purified on the chromatography column (4.0 cm i.d x 45 cm length)
packed with 100 g silica gel. The crude extract was eluted with increasing polarity solvent system by
using four types of solvent which were hexane, dichloromethane, ethyl acetate and methanol. A total
of 100 mL for each solvent were used to fractionate methanol crude extract. Fractions of 25 mL each
were collected in test tube then were subjected to GC-MS analysis. The purpose of fractionation is to
reduce number complexity of compounds present in crude extract. Gas chromatographic analyses of
the samples were carried out on model Shimadzu (QP 2010) plus mass spectrometer (GC-MS) fitted
with BPX-5 capillary column. The compounds were identified by comparing the mass spectrum of
obtained from the analysis with those Spectral Library in the data system.
2.3 Preparation of fungi inoculums
Trametes versicolor, Gloeophylum trabeum and Chaetomium globosum used in this study. Malt
o
Extract Agar (MEA) was the media for fungi growth. Before autoclaving at 121 C for 15 minutes, MEA
solution was stirred with stirrer. MEA solution was pour into sterile disposable Petri dishes and left to
cool and solidified. Inoculation of Trametes versicolor, Gloeophyllum trabeum and Chaetomium
globosum from stock culture onto agar plates were done aseptically in the lamina flow hood. Fungi
were reinoculated after one week to prepare pure cultures. Fungi growths were checked frequently to
make sure there is no contaminations occur. If contamination occurs, new inoculation was done to
replace it.
2.4 Antifungal assay
Antifungal assays were carried out in three replicates and data were averaged. Crude extract were
dissolved in methanol. MEA media mixed with methanol were used as positive control. Methanol
extract were mixed with methanol to become concentrations of 150 mg/mL, 50 mg/mL, 25 mg/mL, 10
mg/mL, 5 mg/mL and 2.5 mg/mL. The mixtures were poured into 9cm Petri dish. Trametes versicolor,
Gloeophyllum trabeum and Chaetomium globosum plugs from the edge of actively growing cultures
o
were transferred onto the centre of the Petri dishes and incubated at 27 C and 70 % relative
humidity. The cultures diameter was measured daily. Antifungal index were calculated when the
mycelium fungi reached the edges of control dishes, the diameter in all experimental dishes was
measured and antifungal index (AI) expressed as % inhibition was calculated by the following
equation:
Antifungal index (%)
Where,
Da is mean diameter of growth zone in experimental dish with extract (cm)
Db is mean diameter of growth zone in control dish (cm)
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3. RESULTS
The total amount of extract isolated with methanol was 8.37 %. The amount of extractives was also
influenced by several species, growth conditions, age of the tree (Gierlinger, 2003) and locality
(Fengel and Wegener, 1989; Negi, 1997; Gierlinger, 2003).
Gas chromatogram from GC-MS analysis combined fraction (CF1) of methanol extract from E. zwageri
is shown in Figure 1. The most abundant compounds was tetratetracontane with the composition of
9.79 % (E1) and 7.52 % (E2), followed by dibutyl phthalate (E3) with the composition of 6.74 %.
E2
E3
E1
Figure 1 Gas chromatogram for CF1 of methanol extract of E. zwageri.
Figure 2 shows gas chromatogram of underivatized CF2 of methanol extract from E. zwageri. The
major compounds detected in CF2 of E. zwageri were isoelemicin (E4), methyl elaidate (E5) and
methyl ester hexadecanoic acid (E6) with the compositions of 33.37 %, 16.63 % and 12.67 %,
respectively.
E4
E5
E6
Figure 2 Gas chromatogram for CF2 of methanol extract of E. zwageri.
CF3 and CF4 were not soluble in dichloromethane for GC-MS analyses which GC-MS just detected
hexane and dichloromethane solvent. Thus, derivative technique was used. Derivatization technique
was done on the CF3 and CF4 by adding tert-butyldimethylsilyl and pyridine.
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Figure 3 shows gas chromatogram of derivatized CF3 of methanol extract from E. zwageri. The major
compound were 1,3-bis(1,1-dimethylethyl)-1,1,3,3-tetramethyldisiloxane (E7) and tert-butyl-[2oxyethoxy]dimethylsilane (E8) with composition of 80.77 % and 12.12 %, respectively. The minor
compounds were nonanoate (E9) and benzoate (E10) with the compositions of 0.73 % and 0.58 %,
respectively.
E7
E8
E10
E9
Figure 3 Gas chromatogram for CF3 of methanol extract of E. zwageri.
Figure 4 shows gas chromatogram of derivatized fraction CF4 of methanol extract from E. zwageri. It
shows high existence of 1,3-bis(1,1-dimethylethyl)-1,1,3,3-tetramethyldisiloxane (E11) and bis ether1,3-propanediol (E12) with the composition of 93.15 % and 2.86 %, respectively. Minor compounds
detected were nonanoate (E13) and dimethyl(tert-butyl)silyl ester-2-ethylhexanoic acid (E14) with the
composition of 0.53 % and 0.34 %, respectively.
E11
E12
E14
E13
Figure 4 Gas chromatogram for CF4 of methanol extract of E. zwageri.
Combined fractions CF1 for methanol extract contain 40.06 % of tetratetracontane. Eicosane was the
second major compound in CF1 with 10.28 %. Heneicosane compound made up of 5.61 %. In CF2,
isoelemicin and methyl elaidate were the major compounds with the compositions of 33.37 % and
17.76 %, respectively. In this study, methyl hexadecanoate and methyl octadeconate can be found in
E. zwageri in CF2 with 12.67 % and 2.56%, respectively. These two compounds were reported have
anti-inflammatory activity (Xavier et al., 2011). In this study, -cadinol compound can be found in E.
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zwageri in CF2 of methanol extract with 0.53 %. These compounds strongly inhibited fungal growth
(Chang et al., 2000).
Antifungal index increased as the concentration of methanol extract of E. zwageri increased (Figure
5). At the concentration of 10 mg/mL, the antifungal index was more than 80 %. The results suggest
that concentration of 10 mg/mL of E. zwageri methanol crude extract has the ability to resist wood
decay fungi especially Trametes versicolor, Chaetomium globosum and Gloeophyllum trabeum.
Figure 5 Antifungal index of different methanol crude extract concentrations from E. zwageri
against Trametes versicolor, Chaetomium globosum and Gloeophyllum trabeum.
Figure 6 showed the growth diameter decreased with increased in methanol extract which indicate the
higher the concentration of extract the higher the inhibitory ability.
(a)
(b)
(c)
Figure 6 Antifungal effects presented for concentration with methanol extracts (from left to right): 0,
2.5, 5, 10, 25, 50, and 150 mg/mL. (a) Trametes versicolor (top row). (b) Chaetomium globosum
(medium row). (c) Gloeophyllum trabeum (bottom row).
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4. CONCLUSIONS
Methanol crude extracts from E. zwageri was 8.37%. Growth diameter of fungi decreased with
increased in methanol extract concentration. The inhibitory ability of methanol extract was evidence at
10 mg/mL. It can be concluded that methanol extract from E. zwageri is toxic to Trametes versicolor,
Gloeophyllum trabeum and Chaetomium globosum. Hexadecanoic acid, methyl hexadecanoate,
methyl octadeconate and -cadinol might be responsible for the antifungal activities in E. zwageri.
References
Chang, S. T., Wang, S. Y., Wu, C. L., Shiah, S. G., Kuo, Y. H., & Chang, C. J. (2000). Cytotoxicity of
extractives from Taiwania cryptomerioides heartwood. Phytochemistry, 55, 227-232.
Eaton, R. A., & Hale, M. D. C. (1993). Wood decay, pests and protection. London: Chapman & Hall.
Fengel. D., & Wegener, G. (1989). Wood: Chemistry, Ultrastructure, Reaction. Berlin: Walter de
Gruyter. p.613.
Gierlinger, N., Jacques, D., Schwanninger, M., Wimmer, R., Hinterstoisser, B., & Paques, L. E.
(2003). Rapid prediction of natural durability of larch heartwood using FIT-NIR spectroscopy.
Canadian Journal of Forest Research, 33,1727-1736.
Ismail, J., & Ipor, I. (2003). Decay Resistance of Extractives-Free Belian (Eusideroxylon zwageri) &
Malagangai (Potoxylon malagangai). Paper presented at the Conference on Forestry and
Forest Research in Malaysia, Sarawak.
Negi, S. S. (1997). Wood science and technology. India: International Book Distributors.
Schultz, T. P., Harms, W. B., Fisher, T. H., McMurtrey, K. D., Minn, J., & Nicholas, D. D. (1995).
Durability of angiosperm heartwood. Holzforschung, 49, 29-34.
Schultz, T. P., & Nicholas, D. D. (2000). Naturally durable heartwood: Evidence for a proposed dual
defensive function of the extractives. Phytochemistry, 54, 47-52.
Xavier, W. K. S., Medeiros, B. J., Lima, C. S., Favacho, H. A., Andrade, E. H. A., Araujo, R. N. M.,
Santos, L. S. S., & Carvalho, J. C. T. (2011). Topical anti-inflammatory action of Caryocar
villosum oil. Journal of Applied Pharmaceutical Science, 1(3), 62-67.
Zabel, R. A, & Morrell, J. J. (1992). Wood microbiology : Decay and its prevention. California:
Academic Press, Inc.
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PHYTOCHEMICAL AND BIOLOGICAL SCREENING OF METHANOL EXTRACTS OF
Enicosanthellum pulchrum
1*
1
1
Noraziah Nordin , Noor Shafifiyaz Mohd Yazid , Koh Sue May , Siddig Ibrahim Abdelwahab
2
and A. Hamid A. Hadi
1
1
Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur.
Department of Chemistry, Faculty of Science, University of Malaya, 50603, Kuala Lumpur.
2
*
Email: aziereality@um.edu.my
Abstract
Natural product extracts have been used for health benefits for many years. In this study, the
methanol extracts of leaves, bark, stem and root of Enicosanthellum pulchrum (Annonaceae) were
screened for the presence of alkaloids, terpenoids, steroids, phenolics and saponins. Two biological
assays in vitro were used to evaluate these extracts for their antioxidant and anticancer activities. All
extracts contain alkaloids, terpenoids and strong saponins. However, only leaves and bark extracts
showed the presence of phenolics content. The biological screening of antioxidant activities were
determined using DPPH scavenging and ferric reducing antioxidant power (FRAP) assays. The bark
and stem samples displayed greater reducing power than quarcetin, trolox and ascorbic acid as
standard drugs. The result for DPPH scavenging assay revealed that bark and stem extracts showed
high inhibitory activity of DPPH with 60 and 56% inhibition, while the IC 50 values of these extracts were
0.43 and 0.64 mg/mL, respectively. Meanwhile, the results for anticancer activity which was analysed
on breast cancer cells (MCF-7) and human normal liver cells (WRL-68) by MTT assay showed no
cytotoxicity activity to all extracts. The results suggest the possibility that this plant may contain
bioactive compounds from the active extracts.
Keywords: Enicosanthelum pulchrum, Annonaceae, antioxidant activity, anticancer activity, plant
methanol extracts
1. INTRODUCTION
Antioxidants in biological systems have multiple functions which include protection from oxidative
damage and in the major signalling pathways of cells (Kannan et al., 2010). However, high amounts of
free radicals can oxidise biomolecules, leading to tissue damage, cell death or degerative processes
and it is also important in oxidative stress (Gulcin 2006; Ak & Gulcin, 2008). Therefore, antioxidants in
cells can prevent damage cause by reactive oxygen species (ROS) which is generated in living
organism during excessive metabolism (Aruoma & Cuppette 1997) and also cancer and a wide range
of other diseases (Prakash et al., 2007).
Cancer is one of the most prominent diseases in humans. Cancer is the uncontrolled growth of
abnormal cells in the body (A.D.A.M 2011). Currently, there is commercial interest of discovery of new
anticancer agents from natural products (Kinghorn et al., 2003). Although there are several synthetic
drugs commercially available and in use but due to the side effects have reinforced the efforts for the
development of alternative from natural origin (Huang & Wang 2004).
Enicosanthellum pulchrum (King) Heusden also known as ‘εempisang’ is a species of the family of
Annonaceae (Burkill, 1966). It was first discovered at the Coteau area on the border of Thailand and
Malaysia. It is a coniferous tree that can grow to about 3-5 meters tall. E. pulchrum produces green
flowers with gentle smell blossom on the top. The propagation of this plant is quite difficult either by
seeding or grafting (David, 1989). This plant is a highland plant which confined to mountain forests at
an altitude of 1,200 – 1,500 m (Ng et al., 1990). Phytochemical study of this plant revealed several
isoquinoline alkaloids such as (-)-asimilobine, (-)-anonaine, (-)-norliridine, liriodenine and (-)-scoulerine
(Lavault et al., 1990). Meanwhile, only one biological activity has been reported so far for this plant
which was anti-PAF (Nordin et al., 2011). In these study, we investigate the presence of chemical
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compounds from each parts of E. pulchrum and its potential as antioxidative and anticancer in
methanol extracts.
.
2. MATERIALS AND METHODS
2.1 Chemicals and reagents
2, 2-Diphenyl-1-picrylhydrazyl (DPPH), fetal bovine serum (FBS), sodium nitrite were purchased from
Sigma-Aldrich. Acetic acid glacial, FeCl3, FeSO4, hydrochloride acid, Mayer reagent, sodium chloride,
Dimetylsulfoxide (DMSO), chloroform, ammonia, ethanol were purchased from Merck. 3-(4,5Dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT) from Invitrogen (Carlsbad, USA). All cell
lines were obtained from American Type culture collection (ATCC).
2.2 Plant material
The plant Enicosanthellum pulchrum was collected from Cameron Highlands Montane Forest,
Pahang. The specimen was identified by a parabotany, Sani Misran, and voucher specimen was
deposited at the Herbarium of the Botany Department, Universiti Kebangsaan Malaysia. Each plant
part, i.e. leaf, bark, root and stem was air-dried and ground to mesh size 40-60 before extracted with
methanol using maceration technique. Evaporation of the solvents using rotary evaporator gave crude
methanol extracts.
2.3 Phytochemical screening
The presence of alkaloids was determined by using the method of Culvenor and Fitzgerald (1963). A
dense, heavy precipitate was designated as 4+, a strong precipitate as 3+, a moderate precipitate as
2+ and a faint cloudy appearance as 1+. The Liebermann-Burchard test was used to determine the
presence of steroids and terpenoids (Said et al. 1990). The formation of a bright purple, red or pink
coloration indicates the occurrence of triterpenes and blues or greens indicate steroids. Saponins
were tested by the froth test (Simes et al. 1959). A froth lasting 30 minutes was designated 1+, 2
hours was designated 2+, 3 hours was designated 3+, and more than 4 hours was designated 4+.
Phenolic test was done according to Sofowora A. (1982). The presence of greens or blues coloration
indicate the presence of phenolic compounds.
2.4 DPPH radical-scavenging activity
The DPPH assay was performed according to the modified method by Orhan et al. 2007 and Brem et
al. 2004. Briefly, 0.02% stable DPPH free radical (50 µL) in methanol (100 mL) was added to
standard/sample/control (20 µL) and methanol (130 µL, total assay volume 200 µL) in a 96-well plate.
Ascorbic Acid (vitamin C) was used as the standard and blank solvent ethanol as the negative control.
The absorbance was read at 517 nm using SUNRISE Microplate Absorbance Reader after 30 min of
incubation at room temperature.
2.5 Ferric reducing antioxidant power (FRAP) assay
The determination of the total antioxidant activity (FRAP assay) is using a modified method of Benzie
and Strain (1999). The stock solutions included 300 mM acetate buffer (3.1g C 2H3NaO2.3H2O and 16
mL C2H4O2), pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl and 20 mM
FeCl3.6H2O solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL
TPTZ, and 2.5 mL FeCl3.6H2O. The temperature of the solution was raised to 37 °C before use.
Methanol extracts (10 µL) were allowed to react with 300 µL of the FRAP solution in the dark.
Readings of the coloured product (ferrous tripyridyltriazine complex) were taken at 593 nm. The
standard curve was linear between 100 and 1000 µM FeSO 4 are expressed in µM Fe (II)/g dry mass
and compared with that of ascorbic acid and quercetin.
2.6 Cytotoxicity assay (MTT assay)
All the cells were maintained in 37°C incubator with 5% CO 2 saturation. Different cell types were used
to determine the cytotoxic effects of E.pulchrum extracts using the MTT assay. For measurement of
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5
cell viability, cells were seeded at a density of 1 x 10 and 5% CO2 cells/mL in a 96-well plate and
incubated for 24 h at 37°C. Cells were treated with the extracts and incubated for 24 h. After 24 h,
MTT solution at 2 mg/mL was added for 1 h. Absorbance was measured at 570 nm. Results were
expressed as a percentage of control giving percentage cell viability after 24 h exposure to test agents
(Cheah et al. 2011).
2.7 Statistical Analysis
All values were reported as mean ± S.E.M. The statistical significance of differences between groups
were assessed using one-way ANOVA. A value of p < 0.05 were considered significant.
2. RESULTS AND DISCUSSIONS
Table 1 showed the results of phytochemical screening of E. pulchrum. The results displayed the
presence of alkaloid at all parts. However, stem and bark samples were found to give strong alkaloid
reactions with indicator 2+. Among the all parts, root sample displayed a very strong phenolic
properties with indicator 4+. Meanwhile, the presence of steroids/terpenoids can be found at leaves,
stem, bark and root sample. A very strong saponins with stable froth lasting for 4 hours were found at
leaves, bark and root.
Table 1. Phytochemical screening for alkaloid, phenolic, steroid/terpenoid and saponin
Species
Parts
Alkaloid
Phenolic
Steroid/terpenoid
Saponin
Enicosanthellum
pulchrum
Leaves
Stem
Bark
Root
+
2+
2+
+
+
+
4+
+
+
+
+
4+
+
4+
4+
The plant extracts of E. pulchrum showed high concentration of saponins content followed by
alkaloids. However, root extract exhibited high concentration of phenolics content as well. These
results indicate that E. pulchrum possess potential compounds as initial for further isolation work.
The methanol extracts i.e leaves, bark, stem and root of E. pulchrum were also investigated for
antioxidant and anti-cancer properties. The antioxidant activity of E. pulchrum extracts for FRAP assay
and DPPH assay are presented in Table 2. In this present study, bark of E. pulchrum had the highest
3+
ability for reducing Fe with 1774.8±0.25 followed by stem and root, 841.9±0.01 and 495.2±0.01,
respectively. These bark extract displayed comparable to the standard drugs, quercetin trolox and
ascorbic acid. The leaves extract exhibited the lowest FRAP value with 122.6±0.01. Therefore, no
further test for leaves extract of DPPH assay.
The percentage inhibition (%) of DPPH results also presented in Table 2. Bark extract showed
significantly higher activity of 60.4±0.02 followed by stem (55.8±0.05) and root (20.2±0.01),
respectively. However, the IC50 of bark and stem exhibited moderate activity of 430.0 µg/mL and 640.0
µg/mL compared to ascorbic acid (70.0 µg/mL) as standard drug (Table 3).
a
b
Table 2. Results of FRAP assay and DPPH assay of E. pulchrum (Mean ±SD )
Extracts/ Drugs
FRAP assay
DPPH assay (%)
Leaves
122.58±0.01
NA
Bark
1774.83±0.25
60.43±0.02
Stem
841.96±0.01
55.77±0.05
Root
495.17±0.09
20.20±0.01
Quercetin 10x
948.29±0.12
68.39±0.02
Trolox 10x
557.13±0.10
NA
Ascorbic acid 10x
14.71±0.01
87.33±0.01
a
b
Values are means of three triplicate determinations; SD, standard deviation; NA-Not analysed
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Table 3. The IC50 values of DPPH assay of active extracts of E. pulchrum
Samples
IC50 (µg/mL)
Ascorbic acid
70.0
Bark
430.0
Stem
640.0
There are some factors contribute to the antioxidant activity such as growth conditions, stability of the
specific antioxidant components, including variations in the process of extraction that can influence the
variation in the antioxidant activity (Price et al. 2006). On the other hand, experiment of anti-cancer
was also performed with the same extracts to determine their activity against anticancer properties.
The cell line used in this study were normal hepatic cells (WRL-68) and breast cancer cells (MCF-7).
The results of each extracts as performed in Table 4. The cytotoxicity assay (MTT) revealed that all
extracts did not produce any toxic effects on WRL-68 and MCF-7 cell lines up to the concentration of
100 µg/mL. Paclitaxel showed high cytotoxicity to all cell lines.
Table 4. Effect of methanol extracts of leaves, bark, stem and root of E. pulchrum and Paclitaxel on
different cell lines in MTT assay
Extracts
MCF-7, 24 h (µg/mL)
WRL-68, 24 h (µg/mL)
Leaves
Bark
Stem
Root
Paclitaxel
>100
>100
>100
>100
0.18
>100
>100
>100
>100
0.10
Although, there are no activities on these two cell lines but the action of these extracts for other cell
lines are currently on-going.
4. CONCLUSIONS
The results indicate that E. pulchrum may contain promising therapeutic agents for antioxidant
properties. At present, the isolation study is being conducted to purify the compounds from the active
extracts.
Acknowledgement
We wish to thank University of Malaya and Ministry of Higher Education for financial support under
research grant HIR-UM-MOHE (F000009-21001).
References
A.D.A.M. (2011). Medical Encyclopedia. www.ncbi.nlm.nih.gov/pubmedhealth. 3 Jan 2011.
Ak, T., & Gulcin, I. (2008). Antioxidant and radical scavenging properties of curcumin. ChemicoBiological Interaction, 174, 27-37.
Aruoma, I.O., & Cuppette, S.L. (1997). Antioxidant methadology: in vivo and in vitro concerps. Illinois:
AOAS Press.
Benzie, I.F.F., & Strain, J.J. (1999). "Ferric reducing/antioxidant power assay: Direct measure of total
antioxidant activity of biological fluids and modified version for simultaneous measurement of
total antioxidant power and ascorbic acid concentration," Methods in Enzymology, 299,15-27.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Brem, B., Seger, C., Pacher, T., Harti, M., Hadacek, F., Hofer, O., Vajrodaya, S., & Greger, H. (2004).
Antioxidant dehydrotocopherols as a new chemical character of Stemona species.
Phytochemistry, 65, 2719–2729.
Burkill, I.H. (1966). A Dictionary of Economic Products of the Malay Peninsular. Kuala Lumpur,
Malaysia: Ministry of Agriculture & Cooperatives.
Cheah, S.C., Appleton, D.R., Lee, S.T., Lam, M.L., Hadi, A.H.A., & Mustafa, M.R. (2011). Panduratin
A inhibits the growth of A549 cells through induction of apoptosis and inhibition of NF-Kappa B
translocation. Molecules, 16, 2583–2598.
Culvenor, C.C.J., & Fitzgerald, J.S. (1963). A field method for alkaloid screening of plants. J. Pharm.
Sc. 52, 303-304.
David, M.J. (1989). Revision of Disepalum (Annonaceae). Brittonia, 41, 359–378.
Gulcin, I. (2006). Antioxidant and antiradical activities of L-carnitine. Life Sciences, 78, 803-811.
Huang, L. & Wang, B.G. (2004). Antioxidant capacity and liphophilic contents of seaweeds collected
from the Qingdao coastline. J Agric Food Chem, 52, 4993-4997.
Kannan, R.R.R., Arumugam, R. & Anantharaman, P. (2010). In vitro antioxidant activities of ethanol
extract from Enhalus acoroides (L.F.) Royle. Asian Pasific Journal of Tropical Medicine, 898901.
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M.C., Wall, M.E., Kroll, N.H., Kramer, R.A., Rose, W.C., Vite, G.D., Fiarchild, C.R., Peterson,
R.W. & Wild, R. (2003). Novel strategies for the discovery of plant-derived anticancer agents.
Pharmaceutical Biology, 41, 53-67.
Lavault, M., Guinaudeau, H., Bruneton, J., Sevenet, T., Hadi, H.A. (1990). (-)-Thaipetaline, a
tetrahydroprotoberberine from a Malayan Annonaceae. Phytochemistry, 29, 3845–3847.
Ng, F.S.P., Low, C.M. & Sanah, M.A.N. (1990). Endemic trees of the Malay Peninsula. Forestry
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Nordin, N., Jalil, J., Jantan, I. & Murad, S. (2011). Platelet-activating factor (PAF) receptor binding
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QUANTIFYING THE DIVERSITY OF AVIFAUNA IN UNDISTURBED, DISTURBED AND LOGGEDOVER PEAT SWAMP FORESTS, BETONG, SARAWAK
Bettycopa Amit
1,2*
2
, Andrew Alek Tuen and Khalid Haron
1
1
Malaysian Palm Oil Board, P.O Box 12600, Kuala Lumpur, Malaysia
Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, 94300 Kota
2
Samarahan, Sarawak.
*Emails: *bettycopa@mpob.gov.my, aatuen@ibec.unimas.my, khalid2mpob.gov.my
Abstract
The avifauna diversity in peat swamp forest is the least studied, probably due to the harsh and
unfriendly environment. The aim of this paper is to present the avifauna diversity in relation to the
different degree of disturbance to peat swamp ecosystem in Betong Division, Sarawak. Sampling of
avifauna was done in October 2010, April 2011, July 2011 and October 2011. Three sites were
selected, site 1 in the Maludam National Park (Undisturbed Peat Swamp Forest), site 2 in the Tanjung
Baru Forest (Disturbed Peat Swamp Forest) and site 3 in the Cermat Ceria Forest (Logged-over Peat
Swamp Forest). Fifteen mist nets were employed each time for a total effort of 540 nets/hours for each
site. A total of 292 birds representing 15 families and 45 species were mist-netted in these studied.
The result of our study showed that Maludam National Park was the most diversified, followed by
Tanjung Baru forest, and Cermat Ceria Forest. Testing for significant difference between sites yielded
hypothesised that Site 1 bird diversity was significantly difference from Site 3 but there was no
significant different between Site 2 from Site 1 and Site 3. Lowland species such as Babblers and
Bulbuls was the dominants species that dominated the understory of peat swamp forest. Eleven
species of mist-netted birds recorded in this studied are categorized as protected animals under the
Sarawak Wild Life Protection Ordinance (1998).
Keywords : Peat Swamp Forest, avifauna, mist net, Betong Division
1. INTRODUCTION
εore than 60 per cent of the world’s tropical peat lands are found in South-East Asia and a few occurs
in Africa and parts of Central America. In South-East Asia, large peat swamp forests (PSF) can be
found on the islands of Borneo (belonging to Indonesia, Malaysia and Brunei Darussalam) and
Sumatra, Indonesia. (UNDP,2006). PSF in Borneo occurs along the coast of Sarawak, Brunei
Darussalam, Sabah and Kalimantan. Sarawak has 1.6 million hectares, followed in order by Peninsula
Malaysia with 0.72 million hectares and Sabah with 0.12 million hectares (Wahid et al., 2009).
Presently, large areas of PSF have been cleared and drained for agriculture, settlement and other
human activities such as logging and hunting. These activities motivated by economic, cultural,
intellectual, aesthetic and spiritual goals are now causing environmental and ecological changes of
global impact (Chapin III et al., 2000).
PSF known as very unique habitats because having a high percentage of endemic species and
provide sanctuary for viable populations of more than 60 animal species listed as globally threaten that
give these areas worldwide attention (UNDP, 2006). Out of 622 species of birds recorded in Borneo
39 are endemic to the island. Many species of forest birds cannot adapt to the disturbances caused by
human activities for these species conserve forested areas is crucial for their survival. Understorey
birds are good indicator species because they are particularly sensitive to changes in habitat
(understorey) conditions.
The bird communities in PSF are the least studied, probably due to the harsh and unfriendly
environment. A study of the avifauna in a PSF adjacent to Universiti Malaysia Sarawak conducted by
Rahman and Tuen (2006) show that a total of 679 birds representing 67 species from 25 families were
mist-netted from 1996 to 1999 and states that PSF are not a low priority habitat in terms of bird
abundance and diversity. This is because less number of fruit trees in PSF and high tannin content of
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most leaves, makes them unpalatable (Laman et al., 2006). Other bird’s surveys were conducted in
conjunction with the joint Malaysian-Netherlands project (2000-2004) states that 192 species of birds
recorded in the PSF at Maludam National Park from the three-year surveys in 1998, 2001 and 2002
through bird observation method. Out of 192 species, 21 species can be categorised as palearctic
migrants (Rahman 2004). Little information is available on the effect of bird diversity in relation to
human activities such as agriculture practice, logging and hunting in PSF. Hence, the aim of this paper
is to present the understory avifauna diversity in relation to the different degree of human disturbance
to peat swamp ecosystem in Betong Division, Sarawak.
2. MATERIALS AND METHODS
2.1 Sites description
Sampling was conducted in three PSF with different degree of disturbance to peat swamp ecosystem
in Betong Division, Sarawak. Two forests were in Maludam area; Maludam National Park (N
o
o
o
o
01 36.058’’ E 111 04.065’’) and Tanjung Baru Forest (N 01 38.733’’ E 111 02.66λ’’) and the other
o
o
forest was located 35 km from Betong town: Cermat Ceria Forest (N 01 23’57.5’’ E 111 24’26.4’’).
Maludam National Park (MNP/Site 1) is situated in the Maludam Peninsula, in Betong Sub-District of
the Sri Aman of Sarawak. Site 1 is categorized as undisturbed PSF because Maludam National Park
is one of the PSF included in the Totally Protected Area (TPA) system besides Loagan Bunut National
st
Park and Medalam River Basin. This park which was gazetted as national park on 31 May 2000
covers an area of 43, 147 ha (Chai, 2005).
Tanjung Baru Forest (TBF/Site 2) is located outside Maludam National Park. This forest is situated
next to orchard and small scale of oil palm plantation own by local people from Titty Longhouse.
Hunting and logging were some of activities practice by local people in this forest. Site 2 is described
as disturbed PSF because various types of anthropogenic activities occur inside and outside the
forest. There are two longhouses situated one kilometre from site 2.
Site
2
Site
1
Site
3
Figure 1. Location of mist-netted sites within the Maludam National Park (MNP/Site 1), Tanjung Baru
Forest (TBF/Site 2) and Cermat Ceria Forest (CCF/Site 2) at Betong, Sarawak.
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Cermat Ceria Forest (CCF, Site 3) is a logged-over peat swamp forest located between two oil palm
estates; Durafarm Plantation belonging to WTK Sdn. Bhd. and Lingga Plantation belonging to
Sarawak Land Consolidation & Rehabilitation Authority (SALCRA). The 1020 ha forest was logged
about 10 years ago.
2.2 Mist-netting
In each forest, 15 mist-nets were deployed randomly for total net-hours were 2160 respectively.
During each samling occasion, the mist-nets were activated from at 0600 hours until 1800 for three
days and were checked every two hours. Captured birds were identified, measured, tagged and
released in the study areas. External morphological measurements of the captured birds were
recorded in a data sheet for further analysis. Species identification was based on Myers (2009). This
list of birds is not extensive because it is limited to birds that use the understory between 0.2 to 3 m
above the ground level (Rahman & Tuen, 2009).
2.3 Data analysis
Shannon’s and Simpson’s indices of species diversity for three sites were calculated using PAST
Software. The differences in Shannon’s diversity t-test between any two sites were tested also by
using PAST Software. ECOSIM Software was used to generate rarefaction curves for species
richness estimation.
3. RESULTS AND DISCUSSION
3.1 Bird species at different sites
A total of 292 birds representing 16 families and 45 species individuals (Table 1) were recorded in all
sites, including 11 species of birds protected under the Sarawak Wild Life Protection Ordinance
(1998). Five protected species were recorded at MNP, seven at TBF and six at CCF. Maludam
National Park recorded high number of bird with 143 birds belongs to 13 families and 33 species,
followed closely by TBF with 93 birds representing 12 families and 24 species and CCF with 56 of
birds belongs to eight families and 18 species.
Family Timalidae (Babblers) is most diverse with 11 species, followed by Pycnonotidae (Bulbuls) six
species, Alcedinidae (Kingfishers) five species and Muscicapidae (Flycatchers and Shama),
Nectarinidae (Spiderhunter and Sunbird) and Picidae (Woodpeckers) with three species each.
Timalidids was also the most diverse family in PSF of Samarahan (Rahman & Tuen 2006) and Logan
Bunut National Park (Laman et al. 2006).
Table 1 Total species of birds mist-netted in Maludam National Park, Tanjung Baru Forest
andCermat Ceria Forest after four times sampling, Betong, Sarawak.
Family
Alcedinidae
Timaliidae
Species Name
Ceyx rufidorsa*
Alcedo meninting*
Lacedo pulchella*
Pelargopsis capensis*
Halcyon coromanda*
Trichastoma rostratum
Stachyris maculate
Stachyris erythroptera
Pellorneum pyrrogenys
Macronous ptilosus
Macronous gularis
Trichastoma malaccanse
Ophrydornis albogularis
Stachyris nigricollis
Malacopteron magnum
432
Undisturbed
Disturbed
Logged-over
MNP
1
6
1
˗
˗
12
19
12
˗
3
˗
1
3
2
2
TBF
6
1
˗
1
2
10
4
7
˗
˗
3
1
˗
3
˗
CCF
1
1
˗
˗
˗
˗
˗
4
3
5
2
8
˗
9
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Species Name
Malacopteron cinereum
Setornis criniger
Pycnonotus brunneus
Pycnonotus plumosus
Pycnonotus erythrophthalmos
Pycnonotus goiavier
Pycnonotus simplex
Rhipidura javanica
Cyornis turcosus
Cyornis banyumas
Rhinomyias ruficauda
Trichixos pyrropyga
Arachnothera longirostra
Hypogramma hypogrammicum
Anthreptes singalensis
Hypothemis azurea
Blythipicus rubiginosus*
Meiglyptes tukki*
Sasia abnormis*
Irena puella
Prionochilus maculatus
Dicaeum trigonostigma
Orthotomus sericeus
Orthotomus ruficeps
Ninox scutulata*
Otus rufescens*
Phodilus badius*
Accipiter trivirgatus
Philentoma pyrhoptera
Harpactes duvaucelii
Undisturbed
MNP
2
18
4
22
2
1
1
1
4
1
1
1
6
1
˗
2
˗
5
1
2
3
1
1
˗
˗
˗
˗
˗
1
˗
Disturbed
TBF
˗
2
˗
3
˗
2
˗
17
5
˗
˗
1
16
˗
˗
˗
˗
˗
1
˗
2
˗
2
1
˗
1
1
1
˗
˗
Logged-over
CCF
˗
4
˗
2
˗
˗
˗
˗
˗
˗
˗
˗
˗
4
2
3
2
˗
1
˗
˗
˗
˗
˗
1
1
˗
˗
˗
3
Total Individuals
143
93
56
Total Species
33
24
18
Total effort
2160
2160
2160
Captured Rate,individual/100 net hours
6.62
4.31
* Protected species under the Sarawak Wild Life Protection Ordinance (1998)
2.59
Family
Pycnonotidae
Rhipiduridae
Muscicapidae
Nectarinidae
Monarchidae
Picidae
Irenidae
Dicaeidae
Cisticolidae
Strigidae
Tytonidae
Accipitridae
Incertae
Trogonidae
3.2 Species richness
The rarefaction curves for mist-netted birds at three sampling sites shown in the figure 3 were
generated by using ECOSIM software. The curve is a plot of the predicted number of species based
on the number of individuals sampled (Hunting, n.d.). The species rarefaction curves at three different
sites have not reached asymptote.
Figure 3 Rarefaction curves for mistnetted birds within undisturbed peat
swamp forest (Site 1), disturbed peat
swamp forest (Site 2) and logged-over
peat swamp forest (Site 3) at Betong,
Sarawak. Rarefraction simulated in
ECOSIM.
4
4
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Hence, additional effort and trapping day is needed which will provide a better insight on the diversity
and abundance of understorey birds in PSF. Based on sample abundance of 44 randomly selected
individuals from Site 3, the rarefaction curves predicted 18 species for MNP, 17 species for TBF and
16 species for CCF.
3.3 Species composition
In MNP, Olive-winged Bulbul was the most dominant mist-netted species with 22 individuals followed
by Chestnut-rumped Babbler (19 individuals), Hook-billed Bulbul (18 individuals) and White-chested
Babbler and Chestnut-winged Babbler (12 individuals each). This result shows that the family Bulbul
and Babbler dominated the understory habitat in Maludam National Park. However, Pied Fantail was
the dominant species in TBF with 17 individuals followed closely by Little Spiderhunter (16 individuals)
and White-chested Babbler (10 individuals).
Figure 2 Bird species composition at three different sampling sites (Maludam National Park, Tanjung
Baru Forest and Cermat Ceria Forest) from high to low number of individuals.
Black-throated babbler was the dominant species in logged-over peat swamp forest, closely followed
by Short-tailed Babblers, Fluffy-backed Tit Babbler, Hook-billed Bulbul and Purple-naped Sunbird.
Babblers, Bubuls, and Sunbirds are lowland species which could be seen, heard or mist-netted at
understory level. This is because availability of food such as insect, small animals, fruits and flowers
for the understory bird’s consumption (δaman et al. 2006).
3.4 Bird diversity
Table 2 shows the values for the Shannon’s and Simpson’s diversity indices and the test of
significance difference between the three sites. Zar t-test on Shannon’s index indicates that Site 1 was
the most diversified site, followed closely by disturbed Site 2 and Site 3.
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Table 2 Diversity indices and tests of significant difference for the three study sites.
Site 1
Site 2
Site 3
(MNP)
(TBF)
(CCF)
Shannon-Wiener's
2.904
2.701
2.659
Site 1 & Site2
(p>0.05)
Simpson's
0.9193
0.9044
0.9152
Site 1 & Site 3
(p<0.05)
Site 2 & Site 3
(p>0.05)
Diversity Index:
Test of Significance
Difference
Testing for significant difference between the three sites with Shannon’s diversity t-test yielded the
hypothesised that there was significant difference between Site 1 bird diversity was significantly
different from Site 3. Based on this result, the bird community at Site 1 was more diverse and
abundant Site 3. This result is not surprising because as mentioned, previous logging activities and
agriculture practices (Laman et al. 2006) influence the bird diversity in Site 3. These results were
supported by Rahman & Tuen (2006) who said that expansion of development to PSF is threatening
some species of birds that represent the last vital refuge for a lowland specialist species. On the other
hand, Site 2 bird diversity was no significant different from Site 1 and Site 3, respectively. This
probably due to TBF located next to Titty Longhouses orchard and also next to Maludam National
Park.
4. CONCLUSION
A total of 292 birds representing 45 species were recorded, including 33 at MNP, 24 at TBF and 18 at
CCF. Different degrees of disturbance cause by human activities such as agriculture practice and
logging seem to have influenced the diversity of avifauna. Lowland species such as Babblers and
Bulbuls dominated the understory of peat swamp forest.
Acknowledgement
The authors would like to express deep and sincere gratitude to the Director General MPOB and
th
Director of Biological Research for allowing this paper to be presented at The 4 Regional Conference
on Natural Resources in the Tropics (NTrop4). The authors also wish to thank the staff of Tropical
Peat Research Institute Unit, MPOB and Institute of Biodiversity and Environmental Conservation,
UNIMAS for their great support and invaluable assistance. The research was supported by a grant
from MPOB.
References
Chai, P.P.K (2005). Management plan for Maludam National Park Betong Division Sarawak. Joint
working group Malaysia-The Netherlands development and management of Maludam National
Park. Forest Department Sarawak, Kuching. JWG/MNP/2004/MP
Chapin III, F.S., Zavaleta, E.S., Eviner, V.T., Nalyor, R.L., Vitousek, P.M., Reynolds, H.L., Hooper,
D.U., Lavorel, S., Sala, O.E., Hobbie, S.E., Mack, M.C. & Diaz, S. (2000). Consequences of
changing biodiversity. Nature Vol:405
Hunting,
E.R.
(n.d.).
Biodiversity
analysis.
Retrieved
from
http://home.medewerker.uva.nl/e.r.hunting/bestanden/Biodiversity_self_tuition_module.pdf on
29 June 2012.
Laman, C.J., Gawin, D.F.A. and Rahman, M.A. (2006) Quantifying the diversity of avifauna at Loagan
Bunut National Park. In: Scientific Journey Through Borneo: Loagan Bunut. A.A. Tuen, A.K.
Sayok, A.N. Toh and G.T. Noweg (Eds). Peat Swamp Forest Project, UNDP/GEF
(MAL/99/G31), Sarawak Forest Department and Institute of Biodiversity and Environmental
Conservation, Universiti Malaysia Sarawak. pp. 163-174
Myers, S. (2009). A field guide to the birds of Borneo. Talisman Publishing Pte Ltd. Singapore.
United Nation Development Programme (UNDP) (2006). εalaysia’s peat swamp forests, conservation
and sustainable use. 33p. ISBN 983-40995-5-X
Rahman, M.A. (2004). A study on fauna of Maludam National Park Betong Division Sarawak. Joint
working group Malaysia-The Netherlands development and management of Maludam National
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Park. Faculty of Resource Science and Technology. Universiti Malaysia Sarawak. Wisma
Printing Sdn. Bhd. Kuching.
Rahman, M.A. & Tuen, A.A. (2006). The avifauna. In:The biodiversity of a peat swamp forest in
Sarawak. Pp 129-136. F. Abang & I. Das (Eds.) Institute of Biodiversity and Environmental
Conservation, Universiti Malaysia Sarawak, Kota Samarahan.
Wahid, O., Aziz, N.A., Mohammed, A.T., Harun, M.H & Din, A.K. (2010). Mapping of oil palm
cultivation on peatland in Malaysia. MPOB TT No. 473. ISSN 1411-7871.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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ISOLATION AND CHARACTERIZATION OF PATHOGENIC BACTERIA FROM SWIFTLET
(Aerodramus spp.) DROPPINGS
1*
1
Leong Sui Sien , Samuel Lihan and Ling Teck Yee
1
2
2
Department of Molecular Microbiology; Department of Chemistry, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
*E-mail: leongsuisien87@gmail.com
Abstract
Swiftlet (Aerodramus spp.) dropping is used as organic fertilizer in agricultural industry because it is
rich in nutrient, nitrogen, phosphorus, potassium and minerals. The bacterial content of the dropping
and the properties associated with the bacteria is uncertain. The aim of this preliminary study was to
isolate, characterize and identify the type of pathogenic bacteria from Aerodramus spp. in Kota
Samarahan, Sarawak. Five samples of swiftlet droppings were collected randomly from each sampling
site of the swiftlet farm. One gram of the dropping sample was diluted in 9ml of 0.85% normal saline
-6
solution. The samples were diluted serially until the dilution of 10 . A 0.1ml of aliquot was plated onto
o
Trypticase Soy agar plates and incubated at 37 C for 24 hours. Biochemical tests were carried out to
identify the isolates from the samples. The swiftlet dropping samples had a total viable count between
7
9
8
1.50 ×10 to 1.46 ×10 cfu/ ml and the mean bacteria viable count was 3.60 ×10 cfu/ ml. Bacteria
isolates from the swiftlet droppings found in this study were gram positive and gram negative bacteria
including Staphylococcus spp. (46%), Bacillus spp. (44%), Streptococcus spp. (4%), Corynebacterium
spp. (2%) and Escherichia coli (4%). Aerodramus spp. droppings may carry various bacterial species
and future analysis should be carried out to confirm the pathogenicity of the bacteria.
Keywords: Pathogenic bacteria, swiftlet, droppings
1. INTRODUCTION
Swiftlets mainly Aerodramus species are birds residing in South East Asia region only. Swiftlet farming
is defined as a modern and commercialized way of building bird’s nest differently as compared to the
traditional harvesting way from caves. Swiftlet farming involved the ways of attracting swiftlets to
inhabit in a man-built building named as swiftlet house. The swiftlet farming industry in Malaysia has
been growing tremendously over the last 8 years. According to Kuan & Lee (2011), Malaysia is the
third largest producer of edible birds’ nests in the world, after Indonesia and Thailand. It was estimated
that there are about 900 plus swiftlet farms in the year of 1998 and increased to 3600 swiftlet farms in
the year of 2006 with the growth rate of 36% annually (Hameed, 2007). The swiftlet farming industry
has high potential in growing into a multi-million ringgit industry mainly due to profitable risk-return
profile and the continuously growing demand for edible birds’ nests by wealthy overseas countries
(Hameed, 2007). Swiflet farming was originated from Indonesia since the year of 1880 in East Jawa
(Swiftlet Eco Park Group of Companies, 2009). In the year of 1970, Indonesia swiftlet industry started
modifications and improvements using in-house farming methods to emulate the cave-like conditions.
Indonesia is the world largest exporter of edible-bird’s nests and has more than 70% global market
share (Swiftlet Eco Park Group of Companies, 2009). Malaysia started swiftlet farming because of the
forest fire happened in Indonesia in the year of 1990. Open burning and haze in Indonesia has led
more than millions of swiftlet migrating to West Malaysia, causing the number of swiftlet farms
increased tremendously. South East Asia is the main region that swiftlets inhabit. Besides, swiflet
farming industry in Malaysia increased sharply due to the Asian economic crisis in the year of 1997 to
1998 (TCL Swiflet Farming, 2010) and full support of the Wildlife and National Parks Department
(Perhilitan) (Jessica and Wilson, 2005). There are an estimation of more than 30,000 shop houses
and commercial premises which have been converted into swiftlet farms in June 2005 throughout
εalaysia, especially the Northern εalaysia (εalaysian Swiftlet Farmers Association, 2005). Swiftlets’
droppings are used as organic fertilizer in agricultural industry because it is rich in nutrient, water,
nitrogen, phosphorus, potassium and minerals (Nyakundi & Mwangi, 2011). Most of the bacteria are
able to grow in the swiftlets’ dropping. According to Susan (2010), swiftlet farmings are causing noise,
smell, property damage and unsanitary conditions in George Town, Pulau Pinang. Beside, swiftlet
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droppings may carry the potential to spread diseases and lung infection within an enclosed area
(Susan, 2010). Therefore, this present study is conducted to isolate and characterize the pathogenic
bacteria from the swiftlet droppings.
2. METHODOLOGY
2.1 Description of the study area
The study area of the swiftlet house was situated at Kota Samarahan. It is located about 30 kilometers
from Kuching.
2.2 Sample collection
Sampling and sample processing procedures for the isolation of swiftlet faeces bacteria were carried
out as described by Nyakundi and Mwangi (2011). Five samples were collected randomly from the
sampling site of the swiftlet farm. The samples were collected by using the spatula and transferred into
storage sterile bag for further analysis in the laboratory. Dilution of sample was made by mixing one
gram of dropping and 9 ml of sterile 0.85% saline solution. The diluted sample was plated on
o
o
Trypticase Soy agar plates and incubated at 37 ± 1 C for 24 hours. The cfu was calculated according
to Nyakundi and Mwangi (2011). Colony forming unit (cfu)= (number of colony per plate) (dilution
factor). Five to 10 colonies were randomly picked for further identification.
2.3 Identification of bacteria
The isolated bacteria from the faeces were identified phenotypically using conventional biochemical
test according to Bergey’s manual (1λ57), involving Gram staining, urease test, hydrogen sulfide test,
motility test, lactose fermentation test, Simmons citrate test, starch hydrolysis test, triple sugar iron,
gas production, indole (tryptone broth) test, mannitol fermentation test, methyl red-Voges-Proskauer
test, oxidase test and catalase test.
3. RESULTS
3.1 Bacteria colony count
The mean total bacteria viable counts in swiftlet dropping samples isolated from Kota Samarahan is
shown in Table 1. It showed that the swiftlet droppings collected from swiftlet house in Kota
7
9
Samarahan had a total viable count between 1.50 ×10 to 1.46 ×10 cfu/ ml and the mean bacteria
8
viable count was 3.60 ×10 cfu/ ml.
Table 1 Mean total bacteria viable counts in swiftlet dropping samples isolated from Kota Samarahan.
Sample
A
B
C
D
E
Total
Mean
Total bacteria count (cfu/ ml)
9
1.46 ×10
7
6.40 ×10
8
1.47 ×10
8
1.12 ×10
7
1.50 ×10
9
1.80 ×10
8
3.60 ×10
3.2 Occurrence of bacteria in swiftlet droppings
The bacteria isolates from the swiftlet droppings collected from Kota Samarahan are shown in Table 2.
Based on the Table 2, it showed that the swiftlet droppings were found to consist of gram positive
bacteria including Staphylococcus spp. (46%), Bacillus spp. (44%), Streptococcus spp. (4 %) and
Corynebacterium spp. (2 %). Besides, the gram negative bacteria namely Escherichia coli (4%) was
isolated from swiftlet droppings .
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Table 2 Bacteria isolated from the swiftlet droppings collected from swiftlet farm in Kota Samarahan.
SAMPLE
BACTERIAL CODE
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
D1
D2
D3
D4
D5
D6
D7
D8
D9
D10
E1
E2
E3
E4
E5
E6
E7
E8
E9
E10
SWF-S-A1
SWF-S-A2
SWF-S-A3
SWF-S-A4
SWF-S-A5
SWF-S-A6
SWF-S-A7
SWF-S-A8
SWF-S-A9
SWF-S-A10
SWF-S-B1
SWF-S-B2
SWF-S-B3
SWF-S-B4
SWF-S-B5
SWF-S-B6
SWF-S-B7
SWF-S-B8
SWF-S-B9
SWF-S-B10
SWF-S-C1
SWF-S-C2
SWF-S-C3
SWF-S-C4
SWF-S-C5
SWF-S-C6
SWF-S-C7
SWF-S-C8
SWF-S-C9
SWF-S-C10
SWF-S-D1
SWF-S-D2
SWF-S-D3
SWF-S-D4
SWF-S-D5
SWF-S-D6
SWF-S-D7
SWF-S-D8
SWF-S-D9
SWF-S-D10
SWF-S-E1
SWF-S-E2
SWF-S-E3
SWF-S-E4
SWF-S-E5
SWF-S-E6
SWF-S-E7
SWF-S-E8
SWF-S-E9
SWF-S-E10
SUSPECTED ORGANISM
Bacillus spp.
Bacillus cereus
Bacillus badius
Corynebacterium xerosis
Bacillus cereus
Bacillus cereus
Bacillus cereus
Bacillus spp.
Bacillus cereus
Bacillus anthracis
Staphylococcus aureus
Staphylococcus aureus
Bacillus anthracis
Bacillus cereus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Bacillus cereus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Bacillus cereus
Staphylococcus aureus
Bacillus cereus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Bacillus cereus
Staphylococcus aureus
Bacillus cereus
Staphylococcus aureus
Bacillus cereus
Bacillus cereus
Bacillus cereus
Staphylococcus aureus
Staphylococcus aureus
Bacillus badius
Streptococcus spp.
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Escherichia coli
Bacillus spp.
Escherichia coli
Staphylococcus aureus
Streptococcus spp.
Bacillus spp.
Staphylococcus aureus
4. DISCUSSION
Bacteria colony count was done to determine the total number of microorganisms present in the
swiftlet dropping samples collected. The total and mean bacteria viable counts in swiftlet dropping
7
9
samples collected from the swiftlet house in Kota Samarahan was between 1.50 ×10 to 1.46 ×10 cfu/
8
ml and 3.60 ×10 cfu/ ml respectively (Table 1). The present bacterial count are higher than the
findings reported by Nyakundi and Mwangi (2011). In their report, the total bacteria viable count was
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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6
6
between 2.4 ×10 and 7.8 ×10 cfu/ g from Marabou stock (Leptoptilos crumeniferus) droppings. The
results of microbiological examination and identification of bacteria in swiftlet dropping samples at
Kota Samarahan are shown in Table 2. A total of 50 bacteria isolates were isolated during the course
of study. Results of this preliminary microbiological examination of the swiftlet samples revealed that
most of the bacteria isolated were gram positive bacteria. Staphylococcus spp. (46 %) was isolated
most frequently, follwed by Bacillus spp. (44 %), Streptococcus spp. (4 %), Eschierichia coli (4 %) and
Corynebacterium spp. (2 %). Staphylococcus spp. and Bacillus spp. were the most frequently
isolated bacteria mainly because they are facultative anaerobes bacteria which are capable of growth
both aerobically and anaerobically (Turnbull, 1996, Wikipedia, 2011). Besides, Bacillus spp. are able
to produce endospores which can stay dormant under stressful environment for long periods (Madigan
and Martinko, 2005) thus able to survive even in dry swiftlet droppings. Contamination of bird feed in
the droppings sample could also be the reason of the occurence of Staphylococcus spp. and
Streptococcus spp. According to Kocijan et al. (2009), the Staphylococcus spp. and Streptococcus
spp. are ubiquitous and commonly found in birds’ feed. This study shows that the bacteria Eschierichia
coli was found in the samples which was normal situation because almost all the wild birds consist of
E. coli (Simpson, 2002). Nyakundi and Mwangi (2011) and Literak et al. (2007) had isolated the E. coli
from the Marabou Stock (Leptoptilos crumeniferus) droppings and rooks droppings samples. The
determination of further bacteria to determine their risk is in progress.
5. CONCLUSION
This preliminary study concluded that there are a number of potentially pathogenic microorganisms
including Staphylococcus spp., Bacillus spp, Streptococcus spp, Eschierichia coli
and
Corynebacterium spp. were found present in the droppings of swiftlet (Aerodramus spp.), thus it may
pose a public health hazard to humans and environment.
References
Hameed, S.M. (2007). The 2007 Malaysia Swiftlet Farming Industry Report. Summary and synopsis.
Publish on 1 June 2007.
Jessica, L. and Wilson, H. (2005). Swiftlet housing boom. NST on 4 September 2005.
Kocijan, I.E., Prukner-Radovcic, Beck, R., Galov, A., Marinculic, A., Susic, G. (2009). Microflora and
internal parasites of the digestive tract of Eurasian friffon vultures (Gyps fulvulus) in Croatia. Eur.
J. Wildl. Res. 55, 71-74.
Kuan, H. and Lee, J. (2011). The complete introductory guide to swiftlet farming. Swiftlet Farming.
pp54-58. Malaysia: Struan Inc Sdn Bhd.
Literak, I., Vanko, R., Dolejska, M., Cizek, A. and Karpyskova, R. (2007). Antibiotic resistant Escheria
coli and Salmonella in Russian rooks (Corvus frugilegus) intering in Czech Republic. Lett. Appl.
Microbial. 45, 616-621.
Malaysia Swiftlet Farmers Association. (2005). Swiftlet Farming in Malaysia. Retrieved on 5 January
2012 from www.hongyunswiftlet.com.
th
Mandigan, M. and Martinko, J. (2005). Brock Biology Of Microorgaisms. 11 Ed. Bacillus. pp. 300.
America: Prentice Hall.
Nyakundi, W.O. and Mwangi, W. (2011). Isolation and Characterisation of Pathogenic Bacteria and
Fungi from Leptoptilos crumeriferu (Marabau Strok) Dropping. Journal of Applied Technology of
Environment Sanitation. 1(1):93-103.
th
Robert, S.B., εurray, E.G.D. and Nathan, R.S. (1λ57). Bergey’s manual. 7 Ed. United States of
America: Baltimore. The Williams & wilkins company.
Simpson, V.R. (2002). Wild animals as reservoirs of infectious diseases in the UK. Veterinary Journal.
163: 128-146.
Susan, L. (2010). Stop the dangerous swiftler houses in George Town! No swiftlet houses in George
Town. Articles on Tuesday, 13 April 2010.
Swiftlet Eco Park Group. (2009). History of swiftlet farm. Retrieved on the 4 January 2012 from
www.swiftletecofarmgroup.com.
TCL Swiftlet Farming. (2010). TCL swiftlet farming design and build birdhouses with cellulose
insulation. Retrieved on the 4 January 2012 from www.tclswiftletfarming.com.
th
Turnbull, P.C.B. (1λλ6). Inμ Barron’s medical εicrobiology. 4 Ed. Bacillus (pp.299). America: Univ of
Texas medical Branch.
Wikipedia.
(2011).
Staphylococcus.
Retrieved
on
12
May
2012
from
http://en.wikipedia.org/wiki/Staphylococcus .
440
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
ABUNDANCE AND NUTRIENT CONTENT OF INSECT AT THE LAKE SIDE OF SARAWAK CLUB
GOLF COURSE AND UNIVERSITI MALAYSIA SARAWAK CAMPUS, KOTA SAMARAHAN,
SARAWAK
Andrew Alek Tuen*, Leow Tze Chin and Sulaiman Hanapi
Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak,
94300 Kota Samarahan, SARAWAK
Email:aatuen@ibec.unimas.my
Abstract
A total number of 743 insects comprising eight Orders, 19 Families and 41 species were collected
from the lake edge of Sarawak Club Golf Course and Universiti Malaysia Sarawak Campus. The four
most abundant insect species collected were Green Rice Leafhopper (N. nigropictus), Yellow Crazy
Ant (Anoplolepis spp.), Sugar Ant (Tapinoma spp.), and Red Tree Ant (Oecophylla smaragdina).
Collectively, they accounted for 61% of the total insect collected. The most diverse insect community
was found at East Campus Lake followed by the West Campus Lake and Golf Course Lake. The
average of nutrient content of four most abundant insect species was 2.82% ash, 62.02% crude
protein, 4.73% crude fat and 21.78 mg/g phosphorus.
1. INTRODUCTION
Insects are the most numerous and diverse living creatures on earth. They form an important
component of the diet of vertebrates such as fish, amphibians, reptiles, birds and mammals (Gullan
and Cranston 2005) thus playing an important role in the prey-predator relationship. In this relationship
the insects prey sustains the predator while some predators provide ecological services by controlling
the population of insect pest.
In order to understand the feeding value of prey to their predator a number of information are required.
This include the relative abundance of the prey items in the environment, which type of prey items are
taken by what animals and how much nutrient the prey supply to the predator. Obviously, the last
information requires that we know how much prey is consumed (food intake) and the nutrient content
and digestibility of the prey (food quality). Nutrient content of selected insect groups, particularly those
known to be consumed by humans, have been analysed by Banjo et al. (2005) and Shen et al. (2006).
A high crude protein content of 589 g/kg DM was reported in Platylomia spinosa (Homoptera:
Cicadoidea) collected from dipterocarp forest of Sarawak (Wilfred 2009). Recent studies by Shahaida
(2011) and Wan Fareez (2012) showed insects to be an important component of the diet of starlings
and egrets in Universiti Malaysia Sarawak (UNIMAS) Campus.
This paper focuses on the relative abundance and nutrient content of selected insects at the lake edge
in Sarawak Club Golf Resort and UNIMAS campus, Kota Samarahan, Sarawak.
2. METHODS
The insects were collected from three sites in Kota Samarahan (Figure 1): Sarawak Club Golf Resort
Lake (Site A), UNIMAS West Campus Lake (Site B) and UNIMAS East Campus Lake (Site C). Site A
is more open while Site B and C have more shade from trees that has been planted as part of the
landscaping project.
Four quadrates, each measuring 1m x 25m, were established along the lake bank about one metre
away from the water edge. The insects within the quadrates were collected by using forceps, bare
hands or aerial net. Insects such as small ants were collected by using a Pooter. A Pooter is an
instrument that is used to collect small insects by sucking them up a tube and into a container. Insects
collected were immediately killed in a killing jar with chloroform and then transferred into a container
and kept in an oven at 70°C.
441
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Figure 1 The three study sites: Study Site A – Golf Course Lake (Left), Study Site B – West Campus
Lake (Middle) and Study Site C – East Campus Lake (Right)
The four most abundant insects were selected for nutritional evaluation. The ash content was
o
determined by incinerating the insect sample in a 500 C muffle furnace for 5 hours and then the ash
was made up to 100 ml using 0.1 M hydrochloric acid for analysis of calcium and phosphorus. Calcium
was determined by inductive coupled plasma technique while inorganic phosphorus was determined
using the metavanadate reduction method. Crude protein was determined by kjedahl method and
crude fat by using Soxhlet ether extraction method.
3. RESULTS and DISCUSSION
The insect community at the edge of the three lakes comprised of eight Orders, 19 families and 41
species (Table 1). Out of 743 individuals collected, 404 or 54.37% were from the Family Formicidae
(Order Hymenoptera). This group of insect tends to aggregate forming large colonies (Bolton 1994) so
that hundreds of them are sometimes encountered at one spot.
In terms of number of species, Order Odonata has the most with 12 species collected. This order
utilizes both aquatic and terrestrial habitat (Norma-Rashid et al. 2001, Kalkman et al. 2008) so it is not
surprising that they are the most diverse group at the lake edge. Borneo has the second highest
number of species in the world (272 species) behind Venezuela (489 species) and second highest in
terms of endemism behind Madagascar (124 vs 135) (Kalkman et al. 2008).
The four most abundant insect species collected were Green Rice Leafhopper (Nephotettix
nigropictus), Yellow Crazy Ant (Anoplolepis spp.), Sugar Ant (Tapinoma spp.), and Red Tree Ant
(Oecophylla smaragdina). Collectively, they accounted for 61% of the total insect collected.
The most abundant insect species at Site A was N. nigropictus with a total number of 132 individuals
recorded while the most abundant insect collected at Site B and Site C were Anoplolepis spp and
Tapinoma spp. with 124 and 32 individuals, respectively. However Tapinoma spp. was recorded more
at Site B (61 individuals) compared to Site C. The leafhopper N. nigropictus was found at Site A only
while O. smaragdina and Anoplolepis spp. were found only at Site B. The tree ant O. smaragdina is a
predator of insects that protects plants and fruits from being attacked by insect pest (Peng, Christian
and Gibb 1995, Offenberg 2004) and its dominance at Site B may explain why this site has less of
other insects.
Site C has the highest number of species (24 species) compared to Site A (19 species) and Site B (16
species). Site C recorded the lowest number of individuals collected (155 individuals) while Site B
recorded the highest with 358 individuals. The high number of individuals at Site B is due mainly to
ants. Site C is much mature having been established in 1994 compared to Site A and B, which were
established in 2005. There is less frequent grass cutting activity carried out at Site C compared to Site
A or B, hence the higher number of species at Site C is expected.
The most abundant insect species at Study Site A was leafhopper but this insect was not recorded at
Site B and C. Site A is more open, the lake being one of the hazard for the golf course, therefore the
grass area was more exposed to the sunlight compared to Site B and C where trees were planted as
part of the landscaping activities. Drier conditions, such as the golf lake edge, seemed to favour the
leafhopper (Hill and Abang 2005).
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Table
1
Number
Order
of
species
and
individual
insects
Family
Description / Species
Lestidae
Dictyoptera
Blattodea
Hemiptera
Coreidae
Lestes spp.
Damselfly spp 2
Agriocnemis femina
Damselfly spp 3
Ischnura senegalensis
Crocothemis servilia
Orthetrum glaucum
Rhyothemis phyllis
Dragonfly spp 1
Acisoma panorpoides
Orthetrum sabina
Neurothermis ramburii
Acheta domesticus
Brachytrupes spp.
Attractomorpha spp.
Locust spp 1
Tetrix spp.
Grasshopper spp1
Oxya japonica
Cockroach spp 1
Cockroach spp 2
Bug spp1
Cicadellidae
Nephotettix nigropictus
Odonata
Coenagrionidae
Libellulidae
Orthoptera
Gryllidae
Tettigoniidae
Pyrgomorphidae
Tetrigidae
Acrididae
Coleoptera
Diptera
Lepidoptera
Coccinellidae
Chrysomelidae
Sarcophagidae
Dolichopodidae
Lycaenidae
Nymphalidae
Formicidae
Hymenopter
a
Xylocopidae
Total Number of Species
Nephotettix virescens
Beetle spp1
Beetle spp2
Sarcophaga spp.
Flies spp2
Flies spp3
Flies spp4
Flies spp5
Zizina otis
Lepidop spp1
Junonia orithya
Anoplolepis spp.
Oecophylla smaragdina
Polyrhachis myrmatopa
spp.
Polyrhachis cyrtomyrma
spp.
Tapinoma spp.
Diacamma spp.
Xylocopa collaris
Number of Individuals
RA = relative abundance
443
collected
at
Three
Study
Study Sites
A
B
C
1
1
4
2
1
4
3
7
1
2
3
4
1
9
2
1
2
2
3
23
1
15
16
1
1
2
2
2
1
2
1
1
13
2
16
5
1
3
1
3
5
2
3
1
18
12
10
1
1
4
12
4
95
Total
No.
1
1
6
1
4
10
3
3
4
1
12
2
28
16
16
1
1
6
1
2
1
1
11
Sites
RA (%)
0.13
0.13
0.81
0.13
0.54
1.35
0.40
0.40
0.54
0.13
1.62
0.27
3.77
2.15
2.15
0.13
0.13
0.81
0.13
0.27
0.13
0.13
132
17.77
16
6
3
1
3
5
5
1
40
1
5
2.15
0.81
0.40
0.13
0.40
0.67
0.67
0.13
5.38
0.13
0.67
124
16.69
95
12.79
11
1.48
36
10
46
6.19
11
61
19
23
0
16
35
8
32
23
1
24
15
5
104
23
1
41
14.00
3.10
0.13
100
743
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
The nutrient content of four most abundant insect is given in Table 2. Generally the ash content is
quite low (1.38-3.95%) indicating the absence of mineralised skeleton in insects. The crude protein
content is high (52.68-71.18%), the value being typical for animal protein source and similar to values
reported for cicadas (Wilfred 2009) but generally higher than the crude protein content of insects in
south western Nigeria (Banjo et al. 2006). In a comprehensive study of the nitrogen content in 152
species of insect Fagan et al. (2002) found predator insects (insects that feed on other insects) to
have 15% more nitrogen content compared to herbivore insects. The crude protein content of predator
insect reported by Fagan et al. (2002) is 68.9% compared to crude protein content of 60.3% in
herbivore insect, implicating dietary nitrogen as major determinant of the nitrogen content of insect.
These researchers also reported recently evolved orders such as Diptera and Lepidoptera have 1525% less nitrogen compared to the more ancient herbivore lineages such as Orthoptera and
Hemiptera. Cuticle structure on the head, thorax and abdomen of the insects are made up of chitin
and protein, which protect and waterproof the outer covering of the insects. Protein served as crucial
material to build exocuticle of the insects (McGavin, 2006).
Table 2 Nutrient content (on dry matter basis) of selected insect collected from UNIMAS campus, Kota
Samarahan (each value is a mean of three replicates).
Nutrient Content
Oecophylla
smaragdina
Anoplolepis
spp.
Tapinoma
spp.
Nephotettix
nigropictus
Ash (%)
Crude Protein (%)
3.95
62.86
3.69
61.37
1.38
52.68
2.25
71.18
Crude Fat (%)
6.81
-
2.65
-
Phosphorus (mg/g)
13.79
-
36.78
14.77
Calcium
nd = not detectable
nd
nd
nd
nd
The calcium concentration in our insect sample was apparently very low and not detectable in the ash
solution but phosphorous was. Similarly Banjo et al. (2006) reported calcium concentration to be about
50% that of phosphorous. Table 2 above shows that the smaller ants such as Tapinoma spp. had
higher phosphorous concentration compared to the larger ant such as O. smaragdina (36.78 vs 13.79
mg/g). The inverse relationship between body size and phosphorous content was reported by Woods
et al. (2004) who also reported higher phosphorous content in recently derived insect orders such as
Diptera and Lepidoptera.
Our study showed that insects are a good source of protein and phosphorous for the predator that
feeds on them. A recent study Wan Fareez (2012) found insect to be the dominant prey consumed by
Little Egret foraging near Site B. Similarly the frugivorous Asian Glossy Starling feeding on Ficus
benjamina at UNIMAS East Campus (near Site C) were found to have insect parts, mainly ants, in
their stomach (Norshahaiza, 2011). Based on their abundance, high protein content and presence in
the stomach of birds, there is no doubt that insect play an important role in the nutrition of birds.
References
Banjo, A. D., Lawal, O. A. & Songonuga, E. A. (2005). The nutritional value of fourteen species of
edible insects in southwestern Nigeria. African Journal of Biotechnology 5: 298-30.
Bolton, B. (1994). Identification guide to the ant genera of the world. Cambridge, Mass.: Harvard
University Press, 222 pp.
Fagan, W.F., Siemann, E. Mitter, C. Denno, R.F., Huberty, A.F., Woods, H.A. and Elser, J.J. (2002).
Nitrogen in insects: implication for tropic complexity and species diversification. The American
Naturalist, 160:784-802
rd
Gullan, P. J., & Cranston, P. S. (2005). The insects: An outline of entomology (3 ed.). United
Kingdom: Blackwell Publishing
Kalkman, V.J., Clausnitzer, V., Dijkstra, K. B., Orr, A.G., Paulson, D.R. and Tol, J.V. (2008). Global
diversity of dragonflies (Odonata) in freshwater. Hydrobiologia 595:351-363
444
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Norma-Rashid, Y., Mohd-Sofian, A. and Zakaria-Ismail, M. (2001). Diversity and distribution of
Odonata (dragonflies and damselflies) in the freshwater swamp lake Tasek Bera, Malaysia.
Hydrobiologia 459:135-146
Offenberg, J., Havanon, S., Aksornkoae, S., Macintosh, D.J. and Nielsen, M.G. (2004). Observations
on the Ecology of Weaver Ants (Oecophylla smaragdina Fabricius) in a Thai Mangrove
Ecosystem and Their Effect on Herbivory of Rhizophora mucronata Lam. Biotropica 36: 344351.
Peng, R. K., Christian, K. and Gibb, K. (1995). The effect of the green ant, Oecophylla smaragdina
(Hymenoptera: Formicidae), on insect pests of cashew trees in Australia. Bulletin of
Entomological Research 85: 279-284
Norshahaiza bt Mohd Zain (2011). Feeding habits of Asian Glossy Starling in UNIMAS, Kota
Samarahan. Final Year Project Report, Faculty of Resource Science and Technology, Universiti
Malaysia Sarawak.
Shen, L., Li, D., Feng, F., & Ren, Y. (2006). Nutritional composition of Polyrhachis vicina Roger
(Edible Chinese black ant). Nutraceutical and Functional Food 28 (Suppl. 1), 107-114.
Wilfred, J. (2009). The nutritional composition of ciciadas (Homoptera: Cicadoidea). Kota Samarahan,
Sarawak : Faculty of Resource Science and Technology.
Wan Fareez (2012) Foraging ecology of egrets in UNIMAS Campus. Final Year Project Report,
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak
445
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
COLONIZATION OF ARTIFICIAL REEF BALLS BY HARD CORALS AT TALANG-SATANG
NATIONAL PARK, SEMATAN, SARAWAK
Nyanti, L.*, Natasha, N.A. and M. Aazani
Department of Aquatic Science, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak (Malaysia)
*Email: lnyanti@frst.unimas.my
Abstract
Artificial reef balls have been deployed for more than a decade at Batu Penyu which is situated within
the Talang-Satang National Park (TSNP), Sematan, Sarawak. However, little published work is
available on the colonization of the artificial reef at TSNP by marine organisms. The objectives of this
study were to determine the composition and colonization rate of hard corals on the reef balls at
TSNP. A self-contained underwater breathing apparatus (SCUBA) was used to collect data at each
reef ball with the aids of quadrate, belt transect and underwater photography. The percentage cover
by hard corals differed among different reef balls and among different levels of reef ball from the
substratum. The highest overall percentage cover by hard corals on reef balls was 38 % at reef ball
No. 3. Different hard corals genera colonized different levels of reef balls. The dominant genus at the
top, middle and bottom levels is Favia. The top level of the reef balls had the highest hard coral
colonization ranging from 21.8 to 45.6%, followed by 2.5 to 14.2% at mid level and 1.0 to 4.8% at level
close to the substratum. Coral genera which consistently show the highest growth rates are Favia and
2
-1
2
-1
Favites with values ranging from 0.3 to 7.1 cm yr and 1.3 to 9.0 cm yr respectively. The
percentage cover by hard coral as well as the dominant genera at each level is determined by the
location of the reef ball which is influenced by the surrounding substrates types and the rate of
sedimentation.
Keywords: Colonization, artificial reef ball, Talang-Satang National Park, corals
1. INTRODUCTION
Artificial reefs act as a potential tool for reef conservation, rehabilitation, maintaince and enhancement
of marine biodiversity (Levner et al., 2007; Relini et al., 2007; Shahbudin et al., 2011). Artificial reefs
are also used in coastal management by protecting coastline and to enhance fish production (Burt et
al., 2009; Carmo et al., 2010). Harris (2009) proposed that artifical reefs support stable bases for coral
attachment as there is a rapid growth in Acropora corals on artificial reef balls in Curacao, Antigua and
Indonesia.
The deployments of artificial reef balls (ARB) begun since the late 1970s throughout Southeast Asia
(Chou, 1997). Artificial reef ball is classified as environmentally friendly because the cement that was
used to construct them are of the same pH as ocean water. The use of special admixtures including
micro silica is designed to increase compression in concrete and flexural strengths, increase durability,
reduce permeability and improve hydraulic abrasion erosion resistance (The Reef Beach Company,
1995). In contrast, artificial reef balls which uses the regular concrete have a surface pH as high as
12. This could inhibit the settlement and growth of marine communities on the surfaces of ARB.
In Sarawak, the earlier ARB was deployed as a tool for conservation of turtles (Ubang, 2001; Awang,
2003). Artificial reef balls possess sharp and rough surfaces which are excellent for prevention of
illegal trawling activities (Ubang, 2001). In 1998, approximately 2,284 units were deployed along the
coast of Sarawak by the Sarawak Reef Balls Working Group. They were also deployed In TalangSatang National Park (TSNP), including at areas near Batu Penyu which is located about two
kilometres from Talang Besar Island.
Ubang (2001) observed visible growth of the early stages of hard corals and other benthic marine
organisms on the ARB at TSNP after two and a half years of deployment. Since this study was done
at the early stage of deployment, identification on the abundant and composition of organisms growing
446
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
on the surfaces of the reef balls was not carried out. A later colonization study by Jantan (2004)
reported that there were no differences in the assemblage of species among ARB deployed in 1998,
1999 and 2002. The percentage cover by hard corals was low at all sites and was classified as poor.
Therefore, the objectives of this study were to assess the composition of hard coral colonising ARB
after 14 years of deployment, determine the hard coral colonization at three vertical zones (top, middle
and bottom) of ARB; and estimate the growth rates of hard corals growing on ARB.
2. MATERIALS AND METHODS
The study was carried out at Talang-Satang National Park (Figure 1). The park consists of Satang
Islands and Talang Islands. Satang Islands consists of Satang Besar and Satang Kecil wheras Talang
Islands consists of Talang Besar and Talang Kecil. The study site was at the west of Batu Penyu
where ARB were deployed in 1988 and the estimated period after deployment was about 14 years.
Samplings were carried out using self-contained underwater breathing apparatus (SCUBA) in July
2011 and April 2012. For the percentage cover of hard corals colonization on the whole reef, ten reef
balls (1, 3, 4, 8, 12, 14, 15, 16, 17 and 19) were selected at random (Fig. 1). Reef balls No. 1, 3, 12
and 14 were located nearer to the natural coral reef while reef ball No. 8, 16 and 17 were farther from
the natural reefs. All other reef balls were located in between the two areas. The percentage cover of
hard corals colonization based on vertical zonations of reef ball (top, middle and bottom) were carried
out at four reef balls (12, 15, 16 and 17) that were chosen at random (Fig. 1). Similarly, the growth
rates for hard coral genera were also carried out at reef balls No. 12, 15, 16 and 17.
Data collections were carried out using two techniques, namely quadrate and underwater
photography. Quadrate frame of two different sizes (0.50 m width x 0.50 m length and 0.23 m width x
0.27 m length) were used in this study. A larger size quadrate was used to determine the percentage
cover by hard corals on the whole reef balls. They were laid on the surface around the reef balls. The
area covered by hard corals was recorded on underwater writing slates (Fig. 2a). The smaller
quadrate was used to collect data for smaller areas at three vertical zones of the reef balls in the same
manner as the larger quadrate (Fig. 2a). In order to ease data collection, a rope was laid across the
reef ball to act as marker to divide the reef ball into half. The three zones (top, middle and bottom) in
each reef ball were divided equally according to the height of the reef balls and ropes were used to
indicate the different zones (Fig. 2a). Data recorded on underwater writing slates were later verified
with the photos taken by underwater camera (Fig. 2b). Hard corals were identified based on Kelley
(2009), Johnston (1986), Wood (1983) and Allen and Steene (1997). The percentage cover of hard
corals present in the quadrate was calculated by using Equation [1]. The growth rate of hard coral was
calculated by using Equation [2].
2
Percentage cover (%) = Surface area covered by the organisms (m ) x 100%
[1]
2
Total surface area of quadrate (m )
2 -1
2
Growth rate (cm y ) = Surface area of coral genus (cm )
[2]
No. of years of deployment
Figure 6 Map of the study area showing the position of artificial reef balls in Batu Penyu. The circled
reef balls were studied.
447
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
(a)
(b)
Figure 2 (a) Quadrate was set up around the reef balls and rope acted as marker was laid on the reef
balls; (b) Underwater photograph of a quadrate and hard corals colonizing artificial reef balls.
3. RESULTS
3.1 Percentage cover of hard corals for the whole reef
The overall percentage cover by hard corals for the whole reef ranged from 6% at reef ball No. 17 to
38% at reef ball No. 3 (Table 1). The genus of hard coral that had the highest average percentage
cover was Favia with 8% and the lowest was Leptoseris with 0.02% (Table 2). The highest percentage
cover of Favia was at reef ball No. 14, with a coverage of 18% and the lowest was on reef ball No. 8,
with percentage cover of 3%.
Table 1 Overall percentage cover by hard corals.
Reef Ball No.
1
3
4
8
12
14
15
16
17
19
Percentage
Cover
25.6
38.0
8.7
2.8
15.6
29.7
10.9
9.5
5.9
13.8
15
16
17
19
3.4
1.3
3.8
2.8
3.3
1.5
7.6
6.2
-
1.5
-
-
Table 2 Percentage of hard corals by genera on each reef ball.
Reef Ball No.
Genus
1
3
4
8
12
14
Favia
11.2 14.5 4.0 2.8 8.5
18.4
Favites
12.2 12.7 4.7 5.2
5.5
Montastrea
Goniastrea
-
-
-
-
0.9
-
-
1.2
0.7
-
Turbinaria
-
-
-
-
0.4
-
-
0.1
0.1
-
Porites
2.3
7.2
-
-
-
2.3
0.2
0.2
-
-
Goniopora
-
-
-
-
0.2
-
-
-
-
-
Acropora
-
-
-
-
-
-
0.2
-
-
-
Lobophyllia
-
-
-
-
0.2
-
-
-
0.2
-
Podabacia
-
-
-
-
0.1
-
-
-
-
-
Oxypora
-
-
-
-
0.1
-
-
-
-
-
Zoopilus
-
-
-
-
-
-
-
-
0.1
-
Psammocora
-
-
-
-
0.04
-
-
-
-
-
Leptoseris
-
-
-
-
0.02
-
-
-
-
-
Astreopora
-
3.5
-
-
-
3.4
-
-
-
-
Encrusted hard coral
-
-
-
-
-
5.7
-
-
-
Total Percentage
25.6
8.7
2.8
15.6
29.7
10.9
9.5
5.9
13.8
38.0
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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3.2 Percentage Cover of Hard Corals by Vertical Zonations
The overall percentage cover by hard corals for the four reef balls (12, 15, 16 and 17) was the highest
at the top zone with 74% (Figure 4). The lowest was at the bottom zone with 7% and the middle zone
had 19% cover by hard corals.
Data for individual reef ball showed that the percentage cover of hard corals was always the highest at
the top zone while the least percentage cover was at the bottom zone (Fig. 5). At reef ball No. 12, the
percentage cover of hard corals at the top, middle and bottom zones were 62%, 33% and 5%
respectively. At reef ball No. 15, it was 58%, 27% and 15% at top, middle and bottom zones
respectively. Percentage of hard corals colonization on reef ball No. 16 was 55% at the top zone, 24%
at the middle zone and 21% at the bottom zone while for reef ball no. 17, it was 78% at the top zone,
15% in the middle and 7% at the bottom zone (Figure 5).
Table 3 shows the percentage cover by hard coral genera for each reef ball at the three different
zones. At the top zone, the highest percentage cover was on reef ball No.12 at 45.6% and the lowest
was on reef ball No.17 at 21.8%. At the middle zone, reef ball No. 12 had the highest coverage by
hard corals at 14.2% and reef ball No. 17 had the lowest coverage at 2.5%. At the bottom zone, the
highest was observed at reef ball No. 16 and the lowest at reef ball No. 17, with coverage of 4.8% and
1.0%, respectively (Table 3). Among the coral genera, Favia and Favites are the two dominant genera
colonizing all the three zones (Table 3).
Figure 4 Mean percentage of hard corals colonization on four reef balls at different vertical zones.
Figure 5 Percentage of hard corals colonization at different zones on reef balls No. 12, 15, 16 and 17.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Table 3 Percentage of hard corals genera colonizing the three different zones of reef balls.
Reef Ball No.
Zone
Genus
12
15
16
17
Top
Favia
21.5
6.8
10.7
10.4
Favites
18.3
5.1
6.9
6.6
Goniastrea
4.4
-
4.8
3.4
Turbinaria
1.4
-
-
0.4
Lobophyllia
-
-
-
1.0
Montastrea
-
-
2.2
-
Porites
-
-
0.1
-
Unidentified hard coral
-
18.1
-
-
45.6
30.0
24.7
21.8
Favia
9.5
2.8
2.3
2.0
Favites
3.3
0.6
2.2
0.4
Montastrea
-
-
1.4
-
Goniopora
0.6
-
-
-
Porites
-
0.5
0.4
-
Lobophyllia
0.3
-
-
-
Podabacia
0.3
-
-
-
Zoopilus
-
-
-
0.1
Psammocora
0.1
-
-
-
Leptoseris
0.1
-
-
-
Turbinaria
-
-
0.01
0.01
Unidentified hard coral
-
4.3
-
-
14.2
8.2
6.3
2.5
Favia
1.1
2.4
1.6
0.9
Favites
0.3
-
1.4
0.1
Montastrea
-
-
1.2
-
Acropora
-
0.5
-
-
Goniastrea
-
-
0.5
-
Turbinaria
0.2
-
0.2
-
Oxypora
0.2
-
-
-
Porites
-
0.1
-
-
Lobophyllia
0.1
-
-
-
Psammocora
0.1
-
-
-
Unidentified hard coral
-
0.8
-
-
2.0
3.8
4.8
1.0
Total
Middle
Total
Bottom
Total
3.3 Growth Rates of Hard Coral Genera
Table 4 shows the range of growth rates of hard corals according to genera. The growth rate for each
genus varies within and among each reef ball. Overall, Favia and Favites consistently showed the
highest growth rates in all the four reef balls. The lowest growth rate was shown by Leptoseris in reef
2
-1
ball No. 12 at 0 - 0.6 cm yr .
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Table 4 The growth rates of hard coral genera at four reef balls.
2
Genus
-1
Growth Rate (cm yr ) for Reef Ball
12
15
16
17
Favia
0.3 - 7.1
0.1 - 6.0
0.1 - 5.0
0.1 - 10.6
Favites
0.3 - 7.1
1.3 - 9.0
0.3 - 20.0
0.2 - 10.4
Leptoseris
0.6
-
-
-
Acropora
-
1.1 - 4.1
-
-
Turbinaria
-
-
0.1 - 2.4
-
Zoopilus
-
-
-
1.6
Goniastrea
0.9 - 7.1
-
0.6 - 17.8
2.4 - 8.5
Goniopora
7.1
-
-
-
Lobophyllia
1.3 - 3.6
-
-
2.9 - 3.6
Oxypora
2.5
-
-
-
Podabacia
3.2
-
-
-
Psammocora
0.6 - 0.7
-
-
-
Turbinaria
0.4 - 7.1
-
0.1 - 2.4
0.1 - 2.7
Porites
-
0.6 - 5.7
0.8 - 2.4
-
Montastrea
-
-
1.4 - 15.9
-
4. DISCUSSION
Colonization rates by hard corals vary among different reef balls and different genera of corals show
different growth rates. Factors that affect the colonization process by marine organisms on artificial
reef included age, depth, spatial orientation and structural design (Perkol-Finkel & Benayahu, 2004) as
well as times scale and sedimentation loads (Perkol-Finkel & Benayahu, 2007; Ming et al., 2010).
Similarly, McClanahan and Obura (1997) reported that coral covers increased with time duration and
growth were dependent on low and high sedimentation.
In the case of reef balls at Batu Penyu in TSNP, with the exception of spatial orientation and
sedimentation loads all other factors such as age, depth, structural design and time scale were similar.
According to Fabricius (2005) and Gleason & Hofmann (2011), sedimentation affects reef structure by
altering the settlement pattern and survival rate of coral larvae and new recruits. Coral larvae were
reported to actively detect and avoid substrata covered with sediment (Gleason & Hofmann, 2011).
This may have explained the lowest hard corals percentage cover at the bottom zone of reef balls
which was affected by higher sedimentation rate, less light penetration and weaker water current to
wash the sedimentation away. Subsequently, the highest percentage cover by hard corals was at the
top zone of the reef ball where sedimentation rate is lower, light penetration higher and stronger water
current helps to wash the sediment away.
Reef balls located nearer to the natural reef has higher percentage cover by hard corals than those
located further away. Those located further from the natural reef sit on more sandy-muddy area with
sediment that is more easily resuspended during rough sea condition. In the study area, Lee (2012)
-2
-1
reported that the rate of sedimentation at the study site was 9.73 - 104.93 mgcm day which is about
10 - 14 times higher than the natural sedimentation rate at coral reef area. Nemeth and Nowlis (2001)
-2
-1
also reported that coral growth will decline when sedimentation rate exceeds 10 mgcm day .
Favia and Favites were the two dominant genera with the highest percentage cover at the top zone of
reef balls. According to Wood (1983), most of Favia and Favites colonies are usually massive and
rounded, which cause higher rate of growth and colonization. Since Favia is one of the sediment
intolerant genus and Favites as the intermediate tolerant genus (McClanahan & Obura, 1997), they
are found more abundant at the top zone because of higher light penetration and stronger water
current to help remove sediment deposited at this zone. Fabricius (2005) reported that sedimentation
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
correlated with the profound changes in coral communities’ structures by altering the frequencies size,
declining mean colony sizes, reduced growth and survival rate and altering the growth formations.
Based on the ranking of Coral Reef Health Criteria (Chou et al., 1994), the status of hard corals
colonizing the reef balls at Batu Penyu, TSNP ranged from poor (1 to 25%) to fair (26% to 50%). Life
coral cover was poor at reef ball No. 4, 8, 12, 15, 16, 17 and 19 but fair at reef balls No. 1, 3 and 14.
5. CONCLUSIONS
The percentage cover by hard corals in artificial reef balls at Batu Penyu in Talang-Satang National
Park ranged from poor to fair after about 14 years of deployment. Favia and Favites were the two
dominant genera colonizing the reef balls. In Batu Penyu, spatial location and rate of sedimentation
were the two main factors affecting the colonization rate and growth of hard corals. Reef balls located
nearer to the natural reef have higher colonization rate by hard corals than those located further away
where the surrounding area consisted mostly of mud and sand.
Acknowledgement
We are thankful to Mr. Zaidi Hj Ibrahim, Mr. Harris Norman, Mr. Mohd Azlan Belly Bujang, Mr. Richard
Toh, Mr. Nazri for assistance in field and laboratory works. The financial support of the Faculty of
Resource Science and Technology of Universiti Malaysia Sarawak is gratefully acknowledged.
References
Allen, G. R., & Steene, R. (1997). Indo-Pacific coral reef field guide. Singapore: Tropical Reef
Research.
Awang, D., & Pit, I. B. (2003). Study on reef ball at Batu Penyu nearby Talang-Talang Island. Bintawa:
Fisheries Research Institute Sarawak Branch.
Burt, J., Bartholomew, A., Bauman, A., Saif, A., & Sale, P. F. (2009). Coral recruitment and early
benthic community development on several materials used in the construction of artificial reefs
and breakwaters. Journal of Experimental Marine Biology and Ecology, 373: 72-78.
Carmo, J. S. A. D., Neves, M. G., & Voorde, M. T. (2011). Designing a multifunctional artificial reef:
studies on the influence of parameters with most influence in the vertical plane. Journal of
Coastal Conservation, 15: 99-112.
Chou, L. M. (1997). Artificial reefs of Southeast Asia- Do they enhance or degrade the marine
environment?. Environmental Monitoring and Assessment, 44: 45-52.
Chou, L. M., Wilkinson, C. R., Licuanan, W. R. Y., Alino, P., Cheshire, A. C., Loo, M. G. K.,
Tangjaitrong, S., Sudara, S., Ridzwan, A. R., & Soekarno. (1994). Status of coral reefs in the
ASEAN region. Proc-Third ASEAN Australia Symp. on Living Coastal Resources. 1: 1-10.
Fabricius, K. E. (2005). Effects of terrestrial runoff on the ecology of corals and coral reefs: review and
synthesis. Marine Pollution Bulletin, 50: 125-146.
Gleason, D. F., & Hofmann, D. K. (2011). Coral larvae: From gametes to recruits. Journal of
Experimental Biology and Ecology, 408: 42-57.
Harris, L. E. (2009). Artificial reefs for ecosystem restoration and coastal erosion protection with
aquaculture and recreational amenities. Reef Journal, 1(1): 235-246.
Jantan, F. (2004). Colonization studies of artificial reef balls around Talang-Talang Islands, Sematan,
Sarawak. (Unpublished Final Year Project Report). Department of Aquatic Science, Universiti
Malaysia Sarawak, Kota Samarahan.
Johnston, N. A. (1986). The Hard Corals of Sabah. Penerbit Universiti Kebangsaan Malaysia, Bangi.
Kelley, R. (2009). Coral finder Indo Pacific. Townsville, Australia: BYO Guides.
Lee, X. L. (2012). Sedimentation rate and nutrients at coral reef at Talang-Talang Island, Sematan,
Sarawak. (Unpublished Final Year Project Report). Department of Aquatic Science, Universiti
Malaysia Sarawak, Kota Samarahan.
Levner, E., Ganoulis, J., Linkov, I., & Benayahu, Y. (2007). Multiobjective risk/cost analysis of artificial
marine systems using decision trees and fuzzy expert estimations. In: Linkov, I., Kiker, G. A.,
and Wenning, R. J. (Eds.), Environmental Security in Harbors and Coastal Areas, Springer,
Berlin, pp.161-174.
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McClanahan, T. R., & Obura, D. (1997). Sedimentation effects on shallow coral communities in Kenya.
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Ming, C. L., Lionel, N. G. C. S., Jeremy, C. S. M., & Angie, S. L. (2010). Natural coral colonization of a
marina seawall in Singapore. Journal of Coastal Development, 14(1): 11-17.
Nemeth, S. R., J. S. Nowlis. (2001). Monitoring the effects of land development on the near-shore reef
environment of St. Thomas, USVI. Bulletin of Marine Science, 69:759-775.
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unplanned artificial reefs in Eilat (Red Sea): comparison to natural reefs. Coral Reefs, 23: 195205.
Perkol-Finkel, S., & Benayahu, Y. (2007). Differential recruitment of benthic communities on
neghboring artificial and natural reefs. Journal of Experimental Marine Biology and Ecology,
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Perkol-Finkel, S., & Benayahu, Y. (2007). Differential recruitment of benthic communities on
neghboring artificial and natural reefs. Journal of Experimental Marine Biology and Ecology, 2539, 340.
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artificial reefs of the Ligurian sea, Italy. Hyrobiologia, 580: 193-217.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
DISTRIBUTION OF Bactrocera dorsalis complex (Handel) IN LUNDU-SEMATAN, SARAWAK
Siti Zuriani Ismail*, Rizoh Bosorang, Siti Nurlydia Sazali and Sulaiman Hanapi
Department of Zoology, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
*Email: isitizuriani@gmail.com
Abstract
A survey on the distribution of Bactrocera dorsalis was conducted along the road of Lundu-Sematan
and a junction to Kampung Biawak, Lundu, Sarawak. Fifty seven pheromone traps were placed at 1
km interval along the Lundu-Sematan and Kampung Biawak roads and the number of trapped B.
dorsalis were counted and recorded. A total of 1204 individuals were successfully trapped. Based on
the capture record, their distribution seemed to increase towards Lundu town as they were more
abundant here compared to the Sematan town with an average capture of 14 and 13 individuals per
trap, respectively, although the difference was not significant (p-value=0.432, 0.05). The average
number of individuals collected at Lundu junction towards Kampung Biawak was lower (9 per trap)
than the number of individuals collected at Lundu-Sematan road with an average 13 per trap. This
could be due to the fact that the area is an oil palm estate. B. dorsalis occupied various types of
vegetation, where ripe fruits are available. The presence of fruit flies indicated that there were fruit
trees in the surrounding areas. Thus, through this survey, the distribution of B. dorsalis along the
Lundu-Sematan road and Kampung Biawak was established and this provides a preliminary base-line
data for future survey on the population fluctuation of B. dorsalis within the study area.
Keyword: Distribution, relative abundance, Bactrocera dorsalis complex, pheromone trap, LunduSematan.
1. INTRODUCTION
Bactcocera dorsalis complex were known to be universal pests that infested many types of fruit trees
and known to be one of the most destructive insect pests of tropical and subtropical fruits and
vegetables (White and Elson-Harris, 1992; Allwood et al., 2001; Trusuta et al., 2005, Siti Zuriani,
2008). This species is known as oriental fruit fly with a black T-shaped mark on the abdomen and a
typical dacine pattern on the wing, predominantly black scutum with lateral yellow stripes, anterior
supra-alar setae, prescutellar acrostichal setae and two scutellar setae on the thorax and facial spots
on the face (White and Elson-Harris, 1992). The species is considered as a serious insect pest that
has been categorised as an organism subject to severe quarantine restriction by most countries in the
world (Zhang and Hou, 2005). Climate, particularly temperature and rainfall, is the main factor
influencing the distribution of fruit fly (Zhou et al., 1996; Jiang et al., 2001). The occurrence of this
species in a particular area could indicate on their potential trait towards fruit and crop in that area.
The result can be used to monitor the pest distribution and to conduct any necessary approach for
controlling their indirect impacts in the community, especially in rural areas. Nowadays, several areas
in Lundu Division had been converted from crop plantation into oil palm plantation in order to boost the
palm oil industry in the state. The conversion of this land might influence the distribution of fruit fly that
concentrates their infestation mainly on fruits and crops. Therefore, this study aims to document on
the distribution of B. dorsalis complex from several areas in Lundu Division, as well as to indicate their
potential threat to the production of fruits and crops, managed by the local communities.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2. MATERIALS AND METHOD
The self-made pheromone traps,
with methyl eugenol used as the
attractant, were placed at 1 km
interval along the Lundu-Sematan
road and the junction to Kampung
Biawak (Fig. 1). The traps were set
up for a duration of one hour before
relocated to subsequent sampling
sites. The trapped B. dorsalis were
counted,
recorded
and
drypreserved
(Fig.
2).
Some
specimens were mounted on card
point as voucher specimens.
Identification was done following
key provided by White and ElsonHarris (1992). Statistical analysis
was conducted using Microsoft
Excel for average value. Critical
value with p = 0.05 was used to
determine significant difference
using Student’s t-test.
Figure 1 Map of Lundu – Sematan road ( ) and Biawak road ( )
(a)
(b)
Figure 2 Adult Bactrocera dorsalis complex: (a) Dorsal view; (b) Ventral view
3. RESULTS AND DISCUSSION
A total of 1204 individuals were successfully trapped and recorded (Table 1). The study indicates that
the distribution of B. dorsalis complex seemed to increase towards Lundu town as they were more
abundant here compared to the Sematan area with average capture of 14 and 13 individuals per trap,
respectively. However, the difference was not significant at 95% confidence interval (p-value=0.432,
equal variance). This finding indicated that B. dorsalis complex was well distributed in the village
areas; assuming people living in the areas were planting a variety of fruit trees around their homes,
which would be attractive to this species. Delrio and Prota (1977; 1988) who conducted survey on fruit
fly in different regions found that the pest is abundant in subtropical or tropical climate where a great
variety of fruits are planted compared to other regions.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Table 1 Data collection of B. dorsalis.
Trap
Coordinate
Total
Trap
Coordinate
Total
Trap
Coordinate
Total
H01
N01°48'07.2"
E109°46'36.8"
290
H20
N01°40'56.4"
E109°47'18.9"
1
H39
N01°44'58.5"
E109°52'02.5"
109
H02
N01°48'21.9"
E109°46'21.5"
27
H21
N01°40'33.4"
E109°47'41.3"
7
H40
N01°43'33.1"
E109°51'54.4"
6
H03
N01°48'22.5"
E109°45'54.5"
3
H22
N01°40'06.3"
E109°47'57.8"
3
N06
N01°37'48.7"
E109°41'03.2"
2
H04
N01°48'06.6"
E109°45'28.7"
1
H23
N01°39'40.2"
E109°48'16.5"
3
N07
N01°38'15.2"
E109°41'19.8"
0
H05
N01°47'49.8"
E109°45'01.3"
0
H24
N01°39'23.0"
E109°48'43.6"
9
N08
N01°38'30.7"
E109°41'48.7"
7
H06
N01°47'29.5"
E109°44'38.0"
3
H25
N01°39'36.0"
E109°49'11.8"
2
N09
N01°38'43.8"
E109°42'19.0"
35
H07
N01°47'01.2"
E109°44'22.2"
12
H26
N01°39'49.6"
E109°49'42.3"
1
N10
N01°38'52.3"
E109°42'50.0"
9
H08
N01°46'31.6"
E109°44'34.6"
9
H27
N01°40'09.8"
E109°50'04.3"
38
N11
N01°38'36.5"
E109°43'17.5"
12
H09
N01°46'02.1"
E109°44'49.0"
21
H28
N01°40'25.0"
E109°50'28.3"
47
N12
N01°38'26.4"
E109°43'48.4"
2
H10
N01°45'33.9"
E109°45'04.1"
29
H29
N01°40'13.2"
E109°50'57.1"
0
N13
N01°38'21.0"
E109°44'17.4"
12
H11
N01°45'07.7"
E109°45'21.1"
11
H30
N01°40'42.9"
E109°50'56.0"
22
N14
N01°38'28.9"
E109°44'48.7"
7
H12
N01°44'46.0"
E109°45'46.9"
4
H31
N01°41'12.5"
E109°50'45.4"
6
N15
N01°38'26.6"
E109°45'20.0"
3
H13
N01°44'21.4"
E109°46'03.8"
0
H32
N01°41'33.5"
E109°51'05.0"
6
N16
N01°38'14.2"
E109°45'47.7"
1
H14
N01°43'49.6"
E109°46'04.4"
9
H33
N01°42'02.2"
E109°51'15.8"
4
N17
N01°38'03.4"
E109°46'21.2"
1
H15
N01°43'19.0"
E109°46'14.6"
48
H34
N01°42'30.5"
E109°51'34.2"
4
N18
N01°38'20.6"
E109°46'44.5"
0
H16
N01°42'49.5"
E109°46'21.3"
6
H35
N01°42'59.8"
E109°51'32.4"
5
N19
N01°38'46.9"
E109°47'00.2"
4
H17
N01°42'18.6"
E109°46'34.9"
10
H36
N01°43'33.0"
E109°51'27.9"
9
N20
N01°39'16.2"
E109°47'11.0"
41
H18
N01°41'51.3"
E109°46'48.8"
33
H37
N01°44'02.1"
E109°51'34.3"
23
N21
N01°39'42.1"
E109°47'31.2"
2
H19
N01°41'27.0"
E109°47'11.3"
34
H38
N01°44'27.1"
E109°51'59.4"
203
N22
N01°40'06.1"
E109°47'54.2"
6
Note: H01 – H40 represent traps located along Lundu – Sematan road respectively, started at
Sematan Town (H01) toward Lundu Town (H29) and start again with H30 toward Kampung Pandan
(H40). N06 – N22 represent traps set at Kampung Biawak (N06) toward main road of Lundu –
Sematan (N22).
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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The average number of individuals collected at Lundu junction towards Kampung Biawak was lower
(with average 9 per trap) than the number of individuals collected at Lundu-Sematan road (with an
average of 14 per trap). However, the difference was not significant at 95% confidence interval (pvalue=0.121, unequal variance). The decreasing number of individuals collected at Lundu junction
towards Kampung Biawak could be due to the fact that the area is an oil palm estate and kerangas
forest. It was noted that the micro environment appeared to be one of the important factors in
determining the population distribution and fluctuation (Qureshi et al., 1975; Fehn, 1982), as less
number of fruit flies were collected at areas occupied by kerangas forest compared to mixed
dipterocarp forest. The study may also correlate with the previous by Murthy and Regupathy (1992)
where distribution of fruit fly was shown to be positively correlated with relative humidity and sunshine.
4. CONCLUSION
This study has documented on the distribution of B. dorsalis complex that is subjected to the
availability of fruit trees in the several areas of Lundu Division. Other factors including the forest types,
relative humidity and sunshine were known to have significant impacts on the distribution and
fluctuation of this species complex. For these reasons, those factors should be highlighted for future
research on the population dynamic and pest management study.
Acknowledgement
The authors would like to acknowledge Faculty Resource Science and Technology, Universiti
Malaysia Sarawak for providing us with the administrative and logistic support during the study. The
research was funded by Fundamental Research Grant Scheme: FRGS/01(24)/767/2010(48).
References
Allwood, A. J., A. Chinajariyawong, R. A. I. Drew, E. L. Hamacek, D. L. Hancock, C. Hengsawad, J. C.
Jipanin, M.Jirasurst, C. Kong Krong, S. Kritsansepaiboon, C. T. S. Leong, and S. Vijaysegaran
(1999). Host plant records for fruit flies (Diptera: Tephritidae) in south-east Asia. The Raffles
Bulletin Zoology. Suppl. 7:1-92.
Delrio, G. and R. Prota (1977). Observations on the population dynamics of Ceratitis capitata Wied. In
some peach and citrus orchards of Sardinia. Infromatore Fitopatologico. 27 (6/7):59-60.
Delrio, G. and R. Prota (1988). Determinants of abundance in a population of the olive fruit fly.
Frustula Entomologica. 11:47-55.
Fehn, L. M. (1982). Influence of meteorological factors on the population fluctuation and dynamics of
Anastrepha spp. Pesquisa Agropecuaria Brasileira. 17(4):533-544.
Jiang, X. L., W.Z. He and S. Xiao. (2001). Study on the biology and survival of Bactrocera dorsalis in
the border region of Yunnan. Journal of Southwest Agricultural University. 23:510-517.
Murthy, J. N. A. and A. Regupathy, (1992). Seasonal incidence of moringa fruit fly, Gitona sp. South
Indian Horticulture. 40 (1):43-48.
Qureshi, Z. A., M. Ashraf, A. R. Bughio and Q. H. Siddiqui. (1975). Population fluctuation and
dispersal studies of fruit fly, Dacus zonatus (Saunders). IAEA; Vienna, Austria: pp. 201-206.
Siti Zuriani, I. and S. Hanapi (2009). Tephritid fruit flies (Diptera; Tephritidae) in Samarahan Division:
Collection from pheromone traps. Proceeding in Symposium Biology Malaysia (SIMBIOMAS).
th
th
Equatorial Hotel, Bangi. 16 – 18 November 2009. Universiti Putra Malaysia (UPM).
Tsuruta, K., H. M. J. Bandara and G. B. J. P. Rajapakse (2005). Notes on lure responsiveness of fruit
flies of the tribe Dacini (Diptera: Tephritidae) in Sri Lanka. Esakia. 45: 179-184.
White, I. M. and M. M. Elson-Harris (1992). Fruit flies of economic significance: Their identification and
bionomics. CAB International Publishing. New York. Pp 53-111.
Zhang, R. J. and B.H. Hou. (2005). Assessment on the introduction risk of Bactrocera dorsalis
(Hendel) through imported fruits with fuzzy mathematics. Acta Entomologica Sinica. 48:221226.
Zhou, Y.S., F.R. Shen. and H. P. Zhao. (1996). Study on the biology of Dacusa (Bactrocera) dorsalis
(Handel) and Synthetical control. Journal of Southwest Agricultural University. 18:210-213.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
CHARACTERIZATION OF BACTERIA ISOLATED FROM FISH AND WATER SAMPLES FROM
SARAWAK RIVER
*Felecia Collick, Samuel Lihan, Kathleen Michelle Mikal, Lesley Maurice Bilung, Kasing Apun
Department of Molecular Biology, Faculty of Resource Science and Technology, University Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak
*Email: felecia_collick@yahoo.com
Abstract
Sarawak River has always been known as the source of food and transportation for local residents
living nearby. Monitoring and identifying bacteria from the aquatic organisms specifically from the
fishes is significant as this will help to understand the condition of the river. Fish and water samples
were taken from the Barrage area of Sarawak River. Isolation of bacteria from fish and water samples
was carried out on non selective agar, Trypticase Soy Agar (TSA). The colony forming unit per millilitre
3
4
5
5
for water and fish samples range from 1.13x10 to 1.39x10 and 2.34x10 to 7.80x10 respectively.
5
The total microbial counts were slightly higher than the specified standard limit (1.0x10 cfu/ml) for
bacteria by ICMSF and USFDA in edible portion of fish flesh. Characterization of the isolated bacteria
was done using GTG5 PCR showed that the isolates were genetically closely related.
Key words: Sarawak River, fish, DNA fingerprinting, GTG5 PCR,
1. INTRODUCTION
Significantly used not only for modes of transportation and recreational activities, Sarawak River also
served as the source of food for some local residents living nearby. Sarawak River specifically Sungai
Sarawak Kanan which is approximately 20km long acts as one of the significant river in Kuching. A
number of the Sarawak population living along the river are still much depending on it to undergo their
daily routine and activities. This river is habitat to numerous types of fish including the family of
Cyprinidae, Gobiidae, Bagridae, and Osphronemidae (Jeanes and Meijaard, 2000). Monitoring river
water quality is important as adequate water supply and good sanitary system will decrease the cause
for water-borne diseases (Liew and Lepesteur, 2006). Pollution is a major threat to our river
nowadays. It may come from various resources such as human activities and animal waste. A survey
of microbiological quality of aquatic organisms has shown that they could harbour pathogenic
organism (Herrera et al., 2006; Mhango et al., 2010). This study aims to analyse total microbial count
of fish muscle and water samples and also its genetic relatedness.
2. MATERIALS AND METHODS
2.1 Sample Processing
Sampling processes were done twice at Sarawak River specifically Barrage area. Samples taken were
the water samples and fish samples. Fish samples were freshly caught and the water samples were
taken with 100ml sterile Schott bottle. Samples transported to the laboratory in cooler box containing
ice and processed immediately.
2.2 Bacterial Isolation
The fish flesh and water samples were homogenized before performing 10-fold serial dilution. The
-1
-5
-1
-3
serial dilution was done from 10 until 10 for the fish flesh samples and 10 to 10 for water sample.
Each dilution was plated on non-selective agar, Tryptase Soy Agar (TSA). After incubation of 24 hours
in 29ºC, colony forming unit on the plates were counted. Based on the colony forming unit counted, 10
colonies were selected randomly from the TSA plates to be preserved and further identified. Coding of
the isolates were done with SAB-W for water samples and SAB-F for fish samples. Biochemical tests
like gram staining, Methyl Red and Voges–Proskauer test, citrate test, oxidase test, sulphide-indole-
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
motility test, catalase test and lysine test were done towards the isolates. Identification was done
referring to Bergey’s εanual of Determinative Bacteriology.
2.3 DNA extraction
DNA of all 36 isolates were extracted using the boiling method (Queipo et al., 2008) with few
modifications. 1.5ml of cultured bacteria in Luria Broth was centrifuge with 10 000rpm for 5 minutes.
The supernatant was discarded, topped up with 500 µl double distilled water and the mixture was
briefly mixed on a vortex mixer. Mixture then placed in boiling water bath for 10 minutes, freezed for 5
minutes before being centrifuged at 10 000 rpm for 10 minutes. A 300 δ aliquot of the supernatant
was transferred to a sterile tube and stored at 4°C until PCR testing.
2.4 GTG5 PCR
GTG5 PCR was carried out in 25µl reaction according to Matsheka (2006) containing 10X Taq Green
Buffer (Promega), 25mM MgCl2, 25mM deoxyribonucleotide phosphate (dNTPs), 25µM (GTG) 5
primer, sterile distilled water, DNA template and Taq DNA polymerase. The conditions of amplification
were as follows;
Condition
Temperature
Time
Number of cycles
Pre-denaturation
95°C
7 minutes
1
Denaturation
95°C
2 minutes
Annealing
36°C
2 minutes
4 cycles
Extension
72°C
2 minutes
Denaturation
95°C
1 minute
Annealing
50°C
1 minute
30 cycles
Extension
72°C
1 minute
Final extension
72°C
5 minutes
1
Electrophoresis of amplified PCR product was done on 1.5% agarose gel in 1X Tris-Borate-EDTA
(TBE) buffer pre-stained with 1 µl ethidium bromide at 90V for 75minutes. The stained gel was then
visualized using UV transilluminator.
3. RESULTS AND DISCUSSION
3
4
5
5
The colony forming unit ranged from 1.13x10 to 1.39x10 and 2.34x10 to 7.80x10 for water and fish
5
sample. The specified standard limit for bacteria concentration in aquatic organism is 1.0x10 cfu/ml
given by the International Commission on Microbiological Specifications for Foods (ICMSF),
(Adebayo-Tayo and Okpo, 2010). Abundance of bacterial colony reported was higher than the
standard limit as location of sampling was located near the downstream of the river render the high
concentration of bacteria gained (Anand et al., 2006). However, bacterial count at any location of
study depends on hydrological and also anthropogenic activities. Located near to the river barrage,
hydraulic movement of the opening and closing of barrage system give rise of sediment to move
upwards into the river basin causing murky environment suitable for growth of bacteria (Ahmadian et
al., 2010; Gorbatkin, 2008)
Diversity of 35 bacterial isolates successfully isolated from the location was done using GTG 5 PCR.
The results of this PCR showed several identical bands as seen in Figure 1.
M 1
2
3 4
5
6
7
8
9 10 11
12 13
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
2500
1500
1000
750
50
Figure 1 Lane M: 1kb DNA ladder (Promega), Lane 1: 2SAB F1, Lane 2: 2SAB F2, Lane 3: 2SAB F3,
Lane 4: 2SAB F4, Lane 5: 2SAB F6, Lane 6: 2SAB F7, Lane 7: 2SAB F8, Lane 8: 2SAB W2, Lane 9:
2SAB W3, Lane 10: 2SAB W4, Lane 11: 2SAB W5, Lane 12: 2SAB W6, Lane 13: 2SAB W7, Lane 14:
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2SAB W8, Lane 15: 2SAB W9, Lane 16: 2SAB W10, Lane 17: SAB F1, Lane 18: SAB F2, Lane 19:
SAB F3, Lane 20: SAB F4, Lane 21: SAB F5, Lane 22: SAB F6, Lane 23: SAB F8, Lane 24: SAB F9,
Lane 25: SAB F10, Lane 26: SAB W 1, Lane 27: SAB W2, Lane 28: SAB W3, Lane 29: SAB W4, Lane
30: SAB W5, Lane 31: SAB W6, Lane 32: SAB W7, Lane 33: SAB W8, Lane 34: SAB W9, Lane 35:
SAB W10.
Table 1: Identified isolates from the fish and water samples
Isolate
Source
Identity
2SAB F1
2SABF2
2SABF3
2SABF4
2SABF6
2SABF7
2SABF8
2SAB W2
2SAB W3
2SAB W4
2SAB W5
2SAB W6
2SAB W7
2SAB W8
2SAB W9
2SAB W10
SAB F1
SAB F2
SAB F3
SAB F4
SAB F5
SAB F6
SAB F7
SAB F8
SAB F9
SAB F10
SAB W1
SAB W2
SAB W3
SAB W4
SAB W5
SAB W6
SAB W7
SAB W8
SAB W9
SAB W10
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Water
Water
Water
Water
Water
Water
Water
Water
Water
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Fish
Water
Water
Water
Water
Water
Water
Water
Water
Water
Water
Bacillus sp.
Enterobacter cloacae
Enterobacter cloacae
Enterobacter cloacae
Bacillus sp.
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus sp.
Bacillus thuriengiensis
Brevibacillus brevis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus thuriengiensis
Bacillus sp.
Bacillus thuriengiensis
Bacillus thuriengiensis
Fingerprinting using GTG5 PCR resulted in multiple identical bands showing less diversity of bacterial
colonies in this location. However, we can detect similar bacteria in the muscle of fish and water
samples. Bacterial isolates gained in the fish muscle could originate from its environment. Muscles of
fish were previously reported to have less number of bacteria compared to their other organs or body
parts. This is also depending on the nutrient intake of the aquatic organisms (Abou-Elela and Farag,
2004; Anand et al., 2006). An open water system with no source of organic waste as food source lead
to less diversity of bacteria as compared to organic waste pollutants affected region (Ampofo and
Clerk, 2010) corresponding to study location which was quite a distant away from industrial activities.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Based on the biochemical tests and 16S rRNA sequencing, only several bacteria were identified which
are Bacillus thuringiensis, Brevibacillus brevis, Bacillus sp. and Enterobacter cloacae.
Bacillus thuringiensis and Brevibacillus brevis isolated from the water samples are soil-dwelling
bacterium (Gerbeva et al., 2003; Vivas et al., 2005) which resulted from the tidal barrage system. The
regular activities of fishing and cargo boats moving along the river also induce budging of soils from
the sediments. Enterobacter cloacae, a human pathogen which can cause a range of infections such
as lower respiratory tract infection, skin and soft tissue infections, urinary tract infections, intraabdominal infections and ophthalmic infections (Ronald, 2002; Jiang et al., 2005) could be harmful as
it was isolated from the muscle of the fish. Both Bacillus thuringiensis and Enterobacter are widely
used in pesticides to control pests (Betz et al., 2000; Jensen et al., 2002; Flores et al., 2012) from
harming the plants. Nearby vegetation by local residences presumed to contribute to the condition.
The safety of consuming products from the river is the main concern for this study. Water quality and
culture practises play an important role in determining quality of the aquatic lives consumed. Proper
sanitation and cooking in preparation of seafood is necessary to avoid undesirable condition.
4. CONCLUSION
Isolation and identification of 35 isolates of bacteria from fish muscle and water samples from Sarawak
River near the barrage region had been done. The number of coliform count was higher than the
standard limit. Few factors presumed to contribute to the condition such as opening and closing of the
barrage system, regular movement of cargo and fishing boats, and nearby vegetation. However, the
bacteria identified were less diverse in terms of species which are only from the genera Bacillus and
Enterobacter.
References
Abou-Elela, J.M. and Farag, A.M. (2004). Bacteriological Quality and Metal Contents of Diplodus
Vulgaris and Siganus Rivulotus in The Eastern Harbour Water: A Comparative Study of
Freshly harvested and Market Fish. Egyptian Journal of Aquatic Research , 30(A): 216-225.
Adebayo-Tayo, B.C. and Okpo, M.A. (2010). Microbiological, Proximate And Heavy Metal
Concentration In Penaeus Sp. (Shrimp) And Calllinectes Sp. (Crab) From Creeks In Niger
Delta, Nigeria. African Journal of Food Agriculture Nutrition and Development, 10(8) 30473064.
Ahmadian, R., Falconer, R. and Lin, B.(2010). Hydro-Environmental Modelling Of Proposed Severn
Barrage,UK. Proceedings of the Institution of Civil Engineers , 163: 107–117.
Ampofo, J.A. and Clerk, G.C. (2010). Diversity of Bacteria Contaminants in Tissues of Fish Cultured in
Organic Waste-Fertilized Ponds: Health Implications. The Open Fish Science Journal , 3: 142146.
Anand, C., Akolkar, P. and Chakrabarti, R.(2006). Bacteriological Water Quality Status Of River
Yamuna In Delhi. Journal of Environmental Biology , 27(1): 97-101.
Betz, F.S., Hammond, B.G. and Fuchs, R.L. (2000). Safety and Advantages of Bacillus thuringiensisProtected Plants to Control Insect Pests. Regulatory Toxicology and Pharmacology , 32(2):
156-173.
Flores, C.C., González, E.G., Ramos, A.V., Valencia, R.T., Méndez, R.D., Salinas, E.S, Godínez,
L.C., Quintana F.R. and Hernández L.O. (2012). Isolation Of The Opde Gene That Encodes
For A New Hydrolase Of Enterobacter Sp. Capable Of Degrading Organophosphorus
Pesticides. Biodegradation , 23(3): 387-397.
Garbeva, P., Veen, J.A. and Elsas, J.D. (2003). Predominant Bacillus spp. in Agricultural Soil under
Different Management Regimes Detected via PCR-DGGE. Microbial Ecology, 45(3):302-316.
Gorbatkin, C. S. (2008). The Distribution of Bacterial Contaminants in Dammed Aquatic Systems.
Dartmouth Undergraduate Journal Of Science, 54-59.
Herrera, F.C., Santos, J.A., Otero, A. and García-López, M.L. (2006). Occurrence Of Foodborne
Pathogenic Bacteria In Retail Prepackaged Portions Of Marine Fish In Spain. Journal of
Applied Microbiology, 100(3):527-536.
Jensen, G.B., Larsen, P., Jacobsen, B.L., Madsen, B., Smidt, L. and Andrup, L. (2002). Bacillus
thuringiensis in Fecal Samples from Greenhouse Workers after Exposure to B. thuringiensisBased Pesticides. Applied and Environmental Microbiology , 68(10): 4900-4905.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Jeanes, K. and Meijaard, E. (2000). Danau Sentarum's wildlife: part 1. Biodiversity Value And Global
Importance Of Danau Sentarum's Wildlife. Borneo Research Bulletin. Borneo Research
Council,
Inc.
Retrieved
July
5,
2012
from
HighBeam
Research:
http://www.highbeam.com/doc/1G1-93827521.html.
Jiang, X., Ni, Y., Jiang,Y., Yuan, F., Han, L., Li, M. and Liu, H. (2005). Outbreak of Infection Caused
by Enterobacter cloacae Producing the Novel VEB-3 Beta-Lactamase in China. Journal Of
Clinical Microbiology, 43(2): 826-831.
Liew, K.B. and Lepesteur, M. (2006). Performance of the rural health improvement scheme in
reducing the incidence of waterborne diseases in rural Sarawak, Malaysia. The Royal Society
of Tropical Medicine and Hygiene, 100, 949—955.
Matsheka, M.I., Lastovica, A.J., Zappe, H. and Elisha, B.G. (2006). The Use Of (GTG) 5
Oligonucleotide As An RAPD Primer To Type Campylobacter Concisus. Letters In Applied
Microbiology, 42(6): 600-605.
Mhango, M., Mpuchane, S.F. and Gashe, B.A. (2010). Incidence Of Indicator Organisms,
Opportunistic And Pathogenic Bacteria In Fish. African Journal of Food Agricluture Nutrition
and Development, 10(10): 4204-4218.
Queipo, M.I.O., Colmenero, J.D.D., Macias, M., Bravo, M.J. and Morata, P.(2008). Clinical And
Vaccine Immunology, 15(2): 293-296.
Ronald, A. (2002). The Etiology Of Urinarytractinfection: Traditional And Emerging Pathogens. The
American Journal of Medicine, 113(1): 14-19.
Vivas, A., Barea, J.M. and Azco´n, R. (2005). Brevibacillus brevis Isolated from Cadmium- or ZincContaminated Soils Improves in Vitro Spore Germination and Growth of Glomus mosseae
under High Cd or Zn Concentrations. Microbial Ecology, 49(3): 416-424.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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MACROBENTHOS STUDY IN SEMATAN ESTUARY RIVER, SARAWAK.
Abang Azizil Fansuri Abang Abdullah*, Shabdin Mohd Long, Harold Tinggang Ngau
Department of Aquatic Science, Faculty of Resource Science and Technology, University Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak
*Email: fansuriaa13@gmail.com
Abstract
The aim of this study is to provide the preliminary data on the macrobenthos community structure at
Sematan River Estuary. A total of 6 sampling stations were established along the estuaries. The
physico-chemical parameters of the water (temperature, turbidity, salinity, conductivity, dissolve
oxygen and pH) were measured in-situ. The macrobenthos samples were collected by using Ekman
Grab sampler and sieved using 500 µm mesh size sieve. The macrobenthos was fixed with 10% of
formalin in the field and preserved with 70% ethanol in laboratory. Results showed that a total of 7
taxa (Bivalvia, Gastropoda, Polychaeta, Oligochaeta, Diptera, Nematomorpha, and Crustacea) were
recorded. The density of macrobenthos was influenced by the river water temperature.
Keywords: Macrobenthos, community structure, estuary, Sarawak
1. INTRODUCTION
Macrobenthos are animals without backbone which can be classified as organisms that can be found
at the surface and inside the sediment with the size greater than 500µm (Govindan, 2002). These
benthos species can live in sediment, on rock, logs, sediment debris and aquatic plants (Eh Rak et al.,
2011). The benthos taxa are including crustacean, molluscs, aquatic worms and insects. Benthos
community such as meio-macrobenthos usually used to indicate the quality of water in the river and
sea (Eh Rak et al., 2011).
Moreover, the abundance of benthic fauna can be used as biological parameter which indicate overall
aquatic productivity of the bottom sediments (Ibrahim et al., 2006). Giere (1993), suggested that
benthos fauna might be used as integrated indicator for water quality within an area. Benthic macrobenthos is good indicator for water shed health (Dinakaran & Anbalagan, 2007; Thompson, 2005;
Davis et al., 2003). . This is due to their short life cycle history, easy to collect and have different
tolerance depends on the amount and types of pollution/habitat alteration.
Benthos community will have considerable response to the small changes in the environment and can
be used to measure degrees of pollution (Fernando, 1981; Coull, 1973). Biological monitoring
techniques have been widely reported using the macrobenthic community as tools in detecting the
benthic environment pollution (Azrina et al., 2005).
Therefore, a benthic study could be used as baseline information to evaluate the existing demersal
stock and serve as a baseline study for future investigation on environmental change in certain area.
Regarding of lack data related to macrobenthos at Sematan River estuary, a preliminary study was
carried out in order to establish a database and guideline for the area.
2. MATERIAL AND METHODS
2.1 Sampling sites
This study was conducted at Sematan River estuary Sematan, Sarawak. The conditions of water in
Sematan River is turbid due to the high suspended solids occur in the water column. The pollution
sources might come from the Sematan area which was dominated by the domestic waste effluents
produced by the resident along the river. The Sematan River estuary vegetation is dominated with few
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
plants such as Rhizophora spp., Bruguiera sp. and Nypa fruticans. Sematan River estuary is
influenced twice a day by tides.
Table 1. The GPS reading, weather condition and habitat description of the sampling sites.
Station
1
GPS Reading
N 01º47.51λ’
E 10λº47.680’
Weather Condition
Sunny
2
N 01º47.210’
E 10λº4.476’
N 01º47.046’
E 10λº46.λ2λ’
N 01º47.502’
E 10λº46.552’
Sunny and Rain
5
N 01º47.22λ’
E 109º 46.241’
Sunny with clouds
6
N 01º47.070’
E 10λº45.771’
Sunny
3
4
Sunny
Sunny
Habitat Description
Type of sediment: sandy (dominant) and
muddy.
mangrove dominated by Rhizophora spp.
Type of sediment: muddy.
mangrove dominated by Rhizophora spp.
Type of sediment: muddy.
mangrove dominated by Rhizophora spp.
Type of sediment: muddy.
mangrove dominated by Rhizophora spp. and
Nypa fruticans
Type of sediment: muddy.
mangrove dominated by Nypa fruticans and
Bruguiera sp.
Type of sediment: muddy.
mangrove dominated by Nypa fruticans
2.2 Field sampling
th
The sampling was carried out on the 19 February 2012. A total of 6 sampling stations were
performed from river mouth up to the upper river and the GPS reading of stations were recorded
(Table 1). The physico-chemical parameters of the water such as pH, salinity, temperature, turbidity,
dissolve oxygen and conductivity were measured (Table 2). The sediment sample for macrobenthos
was collected using Van Veen Grab Sampler (15.5 cm width). Triplicate of sediment samples were
sieved using 0.5 mm sieve. All the retained materials on the sieves were collected and fixed in the
labelled specimen bottles using 10% buffered formalin.
2.3 Laboratory analysis
All samples were sorted in the laboratory and stained with Rose Bengal before preserved with 70% of
ethanol. The Organisms were identified to the major taxa. The identifications steps were done using a
Higgins and Thiel (1988) and Pechenick (2005).
3. RESULTS AND DISCUSSION
There were six parameters recorded in this study namely pH, salinity, temperature, turbidity, dissolved
oxygen (DO) and conductivity (Table 2). Three replicates of reading were collected for each station to
reduce the errors. As shown in table 2, pH value varied within a narrow pH range of mean 7.09 – 8.09
units. The highest value was recorded at station 1 with 8.09 units. This was due to the location of the
station at the river mouth which was influenced by the presence of carbonate during tides (Lau, 2011).
The salinity was higher at station 1 with 28.67 PSU but was significant decreased at station 6. Station
6 was located at the upper stream and the input of freshwater from land influenced the low salinity at
the station. The temperature was higher at station 2 with 29.5ºC. The turbidity was higher at station 1
with 56.41 NTU. Not much differences of temperature been recorded in each stations but the higher
value was at station 2 (Table 2). The DO values ranged from 10.03 to 11.33 mg/l. The DO values in
this study were not much difference between stations but it can be consider as high. The conductivity
value was higher at station 1 and 2 but lowest at station 6.
Results showed (Table 3) that seven major taxa of macrofauna (Bivalvia, Gastropoda, Polychaeta,
Oligocheata, Diptera, Nematomorpha and Crustacea) were recorded at Sematan River estuary. The
most common taxa (six out of seven taxa) were recorde at station 2 and 4. At station 6 only three taxa
was found which can be concluded that station 6 was the least taxa recorded in the Sematan River
estuary.
Table 2: Physico-chemical parameters
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Station/
Parameter
pH
Salinity
(PSU)
Temperature
(ºC)
Turbidity
(NTU)
Dissolve
Oxygen
(mg/l)
Conductivity
(µm/s)
Station 1
8.09±0.02
28.67±0.58
28.9±0.00
56.41±7.25
10.03±0.06
48.42±0.05
Station 2
7.96±0.01
26±0.00
29.5±0.00
34.48±1.03
11.1±0.1
44.33±0.14
Station 3
7.09±0.01
17±0.00
28.4±0.00
18.1±0.28
11.33±0.32
17.04±0.02
Station 4
7.27±0.00
13±0.00
27.83±0.06
19.39±0.18
10.8±0.1
19.47±0.00
Station 5
7.45±0.02
6±0.00
27.3±0.00
21.64±0.24
11.27±0.06
7.49±0.00
Station 6
7.28±0.02
4.33±0.58
27.5±0.00
28.46±0.52
10.77±0.15
3.55±0.02
The common taxa that was found at all the station was Oligochaeta. This shows that Oligochaeta was
the dominant species at Sematan River estuary. Meanwhile Gastropoda and Nematomorpha were
found at two stations only. This show that both taxa were the least groups found but in term of density
the Nematomorpha was higher than gastropoda. The total density was recorded highest at station 4
2
2
(1125 ind/m ). In other hand, the lowest density was recorded at station 6 was (41.67 ind/m ).
2
Table 3 Taxonomic list and density (number of individuals /m ) of macrofauna in the Sematan River
estuary.
Taxa/Station
St.1
St.2
St.3
St.4
St.5
St.6
Gastropoda
Bivalvia
Polychaeta
Oligochaeta
Diptera
Crustacea
Nematomorpha
Total
27.78
41.67
333.33
13.89
416.67
13.89
194.44
263.89
138.89
13.89
13.89
638.89
222.22
138.89
97.22
138.89
597.22
27.78
41.67
388.89
208.33
263.89
194.44
1125.00
13.89
41.67
208.33
180.56
41.67
486.11
13.89
13.89
13.89
41.67
Pearson correlation coefficient of the physico-chemical parameters of the water with density and
number of taxa showed that the density of the macrobenthos was influenced mostly by temperature.
According to Jayaraj et al. (2006), the temperature was found as one of the factors that influence the
density of benthos in tropical countries. Besides that, the turbidity, salinity and conductivity also show
significance values that prove these parameters also influenced the density after the temperature.
Table 4 The Pearson correlation between physico-chemical parameters of the water with density and
number of taxa
Parameters
pH
Salinity
Temperature
Turbidity
DO
Conductivity
Density
-0.104
0.231
0.661*
-0.267
0.165
0.213
No. Taxa
0.218
0.268
0.662*
-0.099
0.275
0.334
*Correlation at (p>0.05)
4. CONCLUSION
There were seven taxa was recorded at Sematan River estuary. The density of macrobenthos was
strongly influenced by water temperature. The data gathere will become the basic of future research
on macrobenthos in the Sematan River estuary.
Acknowledgement
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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We would like to thanks Universiti Malaysia Sarawak for providing facilities, administrative and logistic
supports. Besides that, we would also like to thanks Mr. Zaidi Ibrahim and Mr Mohd Nor Azlan Bujang
Belly for their help during the field sampling. Finally we would like to thank Ministry of Higher
Education for supporting this project through Fundamental Research Grant Scheme
(FRGS/07(03)/786/2010(67).
References
Azrina, M. Z.; Yap, C. K.; Ismail, R. A.; Ismail, A., & Tan, S. G., 2005. Anthropogenic impacts on the
distribution and biodiversity of benthic marcovertebrates and water quality of the Langat River,
Pensular
Malaysia.
Department
of
biology,
faculty
of
science.
UPM.
doi:10.1016/j.econv.2005.04.003
Coull, B. C., 1973. Estuarine meiofauna a review, tropic relationship and microbial ecology, Stevenson
L. H. and Colwell (Eds.). University of South Carolina Press, Columbia, pp449-511.
Davis, S.; Gollady, S. W.; Velllidis, G. & Pringle, C. M., 2003. Macroinvertebrate Biomonitoring in
Intermittent Coastal Plain Streams Impacted by Animal Agriculture. Journal of Environmental
Quality, 32;1036-1043.
Dinakaran, S., & Anabalagan, S., 2007. Anthropogenic impacts on aquatic insects in six streams of
south western ghats, Journal of Insect Science. 7(37), 1-9.
Eh Rak, A.; Said, I., & Mohamed, M., 2011. Effects of land use on benthic macroinvertebrate
assemblages at the three rivers in Endau catchment area, Kluang, Johor, Malaysia. Journal of
Applied Sciences in Environmental Sanitation, 6 (2): 97-103.
Govindan, K., 2002. Marine benthos - a future perspective. Proc. The National Seminar on Creeks,
Estuaries and Mangroves – Pollution and Conservation : 28 -30.
Higgins, R. P. & Thiel, H., 1988. Introduction to the study of meiofauna. Smithsonian Institution Press.
Washington, D.C. London. Pg 238-464
Ibrahim, S.; Wan, M. R. W. H.; Zaleha, K.; Zuliatini, M. J.; Zakaria, M. Z. & Sukree, H., 2006. Seasonal
Abundance of Benthic Communities in Coral Areas of Karah Island, Terengganu, Malaysia.
Turkish Journal of Fisheries and Aquatic Sciences. 6: 129-136.
Lau, S., 2011. Water in the environment: Tainted Life Source Hungers for cures. Universiti Malaysia
Sarawak. Sarawak, Malaysia. Pg 4.
Jayaraj, K. A.; Jayalakshmi, K. V. & Saraladevi, K. 2006. Influence of environmental properties on
macrobenthos in the northwest Indian shelf. Environment Monitoring Assessment. 127: 459-475
th
Pechenik, J. A., 2005. Biology of the Inverterbrates (5 Ed.). Mc Graw-Hill Higher Education United
States of America. Page 207-444
Thompson, J., 2005. Using Benthic Macroinvertebrates and GIS to Assess and Manage Watershed
Health of the Colorado River Basin. City of Austin, Texas.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
WATER QUALITY AND HEAVY METALS CONCENTRATION OF BATANG LUPAR RIVER
ESTUARY, SARAWAK
*Nur Atiqah Mohamad Yusoff and Shabdin Mohd Long
1
Department of Aquatic Science, Faculty of Resources Science and Technology, Universiti Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak.
*Email: atiqnuratiqah@yahoo.com
Abstract
Water quality and heavy metal (Cd, Cr, Cu, Zn, Fe and Pb) studies in water were conducted at Batang
Lupar River estuary on May 2011. This estuary was located in the south-western part of Sarawak. In
order to determine the status of water quality in Batang Lupar River estuary, parameters such as
temperature, pH, dissolved oxygen, turbidity, salinity, Biochemical Oxygen Demand (BOD 5), total
suspended solid (TSS), Chemical Oxygen Demand (COD) and ammonia-nitrogen concentrations were
measured. Water sample was also collected to measure the concentration of selected heavy metals
in Batang Lupar River estuary. The value of selected water quality in Batang Lupar River estuary
were ranged from 30.10±0.00 ºC to 30.60±0.00 °C (temperature), 7.36±0.01 to 7.53±0.00 (pH),
5.77±0.02 to 6.68±0.02 mg/L (dissolved oxygen), 50.55±0.07 to 448.00±12.73 NTU (turbidity),
10.00±00 to 15.00±0.00 PSU (salinity), 2.05±0.30 to 2.48±0.73 mg/L (BOD5), 216.65.42±20.15 to
323.44±50.588.16 mg/L (total suspended solid), 23.00±4.24 to 52.00±8.49 mg/L (Chemical Oxygen
Demand) and 0.110±0.014 to 0.170±0.014 mg/L (ammonia-nitrogen). The heavy metals concentration
were ranged from 0.031±0.004 to 0.043±0.002 mg/L (Cr), 0.012±0.001 to 0.017±0.001 mg/L (Cd),
0.019±0.001 to 0.028±0.001 mg/L (Cu), 0.223±0.001 to 0.310±0.017 mg/L (Zn), 0.038±0.001 to
0.294±0.001 mg/L (Fe) and 0.046±0.001 to 0.076±0.001 mg/L (Pb). The water quality of Batang
Lupar was slightly polluted and concentrations of heavy metal (Zn, Pb and Cr) were within the safe
limit of National Water Quality Standard for Malaysian river (NWQS) threshold level except Cu and Cd.
Keywords: Water quality, heavy metals, Batang Lupar River.
1. INTRODUCTION
Water and life are closely associated with one another. Without water, many organisms including
human beings will not exist. River is usually used by human for transportation, drinking, recreation
activities and food sources. Nowadays peoples are complaining about the situations where the quality
of water is not good enough for normal usage. Many rivers are polluted by many sources such as from
agriculture, developmental of rural area, oil spill and drainage (Azrina et al., 2005). According to the
Department of Environment (2010), there are 80 rivers that were clean, 59 rivers slightly polluted and
7 rivers were polluted. Water can be classified as clean, slightly polluted, or polluted depends on
sources of the water (Department of Environment, 2007). Most people classified the quality of water
by the appearance of the water such as the water color, odor and taste. It is difficult to mention and
quantify the good quality of water by looking at their appearance. Therefore, water quality analysis is
used to describe biological, chemical, and physical characteristic of water (Boyd and Tucker, 1998).
According to Jonnalagadda and Mhere (2000), many rivers at developing countries are exposed to
pollution due to anthropogenic activities such as industrial activities and sewage discharge. Other than
that, water pollution can be resulted from mining and agriculture activities discharged into water bodies
that have lead to deterioration in quality of water bodies and environment (Kambole, 2003). Batang
Lupar River is located at south-western part of Sarawak and one of the well-known rivers in Sarawak.
Batang Lupar River was mostly inhabited by rural villagers due to importance of the river itself to
support their life. Some of the villagers were padi planters because rice grows very well along the
river. They also relied on rubber, which was now the main focus due to its good price. It was reported
that continuous land clearing has exposed the land and drainage alteration may cause further erosion
and sediment run-off into nearby catchment during heavy downfall (Rajali and Gambang, 2000). Due
to the potential environmental concerns associated with the Batang Lupar River, this study aimed to
assess its water quality.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2. MATERIAL AND METHODS
Batang δupar estuary was located at N 01°41.417’ E 10λ° 54.8λ0’ in the state of Sarawak. Five
sampling stations were chosen in the Batang Lupar River estuary (Figure 1). Water samples for
Ammonia-nitrogen, Biological Oxygen Demand (BOD5), Total Suspended Solid (TSS), Chemical
Oxygen Demand (COD) and heavy metals analyses were taken using Van Dorn bottle water sampler.
Physico-chemical parameters of the water such as pH, salinity, temperatures, turbidity, and dissolved
oxygen (DO) were measured in-situ by using Eutech Instrument portable water parameter. Ammonianitrogen, BOD5, TSS, COD and heavy metals were analyzed in the laboratory using standard method
(APHA, 1998). For heavy metals analysis water samples were filtered through 0.45 µm membrane
filter and were acidified with 10 % nitric acid (HNO 3) until pH less than 2 (USEPA, 2001). Then, heavy
metals were analyzed using flame Atomic Absorption Spectrophotometer (AAS). One-way Analysis of
variance (ANOVA) statistical test was performed to check the significant of P value. Computation of
water quality index was determined based on the formula derived by Department of Environment. Only
six (6) parameters were used to calculate the Water Quality Index (WQI). The parameters used to
calculate WQI were DO, pH, TSS, COD, BOD5 and ammonia-nitrogen.
Station
5
Station
4
Station
3
Station
2
Station
1
Figure 1 Location of sampling station along Batang Lupar River estuary. (Source: Google Map)
3. RESULTS AND DISCUSSION
The water quality parameters of five stations in estuary of Batang Lupar estuary were presented in
Table 1. Temperature ranged from 30.10±0.00 °C to 30.60±0.00 °C. The lowest temperature value
was recorded at station 1 while the highest values were recorded at station 4 and 5. Overall, water
temperature was a little bit high as compared to other estuary. This possibly due to the hot wheather
during sampling time where the temperature was measured in the afternoon. pH values ranged from
7.36±0.01 to 7.53±0.00. The minimum value was recorded at Station 5 and the maximum was at
station 1. The pH values at station 2, 4 and 5 were significantly diffrence among each other (p<0.05).
Station 1 and 3 showed no significant difference (p=0.260). pH of the Batang Lupar estuary was
classified into class IIB based on NWQS.
Dissolved oxygen (DO) ranged from 5.77±0.02 mg/L at station 4 to 6.68±0.02 mg/L at station 1.There
was no differences of DO between station 2 and 4 (p=0.530). However, station 1, 3 and 5 showed
significant differences among each other (p<0.05). Dissolved oxygen of above 5 mg/L was considered
good (Aweng et al., 2011). Based on NWQS, the dissolved oxygen value in Batang Lupar estuary was
classified into class IIB.
The highest and lowest values of turbidity were recorded at station 1 (448.00±12.73 NTU) and station
3 (50.55±0.07 NTU) respectively. The turbidity was significantly difference between station 1,4 and 5
(p≤0.001) while there was no significant difference between station 2 and 3 (p=0.λλ8). Based on
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NWQS, the turbidity at Batang Lupar estuary was classified in Class IIB and III. The highest turbidity in
this river was resulted from mining activities from the catchment area which load the suspended solid
into water bodies (Ashraf et al., 2011).
Salinity was ranged from 10.00±0.00 PSU at station 1 to 15.00±0.00 PSU at station 3, 4 and 5. The
salinity values was quite low as compared to other estuary in Sarawak( Gandaseca et al., 2011).This
was due to the influenced of freshwater input due to heavy rainfall at the upper river and the
parameter was measured during low tide.
Total suspended solid (TSS) ranged from 216.65±20.15 mg/L at station 5 to 323.44±50.58 mg/L at
station 2. There was no significant difference among the stations (p>0.05). Based on NWQS, TSS at
Batang Lupar estuary was classified into Class III and exceeded the Class IIB limit. Agriculture
activites and land clearing of mangroves forest were considered as factor that contributes to the high
TSS in this river (Aweng et al., 2011). According to Natural Resources and Environmental Board
(NREB) Sarawak (2006), land clearing activities such as deforestation for agricultural activities and
urban development may increase the sediment input into the water bodies from the water runoff.
Biochemical oxygen demand (BOD5) in Batang Lupar estuary was ranged from 2.05±0.30 mg/L at
station 4 to 2.48±0.73 mg/L at station 3. There was no significant difference of salinity among stations
in this river (p>0.05). The highest BOD5 was recorded at station 3. According to Ling et al., (2010) the
decaying of organic matter in that river will result in lower DO and higher BOD. Based on NWQS,
BOD5 in all stations of this river were classified into Class IIB.
Chemical oxygen demand (COD) ranged from 23.00±4.24 mg/L to 52.00±8.49 mg/L. The highest
value was recorded at station 1 and the lowest value was found at station 5. COD at Station 1 and 2
showed significantly difference with station 5 (p=0.026 and p=0.041). According to NWQS the COD in
all stations of Batang Lupar estuary were classified in Class IIA and Class IV. The high level of COD in
the Batang Lupar estuary was due to the discharge of organic matter and inorganic chemical from
agriculture and settlement areas located along the Batang Lupar River (Gandaseca et al., 2011).
The highest and lowest values of ammonia-nitrogen concentration in Batang Lupar estuary were
0.170±0.014 mg/L (station 4) and 0.110±0.014 mg/l (station 2) respectively. However, there was no
significant difference of ammonia-nitrogen between all stations in Batang Lupar estuary (P>0.05). The
values of ammonia-nitrogen in Batang Lupar estuary was slightly low as compared to other estuary
(Ling et al., 2010).The values fall in Class I and Class IIA under NWQS. The values of ammonianitrogen at all station in Batang Lupar estuary was below the recommended limit of 0.90 mg/L (DOE,
2007).
Table 1 Water quality parameters of Batang Lupar estuary
Parameters
St 1
St 2
St 3
St 4
St 5
Temp (ºC)
pH
DO(mg/L)
Turbidity(NTU)
Salinity (PSU)
TSS(mg/L)
BOD5 (mg/L)
COD (mg/L)
30.10±0.00
a
7.53±0.00
a
6.68±0.02
a
448.00±12.73
10.00±0.00
a
289.75±103.0
a
2.38±0.01
a
23.00±4.24
30.20±0.00
b
7.49±0.01
b
5.82± 0.01
b
52.20±2.26
12.00±0.00
b
323.44±50.58
a
2.13±0.30
a
26.00±2.83
30.30±0.00
a
7.52±0.01
c
6.25±0.06
b
50.55±0.07
15.00±0.00
b
296.05±89.4
a
2.48±0.73
ab
31.00±8.49
30.60±0.00
c
7.39±0.01
b
5.77 ± 0.02
c
115.00±4.24
15.00±0.00
c
291.20±2.26
a
2.05±0.30
ab
39.00±4.24
30.60±0.00
d
7.36±0.01
d
6.01±0.02
d
201.5±2.12
15.00±0.00
d
216.65±20.15
a
2.24±0.17
b
52.00±8.49
NH4-N (mg/L)
0.140±0.014
a
0.170±0.014
a
0.120±0.057
a
0.110±0.014
a
0.160±0.028
a
Note: DO; dissolved oxygen, TSS; Total Suspended Solid, BOD5; Biochemical Oxygen Demand COD;
Chemical Oxygen Demand,*Mean on the same row with the same superscript is not significantly
different at 5% significant level
Table 2 shows the heavy metals concentrations in water at five stations of Batang Lupar estuary. The
decreasing order of heavy metals concentration in water of Batang Lupar was Zn>Fe>Pb>Cr>Cu>Cd.
The lowest concentration of heavy metals in Batang Lupar estuary was cadmium.. The values range
from 0.012±0.003 mg/L (station 1) to 0.017±0.002 mg/L (station 5). However, the value of cadmium in
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the river water was exceeded the recommended safe limit of NWQS (0.01 mg/L). Zinc concentration
was ranged from 0.223±0.002 mg/L (station 2) to 0.310±0.004 mg/L (station 5) while concentration of
iron was ranged from 0.038±0.008 mg/L (station 1) to 0.294±0.080 mg/L (station 5). Zinc was the
highest elements found in Batang Lupar estuary. The value of zinc (5 mg/L) and iron (0.3 mg/L) in this
river were below the recommended safe limit of NWQS. The concentration of chromium was ranged
from 0.031±0.002 mg/L (station 5) to 0.043±0.002 mg/L (station 2). The concentration of copper in
Batang Lupar estuary was ranged from 0.019±0.007 mg/L (station 2) to 0.028±0.012 mg/L (station 3).
Finally, concentration of lead (Pb) was ranged from 0.046±0.007 mg/L (station 1) to 0.076±0.002 mg/L
(station 5). In general, there was significant differences (p<0.05) of chromium, zinc, iron and lead
concentrations in all five stations of Batang Lupar estuary. However, there was no significant
differences (p>0.05) of cadmium and copper concentrations in all stations of Batang Lupar estuary.
Human activities such as fishing, agriculture,and residential area were contributed to the sources of
heavy metals at this study area (Ahmad et al., 2009). The excess of heavy metal load of estuary water
can be attributed to the geology of the river bed, tidal effects and catchment area (Kar et al., 2008).
The comparison of heavy metals with the standard of NWQS showed that all heavy metals
concentration was below the recommended safe limit except for cadmium.
Table 2 Heavy metal concentration in water (mean ± SD) at different stations
Metals (mg/L)
Stations
Cd
Cr
Cu
Zn
Fe
Pb
1
0.012±0.003
0.039±0.001
0.026±0.007
0.228±0.007
0.038±0.008
0.046±0.007
2
0.014±0.006
0.043±0.002
0.019±0.007
0.223±0.002
0.057±0.002
0.049±0.002
3
0.013±0.004
0.033±0.005
0.028±0.012
0.226±0.005
0.108±0.001
0.058±0.001
4
0.016±0.001
0.038±0.002
0.023±0.021
0.247±0.004
0.039±0.004
0.064±0.012
5
0.017±0.002
0.031±0.002
0.022±0.008
0.310±0.004
0.294±0.080
0.076±0.002
Mean
0.014±0.002
0.037±0.005
0.024±0.004
0.247±0.037
0.107±0.108
0.059±0.012
The Water Quality Index (WQI) was calculated based on the Department of Environment - water
quality index (DOE-WQI) formula. The index was shown in Table 3. Batang Lupar estuary was clean
based on the range of BOD. If the index was based on the range of ammonia-nitrogen and suspended
solid, Batang Lupar River was classified as slightly polluted and polluted. The index of water quality of
Batang Lupar estuary was 78 and classified as slightly polluted which in under class II. As compared
to εalaysian’s guidelines, the water quality index for class II represented slightly polluted to polluted
waterand suitable for water supply but conventional treatment are required.
Table 3 DOE classification on water quality parameter based on water quality index.
Sub Index and Water Quality Index
Biological oxygen demand (BOD)
Ammonia- nitrogen (NH3-N)
Suspended Solids (SS)
Water Quality Index (WQI)
Batang Lupar
estuary
91
86
41
78
Index
Range
91 - 100
71 - 91
0-69
60-80
Classification
Clean
Slightly polluted
Polluted
Slightly polluted
4. CONCLUSION
Human activities such as fishing, mining, , agriculture activity and settlement area were contributed to
the water pollution in Batang Lupar estuary. The water quality parameters were compared to NWQS
and all parameters fall in the range of Class I to III. The water quality index of Batang Lupar estuary
was 78 which fall into slightly polluted site. The most abundant element in water was zinc and the least
element found in wáter was Cd. Zn, Cu, Fe, Pb and Cr were within the safe limit except for Cd where
its concentration was exceeded the safe limit of NWQS. Human activities was suggested as one of the
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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sources of heavy metals in water. The increase of heavy metals level in the wáter will give the bad
impacts on the aquatic ecosystem due to its toxicity.
Acknowledgement
The authors are gratefully to Faculty of Resources Science and Technology, Universiti Malaysia
Sarawak for supporting this work and also thankful to laboratory assistant in Department of Aquatic
Science Departmentfor their assistant during sampling. This study was funded by the Malaysian
government through FRGS grant (FRGS/07(03)/786/2010(67)).
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Estuaries Coastal and Shelf Science 71: 218-231.
Department of Environment. 2007. Malaysia Environmental Quality Report 2007. Department of
Environment, Ministry of Natural Resources and Environment Malaysia.
Gandaseca, S., Noraini, R., Johin, N. and Arianto, C.I. 2011. Status of water quality based on the
physico-chemical assessment on river water at Wildlife Sanctuary Sibuti Mangrove Forest, Miri
Sarawak. American Journal of Environmental Sciences 7(3), 269-275.
Jonnalagadda, S.B., and Mhere, G. 2000. Water quality of the Odzi River in the Eastern Highlands of
Zimbabwe. Journal of Environmental Health, 35, 2371-2376.
Kambole, M.S. 2003. Managing the water quality of the Kafue River: Physics and Chemistry of the
Earth, 28, 1105-1109.
Kar, D., Sur, P., Mandal, S.K., Saha, T. and Kole, R.K. 2008. Assessment of heavy metal pollution in
surface water. International Journal Environmental Science Technology 5(1): 119-124.
Ling, T.Y., Rohene, S., Kho, C.P. and Lee, N. 2010. Organic matter, nutrients and trace metals of
Serin River. World Applied Sciences Journal 8(4), 496-502.
NREB. 2006. Environmental Quality Report 2006. Natural Resources and Environment Board,
Sarawak, Malaysia.
Rajali, H., and Gambang, A.C. 2000. Ecological Balance of Batang Lupar estuary: Potential Impacts
and Mitigations. Fisheries Research Institute Branch.
USEPA. 2001. Trace elements in Water, Solids and Biosolids by stabilized Temperature Graphite
Furnace Atomic Absorption apectrometry. U.S Environmental Protection Office, Office of
Science and Technology, Washington, DC. EPA-821-R-01-011.
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PRELIMINARY STUDY ON IDENTIFICATION SPECIES OF BAGWORM ON Michelia champaca
(Magnoliaceae) TREES AT FACULTY OF SCIENCE AND TECHNOLOGY, UNIMAS
Sidi, Mogeret* and Alyani, B.
Department of Plant Science and Enviromental Ecology, Faculty of Resource Science & Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak.
*Email: smogeret@frst.unimas.my
Abstract
Study was conducted to identify species of bagworms that attacked Michelia champaca around
Faculty of Resource Science and Management. Pupation bag of bagworms were collected from
infested trees were then identified by using Key to Common Species of Physicidae Infesting Oil Palm
(Pupation Bag). Result showed pupation bag of bagworm from study site was medium in size, smooth
surface and without large attached leaf fragment as showed by Metisa plana. Therefore the bagworm
was identified belonging to M. plana.
Keywords: Michelia champaca, bagworm, Metisa plana, identify
1. INTRODUCTION
Bagworm is known as leaf eating caterpillars and is serious pest on trees and shrubs. The caterpillars
characterized by the possession of bag, which they built out of tough silk consisted of overlapping
dried plants such as leaves and small twigs from their host plant (Barlow, 1982). In Malaysia,
bagworms are common on many orchard, landscape and ornamental trees (Ahmad and Ho, 1980).
Bagworms attack may not visible at the beginning because they are inconspicuous when young but
the results from their attacks can be seen clearly (Caron, 2004). Newly hatched larvae will build a case
on tree using silk that their produce and twigs or dead leaf and at the same time feed on leaves. Ability
to feed at larvae stage is at high rate because they need to prepare to enter pupae stage. Leaves that
bagworms eat will turn to yellow and after that dead before falling down. In landscapes area,
defoliation caused by bagworms could reduce the esthetic value of arborvitae and landscapes (Raupp
et al., 1988).
The example of serious attack of bagworms can be seen at the surroundings of Faculty of Resource
Science and Technology, Universiti Malaysia Sarawak. Plants that are dominant at the surroundings
of this faculty are Michelia champaca trees and were planted as ornamental plants. These trees
undergo a serious attack of bagworms in March to May every year since 2009 and its appearance as
bad as a dead tree. Their leaves are falling down and most of their branch festooned with bagworms
cases However, the trees survive by producing new buds and new leaves. This situation becomes
worse from time to time because no preventive measures are taken to control them.
Although this pest did not cause the tree to die but it create unhealthy landscape environment
especially to the tree that were planted as ornamental plants. The objective of this study is to identify
the bagworm species that attack M. champaca trees at FRST Universiti Malaysia Sarawak.
2. MATERIALS AND METHODS
2.1 Samples collection
Pupation bag of bagworms contained larvae were collected from infested Michelia champaca trees
within the Faculty of Resource Science and Technology of Universiti Malaysia Sarawak starting from
early of October 2011 until end of January 2012.
2.2 Pupae Identification
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Pupation bag of bagworms were compared with Key to Common Species of Psychidae Infesting Oil
Palm (Pupation Bag) by Norman et al. (1994) as showed in Figure 1.
1. Bag small, 7-12 mm long, suspended on long silky thread ……...............................................2
Bag medium to large, 12-33 mm long, suspended by short silk attachment that is sometimes
hook-shaped …………………………………………………………………………………………...3
2. Bag
10-12
mm
long,
usually
appearing
ragged
or
spiky
….………………………………………………………………………………Brachycyttarus griseus
Bag 7-9 mm long, usually appearing smooth and rounded
……………………………………………………………………............................Pteroma pendula
3. Medium sized bag, 12-13 mm long, smooth surfaced, without large attached leaf fragments,
with
hook-shaped
attachment,
swollen
at
attached
end
……………………………………………………………………………….....................Metisa plana
Large bag, 27-33 mm long, rough-surfaced, with large attached fragments of host plant leaf
……………..………………………………………………………………………...Mahasena
corbetti
Figure 1 Key to Common Species of Psychidae Infesting Oil Palm (Pupation Bag) (Norman et al.,
1994).
3. RESULTS AND DISCUSSIONS
Species identification should be done by adult identifications. Identification by using the bagworms
adults will involves the examination and comparison of various structures such as wing venation, scale
structure and genital morphology of adult males (Norman et al., 1994). These structures are the most
readily accessible features that will clearly differentiated the bagworms species from one another.
However, no adult were caught by using light trap and insect net. As an alternative, bagworms species
also can be identified by using bagworms shape as practiced by Norman et al. (1994). As showed by
Figure 2, each bagworm species have different kind of pupation bag.
(a)
(b)
(c)
Figure 2 Different kind of pupation bag for different species of bagworm (a) Bag of Metisa plana
medium sized bag, 12-13 mm long, smooth surfaced, without large attached leaf fragments, with
hook-shaped attachment, swollen at attached end, (b) Bag of Mahasena corbetti is large around 27-33
mm long, rough-surfaced, with large attached fragments of host plant, (c) Bag of Pteroma pendula is
7-9 mm long, usually appearing smooth and rounded
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Figure 3 Pupation bag of bagworm that attacked M. champaca trees (left) compared to Metisa plana
Pupation bag of bagworm from study site (Figure 3) showed a tendency to have the same shape as
Metisa plana species that was described by Norman et al. (1994). Based on the Key to Common
Species of Psychidae Infesting Oil Palm (Pupation Bag) stated by Norman et al. (1994), the Metisa
plana species have medium size of pupae which in range from 12 to 13 mm long, have smooth
surfaced, without large attached leaf fragments, with hook-shaped attachment and swollen at attached
end. These characteristics match well with the characteristics of the pupation bag that were taken from
the study site. The pupation bag taken from the study site have medium size of pupae, have smooth
surface and without large attached leaf fragment as showed by Figure 3. Therefore bagworm species
that attack M. champaca trees at FRST UNIMAS were identified belonging to Metisa plana species.
References
Ahmad, Y. and Ho, T.H. (1980). List of economic pests, host plants, parasites and predators in West
Malaysia (1920-1978).Ministry of Agriculture, Malaysia, Bulletin No. 153, p. 538.
Barlow, H.S. (1982). An Introduction to the Moths of South East Asia. Art Printing Works Sdn. Bhd.
Kuala Lumpur, p. 305.
Caron, D.M. (2004). Bagworms. University of Delaware, USA. Retrieved September 18 2011, from
http://ag.udel.edu/extension/horticulture/pdf/ent/ent-04.pdf
Norman, K., Robinson, G.S. and Basri, M.W. (1994). Common bagworm pests (Lepidoptera:
Psychidae) of oil palm, with notes on related South-East Asian species. Malayan Nature
Journal, 48: 93-12.
Raupp, M. J., Davidson, J. A., Koehler, C. S., Sadof, and Reichelderfer, K. (1988). Decision making
considerations for pests causing aesthetic damage. Bulletin of the Entomological Society of
America. 34: 27 – 32.
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CUTICULAR HYDROCARBON ANALYSIS OF SELECTED Bactrocera Species (DIPTERA;
TEPHRITIDAE) FROM SOUTHERN SARAWAK
Intan Roszaiyani Zainol Abidin, Rizoh Bosorang*, Siti Zuriani Ismail, Zaini Assim,
Siti Nurlydia Sazali and Sulaiman Hanapi
1
2
Department of Zoology, Department of Chemistry, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia
*Email: rizohbosorang@gmail.com
Abstract
The study was conducted to investigate the variation of hydrocarbon pattern in Tephritid fruit flies
using Gas Chromatography technique. Quantitative and qualitative analyses were performed on nalkanes extracted from the cuticular lipids of three Tephritid fruit flies species, namely, Bactrocera
dorsalis, B. umbrosa and B. cucurbitae. Homologous series ranging from n-nonacosane to ntetratriacontane (C29 to C35) make up the n-alkane fraction. Odd-numbered alkanes are the
predominant compounds in all species. The major n-alkane found in those species is n-hentriacontane
(C31), n-dotriacontane (C32), and n-tritriacontane (C33). There were statistical significant differences in
the pattern and compositions of n-alkane found in this three species. This finding suggests that the
cuticular hydrocarbon analysis is reliable as an alternative marker for identification of Tephritids,
instead of morphological and molecular approaches.
Keywords: fruit flies, gas chromatography, cuticular hydrocarbon.
1. INTRODUCTION
Cuticular hydrocarbon plays important roles in species identification. Examination of the cuticular lipids
of some species in several super families had earlier revealed that there are significant differences in
the composition of the hydrocarbon (Jacob, 1979). Previous study had applied cuticular hydrocarbon
analysis for the recognition of species complex (Carlson and Service, 1979). Chua (1998) had
reported that there may have been taxonomic confusion between Dacus spp. and Bactrocera spp. and
that many of the 500 recorded species of Dacus may, in fact be Bactrocera. According to Clarke et al,
(2004) the B. dorsalis (Diptera: Tephritidae: Dacinae) complex was originally defined to contain 16
species closely related to B. dorsalis. Therefore, using morphological to identified species is very
difficult especially with the closely related species or species complex. This study aims to investigate
the potential of cuticular hydrocarbons as marker to identified fruit flies species by examines the
variation of hydrocarbon pattern in Tephritid fruit flies using gas chromatography techniques.
2. MATERIALS AND METHOD
The adult fruit flies were collected from selected location at Samarahan Division. Most of them were
collected from pheromone trapping. Otherwise the larvae collected from infested fruits were reared to
adult. The specimens were identified using identification key by White and Elson-Harris (1992), and
O
then frozen at -20 C until extraction of cuticular lipid performed. Three replicates were carried out in
each species for each extraction. Adult fruit flies were immersed in 10-20 ml of hexane for 10 minutes
before the extract was spiked with 50 µl of 50 ppm eicosane and then filtrated through a column
chromatography packed with silica gel. The filtrate was collected in 25 ml pear-shaped flask, then
evaporated to dryness under a stream of nitrogen and re-dissolved in 200 ml of hexane for Gas
Chromatographic (GC) analysis which performing with a Shimadzu GC-17A (GC-FID). The
compounds obtained were identified by comparing with the GC retention times and mass
fragmentation patterns of standard reference materials. The standard mixtures of n-alkanes ranging
from C9 to C34 were injected on GC-FID. Cluster analysis (CA) and Principal Component Analysis
(PCA) were done to estimate the importance of genetic similarity and dissimilarity of hydrocarbons
compositions. The Jaccard Index was used to calculate the quantitative and qualitative differences in
the cuticular chemicals between the individuals (Whitlow, 2003).
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3. RESULTS AND DISCUSSION
Quantitative and qualitative analyses were
performed on n-alkanes extracted from the
cuticular of three species of Tephritid fruit
flies; Bactrocera dorsalis, B. umbrosa, and
B. cucurbitae. Generally, genus Bactrocera
show homologous series of cuticular
hydrocarbon ranging from n-nonacosane to
n-pentatriacontane (C29 to C35), make up the
n-alkane fraction. The finding was supported
that n-alkanes is major components detected
in Diptera and bees (Carlson and Bolten,
1984). Odd-numbered alkanes consist of nhentriacontane (C31), n-tritriacontane (C33)
and n-pentatriacontane (C35) are the
predominant
compounds
in
genus
Bactrocera, comprise 60-66 % in all species
examined.
However, there are slightly different in the
pattern of this three species of Bactrocera.
B. umbrosa is a little has shorter range of the
compound from n-triacontane (C30) to the ntetratriacontane (C34) compared to other
species. While, B. cucurbitae produce wider
compound then others which is ranging from
n-nonacosane (C29) to n-pentatriacontane
(C35). There were statistical significant
differences in the pattern and compositions
Figure 1 Chromatograms of Bactrocera species: a)
B. dorsalis; b) B. umbrosa; c) B. cucurbitae
n-alkane found in this three species.
Analysis of variance indicated that there is
significant different between proportion of nalkane among the three species where F=
9.193, p value= 0.002 is less than 0.05. All
the Bactrocera spp. had different chemicals
of chain lengths between C29 and C35, thus
suggests that each species had the different
enzyme systems controlling chain length.
Cluster Analysis had revealed that B.
cucurbitae and B. dorsalis were closely
related but the B. umbrosa was separated
from those species.
This observation supports the study done by
Goh et al. (1993), who analyzed two species
of Malaysian Bactrocera fruit flies using Gas
chromatography-mass spectrometry on their
cuticular hydrocarbons.
Figure 2 A tree diagram from a cluster analysis using
the concentration of the compound in each species.
The cluster analysis was performed with Jaccard Index
as the distance measure and the linkage rule was the
unweighted pair group average.
4. CONCLUSION
Extraction, characterization, and identification of hydrocarbon fractions of cuticular lipid from three
species of Tephritid fruit flies using gas chromatography techniques were success-fully done.
Significant differences between compositions of each compound between species approved that
cuticular hydrocarbon can be used as chemotaxonomic markers since the present of species-specific
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cuticular chemicals in sample. However, more investigations, time, and efforts are needed in order to
document the cuticular hydrocarbon profile for Sarawak fruit flies so that this objective will be achieved
better.
Acknowledgement
The authors would like to acknowledge Faculty Resource Science and Technology, Universiti
Malaysia Sarawak for providing us with the administrative and logistic support during the study. The
research was funded by Fundamental Research Grant Scheme: FRGS/01(24)/767/2010(48). The
authors also wish to thank to Mr. Rajuna Tahir for his kind assistance in laboratory work, especially
with the GC setup.
References
Carlson, D.A. and Service, M.W. (1979). Differentiation between species of the Anophele gambiae
complex (Diptera: Culicidae) by analysis of cuticular hydrocarbons. Ann. Trop. Med. Parasitol 3:
589-592.
Carlson, D. A., and Yocom, S. R. (1986). Cuticular hydrocarbons from six species of fruit flies. Arch.
Ins. Biochem. Physiol. 3: 397-412.
Chua, T. H., 1998. Annonated checklist of the tribe Dacini, subfamily Dacinae in Malaysia (Diptera:
Tephritidae). Serangga 3(1): 49-90.
Clarke, A.R, Armstrong, K.F, Carmichael, A.E, Milne, J.R, Raghu, S., Roderick, G.K and Yeates, D.K.
(2004). Invasive phytophagous pests arising through a recent tropical evolutionary Radiation:
The Bactrocera dorsalis complex of fruit flies. Annu. Rev. Entomol. 2005. 50: 293-319.
Goh, S. H., Ooi, K. E. , Chuah, C. H., Yong, H. S., Khoo, S. G., and Ong, S. H. (1993). Cuticular
hydrocarbons from two species of Malaysian Bactrocera fruit flies. Biochemical Systematics and
Ecology 21(2): 215-226.
Jacob, J. (1979). Chemotaxonomic investigations on the cuticular lipids of beetles. Biochemical
Systematics and Ecology, 7: 141-145.
White, I. M and M. M. Elson-Harris (1992): Fruit flies of economic significance; their identification and
bionomics. CAB International Publishing. New York.
Whitlow, V. V. S., 2003. Recognition in burying beetles (Nichoporus spp., Siphidae, Coleoptera).
Unversity of der Albert Ludwigs. Unpublished report.
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KENAF RETTING USING Ceratocystis paradoxa UMAS-PG3
1*
1
1
Dayang Syahreeny A. M. , Awang Ahmad Sallehin A. H. , Hairul Azman R. and Ismail J.
1
2
2
Department of Molecular Biology, Department of Plant Science and Environmental Ecology,
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, Kota Samarahan
*Email: dyg.syahreeny@gmail.com
Abstract
Kenaf retting using locally isolated pectinolytic and ligninolytic fungi Ceratocystis paradoxa UMASPG3 via flask culture under shake condition was performed and investigated. The evaluation of retting
efficiencies is based on the residual percentages of pectin and lignin left and the study of the fibre
surfaces via scanning electron microscope (SEM) of before and after retting of kenaf fiber. As a result,
the highest removal rate of pectin and lignin in fungal retting of kenaf is 46.90% ± 2.1 and 21.95% ±
2.0, respectively whereas SEM revealed pits along the cell wall of the kenaf fibre surfaces indicating
the removal of pectin and lignin under standard retting conditions.
Keywords: kenaf, retting, pectin, lignin, Ceratocystis paradoxa
1. INTRODUCTION
Kenaf (Hibiscus cannabinus and H. sabdariffa) is a warm season annual hibiscus and among the least
expensive and most versatile of textile fibres. Useful components of a kenaf plant includes the stalks
(bark and core), leaves, and seeds (Webber & Bledsoe, 2002). Kenaf stalks consist of an outer fibre
(bast) and fiber (core) which is comparable to softwood tree fibres and hardwood fibers, respectively
(Kutacova, 1998). The use of a non-tree source such as kenaf for paper production is a cost effective
alternative to make paper without cutting woody trees. It has been demonstrated that kenaf can be
utilized to generate high-quality writing and specialty papers (Liu, 2002). Retting is the major problem
in producing high-grade products from kenaf fibre (Yu & Yu, 2007). It is a process that removes nonfibrous materials (Song, 2006), such as the removal of pectin and lignin, leaving separated kenaf bast
and core fibres by employing enzymatic and microbial retting technology. Apart from that, in paper
manufacturing, retting is a crucial process as the fibre extraction process affects the final product
quality. Improvement in biological or enzymatic retting is user-, cost- and environment-friendly thus
crucial in upgrading the quality of the kenaf bast fibers.
2. METHODOLOGY
2.1 Kenaf
Kenaf (Hibiscus cannabinus) used in this study were generously supplied by Lembaga Kenaf dan
Tembakau Negara in Kubang Kerian, Kota Bharu, Kelantan.
2.2 Kenaf retting by fungal treatment
The samples, consisted of five pieces of kenaf fiber, approximately 15 mm in diameter and 30 mm in
length and weighing between 10 – 15 g. The ratio of fiber to minimal salt media was set at
o
approximately 1:10. All of the medium used were sterilized at 121 C for 20 min prior to fungal
o
inoculation. Retting was done at initial pH5 at 30 C for one week with shaking condition at 120 rpm.
Locally isolated fungus Ceratocystis paradoxa (UMAS-PG3) was employed in this study as the
inoculum. The uninoculated kenaf was used as control in the retting experiments.
2.3 Pectin, lignin and cellulose content determination
The lignin content of untreated and biotreated were determined following the standard methods of
AOAC Official Method 973.18 (AOAC 1990) whereas pectin and cellulose content were quantified
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based on Kratchanova et al. (2004) and Ververis et al. (2007), respectively. All of the analysis were
done in triplicates.
2.4 Scanning Electron Microscopy
The surfaces of raw and retted kenaf were analysed using a Jeol JSM 639OLA scanning electron
microscope with an accelerating voltage of 10kV. The fibers were sputtered with Au/Pd for 5 minutes
prior to imaging. The samples were sputtered using a Jeol JCF 1600 auto fine coater to help eliminate
charging on the samples during imaging.
2.5 Statistical analysis
Paired T-test was performed using SPSS 17.0 to analyse the data obtained.
3. RESULTS AND DISCUSSION
Chemical analyses elucidate the relative loss or changes of kenaf components after bio-treatment.
Figure 1 exhibits the changes in chemical composition of treated kenaf and control after 7 days. Pectin
showed a decreased with 46.90% ± 2.1 removal rate followed by 21.95% ± 2.0 of lignin removed
under shake condition. Even shape and size of simple pits were found along the cell wall of the fibre
(Figure3a). This indicates that lignin, pectin and other polysacharides in middle lamellae were most
likely removed during retting which revealed the pits along the cell wall that were not observed in
unretted kenaf (Figure2).
Figure 1 Residual chemical compositions of retted kenaf by Ceratocystis paradoxa UMAS-PG3 after
7 days. Data are means of triplicates ± SD. Statistical analysis showed that the differences were
significant at 0.05 confidence levels in chemical composition of pectin and lignin, except for cellulose
of untreated and biotreated kenaf after the microbial retting experiments.
On the other hand, an increase in cellulose recovery was also observed (Figure 1) but not significant
at 0.05 confidence level. This contradicted with the SEM results as cellulose degradation is suspected
as the irregular size and shape of pits was also observed along the cell wall (Figure 3b). The
observation may be because of the results of hyphae attack during retting towards the cell wall of
kenaf fibre. Apart from that, the shaking condition also permits a close contact between the hyphae
and kenaf surface thus enabling the enzyme to penetrate deeper into lignocellulosic component as
accessible surface area may be hindered by crystallinity or lignin protection or hemicellulose
presentation or all of the components in ligninocellulosic fibre that resulted in cellulose degradation
(Taherzadeh and Karimi, 2008).
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Figure 2 Unretted kenaf bast fibre. The surface shows the presence of waxes and other encrusting
substance such as hemicelluloses, lignin and pectin.
(b)
(a)
Figure 3 Retted kenaf bast fibre. (a) Regular size and shapes of simple pits. (b) Irregular size and
shapes of pits look alike.
4. CONCLUSION
Physiochemical analysis confirmed positive effects of fungal retting which showed reduction of lignin
and pectin of retted kenaf fibre. In addition, SEM analysis provided a reliable data on main kenaf fibre
characteristic and the efficacy of the fungal retting eventhough limited to the kenaf fibre surface
morphology, as partial purified kenaf fibre bundle was observed supporting the physiochemical
analysis. The fungal kenaf retting experiments should be further optimized under various parameters
to achieve a better retting degree supported by comprehensive fibre characterization and
technological performance assessment.
References
Kratchanova, M., Pavlova, E., and Panchev, I. (2004). The effect of microwave heating of fresh orange
peels on the fruit tissue and quality of extracted pectin. Carbohydrate Polymers, 56, 181–185.
Kutacova, P. (1998). Enzymatic Modification of Kenaf Pulp. (Unpublished master’s thesis). University
of Toronto.
Liu, A. (2002). World Production and Potential Utilization of Jute, Kenaf, and Allied Fibers .
Proceedings of the 2000 International Kenaf Symposium.
Song, K.H. (2006). Chemical and Biological Retting of Kenaf Fibers. Textile Research Journal October
2006, 76, 751-756.
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Taherzadeh, M. J. and Karimi, K. (2008). Pretreatment of Lignocellulosic Wastes to Improve Ethanol
and Biogas Production. International Journal of Molecular Sciences, 9, 1621-1651.
Ververis, C., Georghiou. K, Danielidis, D., Hatzinikolaou, D. G., Santas, P., Santa, R., Corleti, V.
(2007). Cellulose, hemicelluloses, lignin and ash content of some organic materials and their
suitability for use as paper pulp supplements. Bioresource Technology, 98, 296–301.
Yu, H. & Yu, C. (2007). Study on Microbe Retting of Kenaf Fiber. Enzyme and Microbial Technology,
40, 1806-1809.
Webber, C. L. & Bledsoe, V. K. (2002). Kenaf Yield Components and Plant Composition. In Janick, J
& Whipkey, A. (Eds.), Trends in new crops and new uses. Alexandria, VA, ASHS Press.
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CONSTRUCTION AND EXPRESSION OF Isochorismate synthase cDNA IN BACTERIAL SYSTEM
Chai Suk Phin* and Hairul Azman Roslan*
Department Of Molecular Biology, Faculty Of Resource Science And Technology,
University Malaysia Sarawak, 94300 Kota Samrahan, SARAWAK
*Emails: chaisukphin88@gmail.com, hairulroslan@hotmail.com
Abstract
Plants can be exploited as the source for viruses, bacteria, fungi, insects, parasites, and even other
plants for growth and survival. Mostly, plants have the ability to defend against pathogens in their
surrounding environment. Nevertheless, some plants lack of strong and adaptive defense system. For
this reason, plants been evolved other antimicrobial defense routes either in inducible or constitutive
condition. Isochorismate synthase (ICS) is an enzyme that involved in the synthesise of salicylic acid
(SA). Salicylic acid is pathogenesis-related (PR) protein that assists in plant defense system against
pathogens. In previous study of Morinda citrifolia, a partial mcICS cDNA have isolated. To study the
function of this cDNA, the gene is cloned into an expression vector. The construction was undertaken
by cloning of the amplified PCR fragment of the mcICS into pET-41a(+) vector and subsequently
transformed into XL1- Blue competent cell. The mcICS was successfully cloned and formed a plasmid
with a size of 7383bp. The constructed plasmid was later transformed into the expression host
BL21(DE3) competent cells prior for protein expression analysis. In this project, sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS PAGE) was carried out for protein analysis and
approximately 54.98kDa of protein is determined.
Keywords: Isochorismate synthase, salicyclic acid, Morinda citrifolia.
1. INTRODUCTION
Morinda citrifolia or the commercially name as Noni is a type of plant from Rubiaceae (coffe family),
and subfamily of Rubioideae. Morinda citrifolia is a significant source of both traditional and modern
medical applications (Tan & Roslan, 2008). Isochorismate synthase (ICS) is an enzyme from Morinda
citrifolia which catalyse the formation of salicyclic acid (SA). Salicyclic acid is a type of phytohormone
that functions in plant defence (Strawn et al, 2007). In previous studies, it has been shown that
salicyclic can be produced through the formation of isochorismate (Chen et al, 2009). Partial mcICS
cDNA from Morinda citrifolia had been previously isolated (Tan & Roslan, 2008) but have not been
characterised. Therefore this project aims to characterise the cDNA via the construction of an
expression vector containing the mcICS cDNA and expression of the cDNA in an expression host. The
expressed protein is then analysed using sodium-dodecyl sulphate polyacrylamide gel electrophoresis
(SDS PAGE).
2. METHODOLOGY
2.1 Polymerase Chain Reaction Cloning (PCR cloning)
PCR cloning was carried out to amplify sequence by using two unique primers, the 5’ BamH1_mcICS
and 5’Sal1nostop_mcICS. The PCR process was performed using High Fidelity PCR Enzyme Mix
(Termo Scientific) as follow: 1 µl 10x High Fidelity PCR Buffer, 0.6 µl 25mM MgCl 2, 1 µl 2.5mM dNTP
mix (Biorad), 1 µl 5’BamH1_mcICS primer, 1 µl 5’ Sal1nostop_mcICS, 0.2 µl High Fidelity PCR
Enzyme Mix, 1 µl template DNA, and 5µl nuclease free water. The thermal cycling conditions for
synthesis this PCR fragments was shown in Table 3.1. The completed PCRs were analysed by gel
electrophoresis with 1% agarose gel to determine the size of PCR fragments..
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Table 1 Thermal Cycling Conditions for PCR Product Clone into pET-41a(+) Vector
Segment
Initial
Denaturation
Denaturation
Annealing
Elongation
Final Elongation
Final Hold
Temperature (°C)
Duration (minutes)
Number of Cycles
94.0
3.00
1
94.0
60.3
72.0
72.0
20.0
0.30
0.45
1.30
8.00
20.00
40
1
1
2.2 Restriction Enzyme Digestion of pET-41a(+) Vector
The pET vector was digested in serial digestión with BamH1 restriction enzyme followed by Sal1
restriction enzyme Firstly, the restriction enzyme digestión was perfomed by mixing 25 µl DNA, 3 µl
10x BamH1 buffer and 2 µl BamH1 restriction enzyme. The tube was mixed thoroughly and incubated
at 37°C for 5 hours, followed by deactivation at 80°C for 20 minutes and lastly DNA purification before
proceeding to the second digestión. In the second digestión, 30 µl of purified DNA was mixed with 3.7
µl of 10x buffer orange and 3.3 µl of Sal1 restriction enzyme. The tube was mixed thoroughly and
incubated at 37°C overnight followed by deactivation at 65°C for 2 minutes and DNA purification step.
2.3 Restriction Enzyme Digestion of mcICS
The mcICS was digested in serial digestión as in method discussed in Section 2.2. Firstly, the
restriction enzyme digestión was perfomed by mixing 30 µl DNA, 3.7 µl 10x BamH1 buffer and 3.3 µl
BamH1 restriction enzyme. The tube was mixed thoroughly and incubated at 37°C for 5 hours,
followed by deactivation at 80°C for 20 minutes and DNA was purified before proceeding to second
digestión. In the second digestión, 30 µl of purified DNA was mixed with 3.7 µl 10x buffer orange and
3.3 µl of Sal1 restriction enzyme. The tube was mixed thoroughly and incubated overnight at 37°C
followed by deactivation at 65°c for 2 minutes and DNA purification step.
2.4 Cloning of mcICS into pET-41a(+) Vector
T4 DNA ligase (Termo Scientific) was used to lígate the sticky end of both pET vector and mcICS.
Ligation was performed by mixing 4.2 µl of 10x T4 ligase buffer, 5 µl of digested pET vector, 30 µl of
insert DNA, and 4.2 µl of T4 DNA ligase (1u/µl). The tube was mixed thoroughly and incubated
overnight at 4°C.
2.5 Transformation into Expression Host (BL21-DE3)
The vector containing mcICS was then transformed into the E.coli strain BL21(DE3) (Stratagene).
Competent cells of BL21(DE3) were thawed on ice for 10 minutes. Competent cells aliquot was prechilled in an eppendorf tube followed by addition of 20 µl of DNA plasmid, mixed gently and incubated
on ice for 20 minutes. The tube was then placed on water bath at 42°c for exactly 2 minutes and
immediately returned on ice for 10 minutes. A 1ml LB broth at pre-warmed at 37°C was added into the
mixture and incubated at 37°C for 1 hour 45 minutes. The transformed culture was spread on
kanamycin plates (100mg/ml) and incubated overnight at 37°C.
2.6 Induction of Target Protein Using IPTG
A single colony of pET-41a(+)/ICS and pET-41a(+) was picked from streaked plates. Each colony was
grown in 6 ml LB containing kanamycin (100 mg/ml) at 37°C overnight. The overnight cultures were
then added into 11 ml LB containing kanamycin and grown at 37°C with shaking at 220-250 rpm to
OD600 = approximately 0.5-0.6 (about 1 hour 30 minutes). Total volume of each culture was divided
into two cultures. IPTG was added into the culture to the final concentration of 1 mM in one flask and
the other culture served as the non-induced control. All of the induced cultures were incubated at 37°C
with shaking 220-250rpm for 4 hours. The non-induced control cultures were placed on ice until
needed in SDS-PAGE analysis. After the induction period, the cells was harvested by spinnning down
at 9000xg for 15 minutes. Supernatants was discarded and the cells was suspended with 2x SDS gel
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sample buffer. Cells were then lysed by incubation at 95°C for 5 minutes and spun down at 6000xg for
15 minutes.
2.7 Analysis The Protein Expression of ICS
The protein samples was analysed on denaturing (SDS-PAGE). Firstly, the PAGE was prepared by
pouring 12% resolving gel into gel cast until estimately reached 1.5 cm below the notched plate
followed by layered with approximately 1ml of ddH2o and left to polymerise about 40 minutes.
Secondly, once the gel was polymerised, the ddH20 was dried with filter paper and laid with 6 %
stacking gel. Then the comb was placed and left to polimerise for 40 minutes. The comb was then
taken out and gel was placed into an electrophoresis chamber (Bio-RAD). A 1 x running buffer was
added into the electrophoresis chamber until the gel was fully immersed. Protein sample was prepared
by adding 13 µl protein sample with 13 µl sample buffer. Samples were loaded into the well and
electrophoresis was carried out at voltage of 120V about 1 hour 30 minutes. Lastly, gel was placed
into fixing solution for 15 minutes followed by staining for 45 minutes and lastly destained for a further
45 minutes.
3. RESULTS
3.1 Agarose Gel Electrophoresis Analysis of mcICS
A 1 % agarose gel was used to analysis the size of mcICS that amplified by PCR (Figure 1) and the
amplified mcICS was approximately 1500 bp.
1500bp
Figure 1 The Size of mcICS Amplified by PCR
3.2 Agarose Gel Electrophoresis Analysis of Recombinant Plasmid
The size of recombinant plasmid was analyzed after cloning into pET vector and analyzed by 1 %
agarose gel electrophoresis. Based on the Figure 2, there are different of size between pET vector
without any insert (5933bp) and pET vector containing the mcICS (7384bp).
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6000 bp
5000 bp
Figure 2 The Size of pET Vector and Recombinant Plasmid
3.3 SDS PAGE Analysis of ICS
Figure 3 is a picture of SDS PAGE, lanes 1 to 4 are samples of pET-41a(+)/ICS and lanes 5 to 8
contains samples of pET-41a(+). Sample of pET-41a(+)/ICS was replicated, as induced samples (lane
1 and 2) and non-induced samples act as control (lane 3 and 4). The parameter was repeated for
pET-41a(+), as induced samples (lane 5 and 6) and non-induced samples act as control (lane 7 and
8). From the SDS PAGE analysis, it can be seen that in the induced samples, a fragment can be seen
to be overexpressed and determined to be 54.98 kilo Dalton in size.
~75
kDa
~63
kDa
Figure 3 The Expression and Determination of mcICS Molecular Weight
4. CONCLUSION
Recombinant pET-41a(+) plasmid with a size of 7483 bp was constructed containing the mcICS. The
vector was then expressed and approximately 54.98 kilo Dalton of mcICS molecular weight was
determined.
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References
BIO-RAD. Mini-PROTEAN Tetra Cell. Retrieved on Sepember, 7, 2012 from http://www.biorad.com/webroot/web/pdf/lsr/literature/10007296.PDF
Chen, Z., Zheng, Z., Huang, J., Lai, Z., & Fan, B. (2009). Biosynthesis of Salicyclic Acid in Plants.
Plant Signaling & Behavior, 4(6), 493-496.
Termo Scientific. High Fidelity PCR Enzyme Mix. Retrieved on September, 7, 2012 from
http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0191.pdf
Termo
Scientific.
T4
DNA
Ligase.
Retrieved
on
Septermber,
7,
2012
from
http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_el0016.pdf
Stratagene. BL21(DE3) Competent Cells, BL21(DE3) pLysS Competent Cells, and BL21 Competent
Cells. Retrieved on September, 7, 2012 from http://www.molecularinfo.com/MTM/A/A3/A31.pdf
Strawn, M. A., Marr, S. K., Inoeu, K., Inada, N., Zubieta, C., & Wildermuth, M. C. (2007). Arabidopsis
Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits
Properties Consistent with a Role in Diverse Stress Responses. The Journal of Biological
Chemistry, 282(8), 5919-5933.
Tan. S. H.,& Hairul. A. R. (2008). Amersham.Cloning of a Near Complete Isochorismate Synthase
(ICS) cDNA from Morindacitrifolia L. Pertanika J. Trop. Agric. Sci, 31(1), 101-105.
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IN VITRO ORGANOGENESIS OF GINGER (ZINGIBER OFFICINALE ROSC.)
Shazatul A.S. and Retno Andayani B.M.*
Kulliyyah of Pharmacy, International Islamic University Malaysia,
Bandar Indera Mahkota, 25200 Kuantan, Pahang.
*Email: retno@iium.edu.my
Abstract
Ginger (= halia, Zingiber officinale Rosc.) is widely used as a spice in many Asian cuisines and well
known as an important medicinal plant. These correspond to the ginger active constituent gingerols
and its volatile oils. In our previous work on the GC-MS profiling of ginger volatile oils from Bukit Tinggi
(Bentong, Pahang) cultivar has revealed a presence of high level of sesquiterpenes hydrocarbon,
namely zingiberene ( 22%) and -farnasene ( 12 %). In contrast, a considerable amount of
monoterpenes, i.e. 1,8-cineole ( 29%) and camphene ( 10%) are presence in Tanjung Sepat cv. In
preparation for somatic hibridation study to obtain a new ginger strain abundant in both type of the
terpenes, we carried out in vitro organogenesis of Bukit Tinggi and Tanjung Sepat ginger cultivars. A
young ginger bud was used as explants and grown on Murashige and Skoog (MS) media,
supplemented with 1-naphtaleneacetic acid (NAA) and indole acetic acid (IAA) in combination as a
growth hormones, 3 percent (w/v) of sucrose under 16 h illumination.
For full paper, please contact the correcponding for further details.
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NOTES ON A NEW NOXIOUS WEED, Paspalum dilatatum Poir. IN SARAWAK, MALAYSIA
I.B. Ipor*, M. Minggu & C.S. Tawan
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, University Malaysia Sarawak, 94300, Sarawak, Malaysia
*Email: ibipor@frst.unimas.my
Abstract
Paspalum dilatatum. Poir. is a potential noxious weed species which introduced from Australia in the
oil palm plantations in Miri Division, Sarawak. It was mainly introduced as pasture in cattle rearing at
Karabungan Centre, Department of Agriculture, Sarawak. It dispersed efficiently through effective
germination and quickly formed phalanx clumps. This distribution pattern and growth pattern of P.
Dilatatum in various areas such opened areas and different stages of oil palm plantations is essential
to determine effective control of this noxious weed.
1. INTRODUCTION
P. dilatatum Poir. is a perennial plant which grown from the solid hairy base that can reach up to 2m
height. It is easily recognised from its short, thick rhizomes. It has hairy leaves lower sheaths while the
leaf blades are hairless (Justin, 2005). The leaf blades are long and narrow, bright green in colour and
more hairy around where it meets the stem. The leaves also have distinct midrib and have rough and
sharp edges (McCarty & Yelverton 2005). Its inflorescence has spike like racemes. The stem has
three to seven racemes commonly, and sometimes may up to 11 racemes.
P. dilatatum grows in clump as perennial. The stems are slender and usually as the seed growth
during summer. It is a fast growing plant and has the ability to grow successfully on a wide variety of
soil. P. dilatatum Poir. has the ability to grow best on disturbed moist soil, fertile clay and loam
bottomland (εcCarty & Yelverton 2005). It’s well known habitat include gardens, turf, vineyards,
orchards, irrigation and drainage ditches, canals, pond and reservoir margins, rice and fields and
stream banks.
Numerous introduced herbaceous species have been frequently referred as important and problematic
weeds in oil palm plantation. Recently establishment of P. dilatatum which introduced through import
of cattle from Australia is predicted to cause serious problem in weed management in oil palm
plantation in Sarawak. In this study, the determination of P. dilatatum distribution and growth pattern in
oil palm plantation is important so that further dispersal of the weed can be avoided. The plant is
believed to be aggressive species that may attack oil palm plantation. This can reduce the yield of the
plantation.
2. MATERIAL AND METHODS
Survey on the occurrence of P. dilatatum was conducted at open areas (Ladang Tujuh and
Karabungan), 5 years old oil palm plantation and 20 years old oil palm plantation of nearby Ladang
Tujuh and Karabungan in Miri Division. Quadrates of 5mx5m were established randomly in the study
areas. Number of clumps in every quadrate was recorded. Every clump was severed to determine the
number of tillers and the total dry weight of the vegetative parts such as leaf blades, stems and roots.
In another assessment, five plants were harvested from each area for biomass allocation assessment.
Leaves, stems and roots were separated from each other. The separate vegetative parts were oven
dried at 60 C for 72 hours to determine their dry weight. The growth analysis and biomass allocation
pattern were assessed by using method described by Patterson (1980).
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3. RESULTS
Field observation revealed that there was different morphological variation of P. dilatatum grown from
open areas (Ladang Tujuh and Pusat Pertanian Karabungan), 5 years old oil palm plantation and 20
years old oil palm plantation. The variations are clearly in terms of height, longest leaves, shortest
leaves and number of leaves in the field. Based on the observation made, not all tillers were able to
produce inflorescence. Tall tillers with more than 170 cm height managed to produce inflorescence.
P. dilatatum at the residential area of Ladang Tujuh has the percentage of coverage ranging from
35% to 82% with the total clumps ranging from 36 to 96. Number of tillers was from two to 82 tillers.
At Karabungan, the percentage of coverage was from 90% to 98% with the total clumpsranging from
12 – 23 and 96 to 315 tillers. The coverage was from 63% to 68% under 5-year old oil palm plantation.
Total clumps was from 4 to 14 with 7 – 65 tillers per clump. The growth of P. dilatatum drastically
decreased with the coverage of 30% to 55%, total clump of 2 to 7. The clumps had total tillers of 7 to
42.
400
a
Total number of clump
350
300
250
200
Number of clump
150
b
100
b
b
50
0
Ladang Tujuh Karabungan
5 YO OP
20 YO OP
Area
Figure 1 Total number of clump for each area and the letter above each bar shows the significant
difference between each area.
1800
b
total number of tillers
1600
1400
1200
1000
Number of tillers
800
600
400
a
a
a
200
0
Ladang Tujuh
Karabungan
5 YO OP
Area
20 YO OP
Figure 2 Figure shows number of tillers for different areas.
Table 1 Growth Pattern and Biomass Allocation of P. dilatatum Poir.
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Area
Ladang Tujuh
Karabungan
5 YO OP
20 YO OP
Number of tillers
5
5
5
5
Plant Dry Weight(g)
90.96
173.83
113.28
33.32
Leaf Dry Weight(g)
44.55
68.4
36.92
12.08
Stem Dry Weight(g)
45.29
69.66
66.66
18.28
Root Dry Weight(g)
1.13
7.18
7.9
2.96
Leaf Weight Ratio(g/g)
0.48
0.39
0.32
0.36
Stem Weight Ratio(g/g)
Root Weight Ratio(g/g)
0.49
0.01
0.40
0.04
0.58
0.06
0.54
0.08
Table1: The biomass allocation for 4 different area.
Karabungan area contributed highest dry weight of stem (69.99 g) and leaves (68.4 g). Root part was
highest at 5-year old oil palm plantation. Leaf weight ratio (LWR) was highest at Ladang Tujuh while
lowest at 5-year old plantation. SWR was highest at 5-year old oil palm plantation and lowest at
opened area at Karabungan. RWR was highest at 20 years old oil palm plantation and lowest at open
area(Ladang Tujuh).
P. dilatatum has its significance as important weed mainly attributted to their aggressiveness and
extensive formation of tillering system and reproduced prolifically from seeds. Due to P. dilatatum is
an excellent colonizer and ruderal competitor, this weed can rapidly regenerated and eventually
dominated the early successional stage of vegetation particularly in open areas, on abandoned
cultivated lands, fallows of shifting cultivation, overlogging and annual burning and plantations crops
such as rubber, oilpalm, coconut, coffee, tea and cocoa. P. dilatatum is typically characterized by rapid
emergence, vigorous shoot growth and unpenetrable formation of canopy. Rapidly growing plants can
use a disproportionate share of resources to the detriment of neighbouring plants.
References
Justin,
K.Y.,Chu, (2005). Paspalum dilatatum, retrieved on October 4, 2011 from
http://www.iewf.org/weedid/Paspalum_dilatatum.htm
McCarty, B. & Yelveton, F., (2005). Dealing with Dallisgrass.82.
Patterson, D.T. (1980). Shading effects on growth and partitioning of plant biomass on cogongrass
(Imperata cylindrica) from shaded and exposed habitats. Weed Sci. , 28 : 735 – 740
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ECOPHYSIOLOGY OF Amorphophallus brachyphyllus (Hett.)
I.B. Ipor*, C.S. Tawan, Meekiong, K. and P.M. Lily
Department of Plant Science and Environmental Ecology, Faculty of Resource Science & Technology,
Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak
*Email: ibipor@frst.unimas.my
Abstract
The study on the effect of light intensity on the vegetative growth and biomass partitioning of
Amorphophallus brachyphyllus (Hett.) was carried at Faculty of Resource and Science Technology
greenhouse, University Malaysia Sarawak (UNIMAS). The growth of A. brachyphyllus was better
under the 75% and 50% shading, in which the 75% shade level showed the highest values in petiole
length, tuber diameter, leaf weight ratio (LWR), petiole weight ration (SWR) and leaf area ratio (LAR).
The 50% shade level also showed the highest values in number of leaflets, plant dry weight (W), leaf
area (A), root weight ratio (RWR), specific leaf area (SLA) and dry matter production (DMP). The 0%
shading showed the highest values for tuber weight ration (UWR), net assimilation rate (NAR) and leaf
area duration (LAD). The study on growth pattern and biomass allocation of A. brachyphyllus was
conducted at limestone forest area of Mount Kasat, Sarikin, Bau, Sarawak. Twenty samples ranged
between 11.31 - 34.64 mm above ground surface stem diameter with their height recorded within the
ranged of 26 - 65 cm. The dry weights mean for twenty plant samples was 34.31 g. Among the plant
2
vegetative parts, the petiole diameter and leaf area (R =0.8524, which y = 416.14x - 4700.9) indicated
a significant relationship with the strongest interaction occurrence.
Keywords: Amorphophallus brachyphyllus, shading, biomass allocation, relationship, important value
1. INTRODUCTION
Amorphophallus species from the family of Araceae or aroid family (Hetterscheid and Ittenbach, 1996)
is commonly known as the devil’s tongue, voodoo lily, or corpse flower (Talt, 2002). It refers to the
horrendous odor of decaying flesh, similar to ‘bunga bankai’ by the Indonesian community, which
means corpse flower (Richerson, 1999). It is perennial and herbaceous foliage with an underground
storage organ, which is usually a tuber of various size and shape that need to be sufficiently anchored
to support the tall petiole or inflorescence (Talt, 2002). Besides, the plant species is considered to be
the largest unbranched inflorescence in the world as its ability to attain 6.5 to 10.75 foot height
(Richerson, 1999). For example, A. titanum or giant titan arum is the largest of all Amorphophallus
species. The health of these plants is measured by the seasonal increased in tuber size especially for
young plants. It is estimated to encompass some 170 species of Amorphophallus that mainly covers
the vast of distribution in the tropical of the old world - ranging from West Africa eastward into
Polynesia (Hetterscheid and Ittenbach, 1996; Talt, 2002). Some could be found throughout the
subtropical zones. Thus, within this generic distribution of Amorphophallus species show a very high
degree of endemism. For example, A. brachyphyllus is endemically found in Sarawak. This
herbaceous plant is highly potential of its extinction, as the plant is easily exposed to human activities
disturbance such as deforestation and developments. Exploitation of this plant adversely effects its
population. As the rate of the plant habitat destruction is increasing, the present study is conducted to
determine and understand the ecology and its response to environmental factors such as light,
besides possible cultivation of these plant species. The results obtained will be essential for detail
scientific documentation and conservation of Amorphophallus species.
2. MATERIALS AND METHODS
The survey and seed collection of Amorphophallus brachyphyllus were conducted at the area of
Mount Kasat, Sarikin, Bau while the effect of light to the plant species vegetative growth developments
was conducted at FRST greenhouse, UNIMAS. The selected seeds of the plant species were
germinated in a seedbed sized 1.1 to 1.2 m (3.5 to 4 feet) wide with the propagation media of pure
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sand, under the greenhouse conditions (Hartmann, Kester, Davies and Geneve, 2002). Two weeks
after germination, the seedlings were transplanted in (10 x 12) cm² polybegs with propagation media
ratio of 3:1:1 (soil: sand: organic matter). After two weeks, the young seedlings were then transplanted
under three different shade levels that was the 0% shade (open shade or direct sun exposure), 50%
shade and 75% shade. The 50% and 75% shade regimes were obtained by using different intensity of
lathe netting sized (2 x 2x 2) m³.
3. RESULTS AND DISCUSSIONS
Plants at 50% shading were initially higher compared to those plants placed under 0% and 75% of
shade level after the first 8 weeks (56 days) of transplanting. But the following 4 weeks, in which after
12 weeks (84 days) of transplanting resulted an insufficient increased in rate of petiole length for
plants at 75% shade level (18.8229 + 8.21885). In the intervening period, the shortest plants in length
were recorded at 0% shading (11.7314 + 2.03269). The values of petiole length were significantly
different between the 75% and 0% shading (p=0.006). Similar results between 0% and 50% shading
values were also analysed significantly different (p=0.005) but insignificant for 75% and 50% shade
level (p=0.463). This was obviously showed by the graph plotting in Figure 1a. Thus, this showed that
the plants under 75% shading were great in growth, followed by 50% shading (22.3200 + 4.00310).
Besides, the fertility of the plant species was showed under higher rate of shade level (Hetterscheid
and Ittenbach, 1996). The level of shading was correlationally with the degree of light intensity and
plant humidity, in which the reduction of light intensity increased the humidity of the plant habitat and
resulted the increasing of the petiole length.
The level of shading was significantly influenced the number of leaflets as it was the main organ for
plant in food production through the process of photosynthesis, which affected the growth and plant
developments (Barden, Halfacre and Parrish, 1987). The plant seedlings for each shade level showed
an insufficient rate of increased in number of leaflets after 12 weeks of transplanting (Figure 1b). The
first 2 weeks (14 days) of transplanting resulted a swifter increased in number of leaflets for plants at
50% shading compared to the other two shade levels. But, the result altered on the fourth weeks (28
days) of transplanting, after the emergence of new leaflets for plants under 75% of shade level. By the
end of the transplanting (12 weeks) resulted higher leaflets production for plants under 50% shade
level. Meanwhile, plants grown at 0% shading (5.1429 + 0.22254) had shown a slow increased in leaf
production, in which none emergence of new leaflets were recorded from 0 to 6 weeks (42 days) of
transplanting. Eventually, a slow increased of leaves productions were shown after 8 to 12 weeks (56
to 84 days) of transplanting.
The production of leaves for plants under 50% shading were significantly different with plants at 0%
shading (p=0.036) but insignificant different if compared to 75% shading (p=0.360). If the lowest and
highest percentage of shade level were compared, an insignificant different analysis was obtained for
those shade levels (p=0.405). The maximum production of new leaflets was recorded for plants at
50% shading (6.0286 + 0.90501), followed by 75% shading (5.5714 + 0.49570). Increasing shade
level to 75% had significantly affected the tuber diameter of A. brachyphyllus, in which recorded the
largest size of tuber compared to 50% and 0% of shade level after 12 weeks of transplanting (Figure
1c). The eighth week (56 days) of transplanting showed the tuber size for plants at 50% shading were
the largest in value but at the last 4 weeks, the tuber diameter for both 50% and 75% shade level were
almost similar. The 12 weeks of transplanting resulted plant tubers at 75% shading (19.3929 +
3.35289) were the largest among those at 0% and 50% shade level (20.6143 + 1.70934). The
diameter rate for plant tubers at 0% shading (16.1257 + 0.89686) were recorded the smallest with a
slow increased, each week of the transplanting.
The tuber diameter was significantly different between the 75% and 0% of shade level (p=0.035). The
similar result also recorded between the 0% and 50% shading (p=0.004) but not between 75% and
50% shade level (p=0.572). Plant tuber was purposed of storage organ that involved in the plant
growth and developments, in which influenced the length and leaflets production of the plant (Talt,
2002). Besides, the health of the plant species especially the young plants was based on the size or
tuber diameter (Richerson, 1999). Each comparison done above that included the petiole length,
number of leaflets and tuber diameter showed a similar result that reported by Roland Charles Singka
(2004) for A. borneensis. This showed that Amorphophallus species were likely to live under lower in
light intensity and humid area for better growth development (Hetterscheid and Ittenbach, 1996).
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Besides, through observations showed that the higher percentage of light intensity or direct sunlight
exposed to the plant species would cause scorches, wilting and injury or damages.
1b
35
9
30
8
Number of Leaflets
Petiole length (cm)
1a
25
20
15
10
5
7
6
5
4
3
2
1
0
0
2
4
6
8
10
0
12
0
Weeks after transplanting
2
4
6
8
10
12
Weeks after transplanting
Tuber Diameter (cm)
1c
30
25
20
Figure 1 Effect of light on vegetative growth
of A. brachyphyllus after 90 days of
transplanting under 3 different shadings; 1a)
Petiole length, 1b) Number of leaflets and 1c)
Tuber diameter. 0% shading 50% shading
75% shading
15
10
5
0
0
2
4
6
8
10
12
Weeks after transplanting
Plant grown under 50% shading produced the highest total of dry weight (W) contrast to those plants
from 75% and 0% of shade level (Table 1). The similar growth pattern was also reported for A.
borneensis. Besides, the plant species also showed an increased in the biomass production when
shaded. The highest and lowest value of (W) was significantly different. Plants from the 50% shading
recorded the highest leaflet area (A) value, followed by 75% and 0% shade level. The increased of
total leaflets area (A) at 50% shading was possible because of the increased in total number of leaflets
at the shade level. The (A) values showed in Table 1 were significantly different for plants under
different level of shadings. The highest value of leaf weight ratio (LWR) was recorded at 75% of shade
level compared to 50% and 0% shading with a significant difference between the highest and lowest
value. But, no significant difference was recorded between 0% and 50% shade level.
The increased and decreased of (LWR) values clearly influenced the values for leaf area ratio (LAR)
with the highest value was noted by plants at 75% shade level, followed by 50% shade and the lowest
was recorded at 0% shading. The similarity of the result was also reported in the study of A.
borneensis. The (LAR) values also showed significant differences between the shade levels. Plants at
50% of shade level had the greatest specific leaflet area (SLA) contrast to those plants at 75% and 0%
shading with a significant difference as the plants under a light-limited regime invested more to the
production of light-harvesting apparatus rather than other component of plant biomass (Imransyah,
2003). Plants under 0% shading was also significantly different compared to plants at 75% shade
level.
As for the petiole weight ratio (SWR) were significantly different among the shade levels, in which the
highest value was recorded for those plants at 75% shading while the lowest was for plants under
50% shade level. This showed that the stem partitioning biomass was significantly influenced by
shade level (Imransyah, 2003). Comparison of values between the levels of shading showed the
reduction of tuber weight ratio (UWR) parallel to the percentage increased of shade level. But, the
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(UWR) values were significantly different between the shade levels. Root weight ration (RWR) for 50%
shade level recorded the highest value among the shade levels, in which showed significant
differences compared to 0% and 75% shading, while the comparison between 0% and 75% was also
significantly different. The result was similar reported for A. borneensis.
Table 1 The effect of light on vegetative growth, leaf area production and biomass allocation in A.
th
st
brachyphyllus seedlings (60 days or 1 harvest).
Shade
level
Plant dry
weight (W)
Total leaf
area (A)
g/g
cm²
0%
0.78a
50%
1.88c
75%
1.55b
LWR
SWR
RWR
UWR
--------------------- g/g --------------------
SLA
LAR
-------- cm²/g --------
13.75a
0.29a
0.22b
0.16b
0.33c
91.97a
22.38a
47.00c
0.30a
0.17a
0.22c
0.30b
104.88c
27.14b
42.75b
0.33b
0.28c
0.13a
0.27a
97.07b
28.66c
Within each column, values sharing the same letter are not significant different at 5% level, according
to Fisher Least Significant Different test.
Plant species at 50% shading recorded the highest value in dry matter production (DMP) while the
lowest was for plants under 0% shading (Table 2). A similar result was also reported for A. borneensis.
As for the net assimilation rates (NAR), the lowest percentage of shade level showed the highest
value compared to those in two different level of shadings. The value reported for A. borneensis was
also similar with the result for A. brachyphyllus. Besides, the analysis showed insignificant differences
between the shade levels. Meanwhile, the leaf area duration (LAD) for 0% shade level was the highest
value recorded and the lowest value was for plant at 50% shading. Analysis for DMP and LAD values
showed significant differences between the shade levels.
The mean value for petiole length recorded for twenty plant samples measured between the ranges of
28 to 65 cm was 43.65 + 8.15 cm. Meanwhile, the mean revealed for total of leaflets was 47.65 +
2
32.78 with the mean value of 3650 + 3059 cm for total leaflet area per plant. Besides, the mean value
for other vegetative measurements that included the petiole and tuber diameter resulted 20.07 + 6.79
mm and 55.17 + 27.23 mm respectively. As for the fresh weight mean measurements jotted for the
plant vegetative parts such as the leaflets, petiole, tuber and root, resulted 47.22 + 30.75 g for leaf
fresh weight, 92.07 + 73.16 g for petiole fresh weight, 120.22 + 119.49 g for tuber fresh weight and
4.10 + 2.14 g for root fresh weight.
Table 2 The effect of light on dry matter production (DMP), net assimilation rates (NAR) and leaf area
th
th
nd
duration (LAD) in A. brachyphyllus seedlings during the 60 to 90 days interval (2 harvest).
Shade level
DMP (g)
NAR (mg/dm²/day)
LAD (dm²/day)
0%
0.495a
0.011a
15.470c
50%
1.860c
0.005a
12.605a
75%
1.745b
0.006a
14.385b
Within each column, values sharing the same letter are not significant different at 5% level, according
to Fisher Least Significant Different test.
Adversely to the mean dry weight values resulted for the plant vegetative parts, the plant leaflets
recorded 7.50 + 5.57 g of dry weight mean, whilst the petiole dry weight mean was 6.66 + 4.41 g.
Meanwhile, the plant tuber and root dry weight mean each resulted 18.10 + 20.27 g and 2.06 + 2.14 g.
The growth pattern of A. brachyphyllus was varied between plants and different established plots, in
which the morphological variation of the plant species was unmatched by any other genera of the
family. The excess of Amorphophallus species variation was found in the spathe (dimension, color and
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shape), the appendix (dimension, shape and sculpturing), odors, tuber (shape), petiole (dimension,
color and pattern) and finally the individual female flower (Hetterscheid and Ittenbach, 1996).
Besides, the other possible factors such as age, maturity and growth development period actuated the
variation of the plant species. The competition that occurred between the plant species in the same
habitat also stimulated the biological production, which caused variability in the vegetative growth of
the plant. The similar statement also reported by Rosmawatti Zaini (2004) in her study on A.
borneensis at Mount Gayu, Padawan. The total dry weights mean for twenty samples of A.
brachyphyllus ranged between 5.43 to 105.75 g was 34.31 + 29.41 g (Table 4).
Apart from that, the mean showed for Leaflets Weight Ratio (LWR) value was 0.2579 + 0.0973, whilst
the value of mean for Petiole Weight Ratio (SWR) was 0.2482 + 0.0945 with the largest value for both
analyses was recorded by plant sample 11. Both (LWR) and (SWR) values for plant sample 11 were
0.4502 and 0.4036 respectively. Apparently, plant sample 19 recorded the highest Tuber Weight
Ratio (UWR) value compared to other plant samples of A. brachyphyllus with the value of 0.7375.
Entirely, the (UWR) mean value for the plant species was 0.4246 + 0.1928. As for the Root Weight
Ratio (RWR) mean value, the assess showed was 0.0739 + 0.0460 g with 0.2061 as the highest value
of (RWR) recorded by plant sample 7.
Based on the similar table, the data analysis for Specific Leaflets Area (SLA) and Leaflets Area Ratio
2
2
(LAR) mean values were 469.95 + 103.49 cm /g and 120.49 + 59.16 cm /g respectively. The highest
2
value of (SLA) and (LAR) were recorded by plant sample 18 with the (SLA) value was 656.45 cm /g,
2
whilst plant sample 20 with the (LAR) value was 230.71 cm /g.
The interaction between the plant vegetative parts such as the petiole length and diameter with the
tuber fresh and dry weight, tuber diameter and leaf area for twenty randomly selected A. brachyphyllus
were analysed using regression analysis. It was for the purposed of determining the relationships
among the plant vegetative parts.
The tuber fresh weight of the plant species was significantly influenced by the petiole diameter with the
2
regression value of R =0.589, under the equation of y = 13.512x - 150.93 (Figure 2a). This explained a
significant relationship that occurred between the two vegetative parts, in which the growth of the
petiole diameter was positive in interaction with the fresh weight of the plant tuber diameter
(p=7.83513E-05). The similar analysis was indicated for the relationship between the petiole diameter
2
and tuber dry weight (p=0.0005). The regression value analysed was R =0.5023, in which y = 2.1169x
- 24.382 (Figure 2b).
The regression value that indicated in Figure 2(c) for the relationship of petiole diameter and petiole
length showed opposite analyses. The analysis obtained was insignificant (p=0.13903) between the
2
two vegetative parts with the regression value of R =0.1175, under the equation of y = 0.4118x +
35.381. There was no significant relationship occurred as the interaction was poor.
Table 4 The total dry weight, leaflets weight ratio (LWR), petiole weight ratio (SWR), tuber weight
ration (UWR), root weight ratio (RWR), specific leaflets area (SLA) and leaflets area ratio (LAR) of A.
brachyphyllus at Mount Kasat, Sarikin, Bau.
Plant number
1
2
3
4
5
6
7
8
9
10
11
12
13
Total dry
weight (g)
LWR
SWR
UWR
RWR
SLA
2
(cm /g)
LAR
2
(cm /g)
12.47
80.89
105.75
12.88
25.11
12.43
22.37
7.81
15.49
17.08
7.73
17.78
5.43
0.3825
0.1234
0.1972
0.1832
0.2091
0.2679
0.1954
0.3624
0.3473
0.3302
0.4502
0.2908
0.3112
0.2960
0.1061
0.1424
0.2593
0.2071
0.2977
0.1846
0.3662
0.3744
0.2933
0.4036
0.2745
0.3886
0.2294
0.6924
0.6183
0.5311
0.5297
0.3274
0.4140
0.1667
0.2247
0.2617
0.1126
0.3903
0.2320
0.0922
0.0781
0.0421
0.0264
0.0541
0.1070
0.2061
0.1050
0.0536
0.1148
0.0336
0.0444
0.0681
467.30
439.48
396.40
467.79
290.48
442.64
528.60
551.94
408.92
639.01
408.62
338.30
502.96
178.75
54.22
78.16
85.71
60.73
58.70
103.26
200.00
142.03
211.01
183.96
98.37
156.54
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14
15
16
17
18
19
20
Total
Mean
Std. Error
32.59
12.55
59.14
56.38
67.24
74.98
40.05
686.15
34.31
29.41
0.1329
0.1387
0.2410
0.2485
0.2097
0.1404
0.3968
5.1588
0.2579
0.0973
0.1461
0.1857
0.1556
0.1862
0.2595
0.1118
0.3253
4.9640
0.2482
0.0945
0.6879
0.6088
0.5550
0.4228
0.5086
0.7375
0.2420
8.4929
0.4246
0.1928
0.0331
0.0670
0.0485
0.1424
0.0222
0.1027
0.0360
1.4774
0.0739
0.0460
357.97
439.08
635.44
473.02
656.45
363.06
581.50
9388.96
469.45
103.49
47.56
60.88
153.11
117.54
137.66
50.99
230.71
2409.89
120.49
59.16
Throughout observations showed that most of the petiole diameter with larger size had petiole shorter
in length. This explained that the plant parts were not influencing each other growth developments.
Oppositely, a strong relationship was shown between the petiole diameter and leaflets area. The
2
regression value obtained was R =0.8524 with the equation of y = 416.14x - 4700.9 (Figure 2d). This
indicated that both of the vegetative parts were significantly interacted to each other (p=6.639E-09).
This explained that both of the vegetative parts were significantly influenced each other growth
development as larger petiole diameter indicated larger area for leaflets. According to Barden,
Halfacre and Parrish (1987), the plant petiole functional as water and mineral transporter to the overall
plant parts, whilst the leaflets contained green pigmentation called chlorophyll that involved in the food
production process; the photosynthesis. Each of the vegetative parts was interacted to each other by
supplying food and water for growth developments. Additionally, both of the vegetative parts were
closely attached to each other that reasonably explained the strong relationship occurrences.
As for the relationship between the petiole and tuber diameter showed in Figure 2(e), resulted a
positive interaction occurrence between the vegetative parts. The regression value analysed was
2
R =0.5869 with the equation of y = 3.074x - 6.5124. The growth of the petiole and tuber diameter
was significantly interacted by influencing each other growth developments (p=8.20044E-05). As
the both of the vegetative parts were fused to each other, the plant tuber functional as storage
organ that supplied reserved food in a form of starch to other plant parts through petiole that acts
as the plant transporter (Barden, Halfacre and Parrish, 1987). Thus, this explained the significant
relationship in growth between the petiole and tuber diameter. Meanwhile, the relationship between
the petiole length and tuber fresh weight was insignificantly influenced each other growth
2
developments (p=0.5186) as the regression value obtained was R =0.0235, under the equation of
y = 2.2474x + 22.127 (Figure 3a). This indicated that no significant relationship occurred, as the
vegetative parts were poor in interaction. The similar result was showed for the relationship
2
between the petiole length and tuber dry weight (p=0.6332). The regression value was R =0.0129,
in which y = 0.2826x + 5.762 (Figure 3b).
As for the relationship showed between the petiole length and petiole diameter, the petiole length
was insignificantly influenced the growth of the petiole diameter (p=0.13903). There was no
significant relationship between the vegetative parts as the value for regression analysis was
2
R =0.1175, in which y = 0.2853x + 7.6161 (Figure 3c). The leaflets areas of the plant species
showed no significant relationship with the petiole length as the interaction occurred between the
vegetative parts were poor. The analysis of the regression value was not significant (p=0.1305)
2
with R =0.1226, in which y = 131.36x - 2083.6 (Figure 3d). The regression value analysed for the
2
relationship between the petiole length and tuber diameter was R =0.0187, in which y = 0.457x +
35.227 (Figure 3e). This showed that the interaction occurred between the vegetative parts was
poor as no significant relationship was indicated by both of the vegetative parts (p=0.5651).
Growth was the process by which a plant increased in the number, size and weight of a cell, organ
or whole organism (Rayburn, 1993). Whole plant development was the orderly and progressive
changed from seed germination through juvenility, maturity, flowering and fruiting, in which
environmental factors may influenced development times or block particular stages altogether
(Ames and Johnson, 2004). According to Rofidza Sendi (2004), plant developed as long as it lived.
The interaction between the petiole diameter and other vegetative parts such as tubers fresh and
dry weight, tuber diameter, petiole length and leaflets area was significant in relationships. More
than 50% of the growth developments that indicated by the plant vegetative parts were significant
(p<0.05) except for petiole diameter and length. Even so, the relationships were still considered to
be poor with less than 70% of interaction occurred among the vegetative parts. The petiole
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diameter and leaf area indicated the greatest of interaction among the plant vegetative parts with
more than 80% of the growth development was significantly related. Meanwhile, the plant length
interaction with other plant vegetative parts showed no significant relationship, as the relationships
among the plant vegetative parts were less than 15%. This indicated that the petiole length
influenced none of the vegetative parts growth developments. These differences were possibly
because of the competitions occurred between the plant species in their habitat. Usually, plants
such as A. brachyphyllus that grew in a cluster and in the same habitat competed for light, space,
nutrient or water would affect the growth of the vegetative parts. According to Bannister (1980), two
(or more) species grown together at the same overall density but varying proportions still
contributed to competition for the purpose of survival.
2b
y = 13.512x - 150.93
R2 = 0.589
500
400
Tuber dry weight (g)
Tuber fr esh weight (g
2a
300
200
100
0
0
10
20
30
40
y = 2.1169x - 24.382
80
R2 = 0.5023
60
40
20
0
-20 0
10
Petiole diameter (cm)
30
40
Petiole diameter (cm)
2d
2c
y = 0.4118x + 35.381
80
2
R = 0.1175
Leaf area (cm2)
Petiole length (cm)
20
60
40
20
0
0
10
20
30
40
y = 416.14x - 4700.9
12000
10000
8000
6000
4000
2000
0
R2 = 0.8524
0
Petiole diameter (cm)
10
20
30
40
Petiole diameter (cm)
Tuber diameter (cm)
2e
y = 3.074x - 6.5124
150
R2 = 0.5869
Figure 2 The relationship between petiole
diameter and other vegetative parts of A.
brachyphyllus; 2a) Petiole diameter and
tuber fresh weight, 2b) Petiole diameter and
tuber dry weight, 2c) Petiole diameter and
petiole length, 2d) Petiole diameter and
leaflet area and, 2e) Petiole diameter and
tuber diameter.
100
50
0
0
10
20
30
40
Petiole diameter (cm)
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3b
y = 2.2474x + 22.127
R2 = 0.0235
500
400
Tuber dry weight (g)
Tuber fr esh weight (g
3a
300
200
100
0
0
20
40
60
80
Petiole length (cm)
R2 = 0.0129
60
40
20
0
0
20
40
60
80
60
80
Petiole length (cm)
3c
3d
y = 0.2853x + 7.6161
40
30
20
10
0
0
20
y = 131.36x - 2083.6
10000
R2 = 0.1175
Leaf area (cm2)
Petiole diameter (cm)
y = 0.2826x + 5.762
80
40
60
R2 = 0.1226
8000
6000
4000
2000
0
0
80
20
40
Petiole length (cm)
Petiole length (cm)
Tuber diameter (cm)
3e
y = 0.457x + 35.227
150
R2 = 0.0187
100
50
0
0
20
40
60
80
Figure 3 The relationship between petiole
length and other vegetative parts of A.
brachyphyllus; 3a) Petiole length and tuber
fresh weight, 3b) Petiole length and tuber dry
weight, 3c) Petiole length and petiole diameter,
3d) Petiole length and leaflet area and, 3e)
Petiole length and tuber diameter.
Petiole length (cm)
The determination of plant growth developments either under different level of light intensity or the
plant interaction to the environment surrounding and condition was extremely important. This was for
the purpose of gaining more knowledge on the plant species background and growth characteristics
as it was considered to be new in the field. The manifestation of the study conducted on the effect of
light on vegetative growth of A. brachyphyllus under different level of shadings showed that the plant
species responded well under higher percentage of shade level for better developments. The result of
the study showed that the plant species were highly fertilized under 75% and 50% shading compared
to direct sun exposure (open shade) or 0% shade level. The fertility of A. brachyphyllus was
determined and observed through the changes occurred throughout the period of the plant growth
developments. The inclusive of changes occurrences were the increased of leaflets number, petiole
length and tuber diameter. Besides, the level of shading was also significantly influenced the total of
dry weight, leaf area and plant biomass.
The growth pattern of A. brachyphyllus at Mount Kasat, Sarikin, Bau showed that most of the plant
ground surface petiole diameter were ranged between 11.31 mm to 34.64 mm with the length
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measured between the ranged of 28 cm to 65 cm. Besides, the plant species indicated a wide range
of variation based on the biological production. Meanwhile, the petiole diameter was significantly
related (p<0.05) with other vegetative parts except the plant petiole length. The strongest interaction
2
was indicated between the petiole diameter and leaflet area (R =0.8524, which y = 416.14x - 4700.9).
Based on the study and observations conducted at the study area showed that A. brachyphyllus often
grew well with the response of higher in petiole length, from the largest trees such as durian, buah
terap, asam mempelam and others. Thus, this indicated that the plant species grew better in moderate
shading. The forest structure of Mount Kasat showed that the five dominant species occurred in the
habitat of A. brachyphyllus were Mallotus korthalsii, Artocarpus sericicarpus, Aglaia discolor,
Goniothalamus uvarioides and Durio zibethinus with their important value (IV) of 18.36, 10.42, 9.55,
8.90 and 8.50 respectively.
Throughout the study conducted, Amorphophallus species showed a unique and interesting
characteristic from the aspect of growth pattern, flower and fruit productions, leaf arrangement and
propagation. Thus, the unequalled of Amorphophallus species was potentially grew as landscape
plant. Besides, the tuber of the plant species was economically important especially for food resource.
This plant storage organ also contributed for medical purposes, in which the contain of the plant tuber
was low in calorie and suitable for balance diet. So, the importance of the plant species should be
exposed to the community especially the local resident, besides to conserve the plant species from
extinction and as an addition to the income or earnings of the residents. As suggestion, the
understanding of Amorphophallus species lifecycle was extremely important in requirement as the
purpose of ensuring the survival of the plant species. The study on the environment, climate
compatibility and adaptation of the plant species was recommended while the biomass allocation in
control parameter condition such as in the greenhouse was essential for addition knowledge on the
plant growth pattern.
Besides, the study on the forest structure and the relationships of the plant species to the environment
area or its habitat should be conducted, for detail and precise information. Relatively, the study such
as the level of light intensity, optimum temperature requirement, soils type and composition was
important in determining the factors that influenced the rate of plant growth and successfulness.
Importantly, the facts and information on the plant species background could be added as lack of
study and research conducted especially in the land of Sarawak.
References
Anderson, J.A.R. 1980. A Checklist of the Trees of Sarawak. Dewan Bahasa dan Pustaka, Sarawak.
Barden, J., Halfacre, G. and Parrish, D. 1987. Plant Science. McGraw-Hill Publishing Company.
HARTMANN, Hudson T., KESTER, DAVIES, Jr. Fred T. and GENEVA, Robert L. 2002. Plant
th
Propagation: Principles and Practices. 7 ed. Prentice Hall Career & Technology, Upper
Saddle River, New Jersey 07458 U.S.A.
Hetterscheid, W. and Ittenbach, S. 1996. Everything You Always Wanted to Know About
Amorphophallus, but Were Afraid to Stick Your Nose Into!, Aroideana. 19: 7-131.
εcεAHON, εargaret J., KOFRANEK, Anton ε. and RUBATZKY, Vincent E. 2002. Hartmann’s Plant
rd
Science: Growth, Development, and Utilization of Cultivated Plants. 3 ed. Prentice Hall
Career & Technology, Upper Saddle River, New Jersey 07458 U.S.A.
Mohd. Imransyah (2003). Response of Rottboellia exaltata (Lour) to Shading. Faculty of Resource
Science and Technology, University Malaysia Sarawak, Kota Samarahan.
Rofidza Sendi (2004). Kajian Corak Populasi dan Allokasi Biojisim Amorphophallus Hewittii (Aldrew.)
dan Amorphophallus Brachyphyllus (Hett.) Di Gunung Aup, Sarikin, Bau. Fakulti Sains dan
Teknologi Sumber, Universiti Malaysia Sarawak, Kota Samarahan.
Roland Charles Singka (2004). Response Amorphophallus Borneensis (ENGL) Terhadap Lindungan.
Fakulti Sains dan Teknologi Sumber, Universiti Malaysia Sarawak, Kota Samarahan.
Rosmawatti Binti Zaini (2004). Ecology of Amorphophallus Borneensis at Gunung Gayu, Padawan.
Faculty of Resource Science and Technology, University Malaysia Sarawak, Kota Samarahan.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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TAXONOMIC REVIEW ON SELECTED VATICA SPECIES (DIPTEROCARPACEAE) OF SARAWAK
BASED ON LEAF MORPHOLOGICAL
Meekiong, K.
1, 2
2
, A. Latiff , C.S. Tawan
1
1
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
2
School of Environmental and Natural Resources Sciences, Faculty of Science and Technology,
Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor
Email: aqmuzzammil@frst.unimas.my
Abstract
The genus Vatica L. is the third largest natural groups in the family Dipterocarpaceae and Borneo with
thirty-five species has the largest representative and being considered as centre of diversity of this
genus. The Vatica species are the most difficult dipterocaros to recognize and this explain why this
common genus in our forests remains so poorly known. These project aiming to employ the details of
leaf characteristics to be used for identification purposes. Fifteen Vatica species were selected as
preliminary examination. Initial results showed a significant used of the leaf characteristics for
identification.
Keywords: Dipterocarpaceae, Vatica, Scanning Electron Microscopic (SEM), leaf morphological
1. INTRODUCTION
The genus Vatica L. is the third largest natural groups in the family Dipterocarpaceae, after genus
Shorea (169 spp.) and Hopea (104 spp.) with 71 species have been botanically described. This
valuable genus distributed from India, Sri Lanka to Myanmar, Indochina, Peninsular Malaysia,
Indonesia, Borneo, Papua New Guinea and the Philippines (Srinual and Thammathaworn, 2008).
Borneo with thirty-five species has the largest representative (Symington, 2004) and being considered
as centre of diversity of this genus. In Sarawak, 32 species have been recorded that occurring
especially in mixed and upper dipterocarp forests and also non-saline habitats at elevation from sea
level up to about 1,800 m altitude (Ashton, 2004). The Vatica species are distinguished from other
genera of the Dipterocarpaceae by the absence of looped intra-marginal nerves on the leaves, the
winged fruits enclose less than half of the nut, anthers are glabrous and the style is stout (Ashton,
1982; Pooma and Newman, 2001; Srinual and Thammthaworn, 2008).
Systematic study on the genus Vatica have been carries out by many researchers, among them are
Ashton (1982; 2004); Maury-Lechon and Curtet (1998); Smitinand et al., (1980); Srinual and
Thammthaworn, (2008) and Symington (2004). Molecular phylogeny studies also have been address
by i.e. Indrioko et al., (2006) and Cao et al. (2006) to add on valuable taxonomic information on the
family Dipterocarpaceae. Anatomical features of the leaves in the Dipterocarpaceae in terms of
paracytic stomata; the presence of large crystals and resin canals in the mesophyll was first
highlighted by Solereder (1908). Metcalfe and Chalk (1957) reported the important of stomata types,
distribution and types of trichomes, the resin canals, the crystals in the mesophyll as informative
characteristics. Studies by Tewary and Sarkar (1985); Rojo (1987) and Srinual and Thammthaworn
(2008) added on the important characteristics on the leaf features in the genus Vatica. Recent study
by Meekiong et al. (2012) also highlighted an important of the micro morphological of the leaves
characteristics to supporting identification purposes, particularly into intra species level.
2. MATERIALS AND METHODS
Fresh specimens used for this work were obtained from various places in Sarawak, e.g. Matang,
Lundu, Sarikei, Lambir and Mukah. Herbarium specimens were obtained from the Herbarium of
Sarawak Forest Department (SAR) and Herbarium of Universiti Malaysia Sarawak (HUMS). The leaf
characteristics were examined and compared among the fifteen selected Vatica species. Among the
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parameters measured were includes shaped of lamina, leaf-base, apex, length and width, number of
secondary veins, number of gland marks and appearances such as indumentums on the leaf surfaces
and petioles. For Scanning Electron Microscopic (SEM), leaves of fresh specimens of the selected
species that were collected in the field were fixed in 80 percent of Formalin-acetic acid-alcohol (FAA)
(modification from Matinez-Cabrera et al., 2009). While leaves from the herbarium specimens were
rehydrated in 5 percent NaOH at 60º C for one hour and fixed in FAA for 24 hours (Martinez-Cabrera
et al., 2009). Five critical points on the leaf, e.g. petiole, midrib, margin, lamina and glandular mark
were selected and cut into smaller potions, 1 cm squares for observations under the Scanning
Electron Microscopic (SEM). The samples then were undergoes into process of Critical Point Drying
(CPD) before coated with the Pallidium (Pd) fine particles using Auto Fine Coater (model JEOL JFC1600). The coated samples then placed into the Analytical Scanning Electron Microscopic (model
JOEL JSM-6390LA). The micro morphological on the leaf surface was observed via various
magnifications. The presence of indumentums and suspicious textures on the leaf were observed,
noted and analysed.
3. RESULTS AND DISCUSSION
The results showed comparatively significant on the leaf morphological for identification purposes.
Based on field observations and details laboratory examination, generally the leaves of Vatica species
divided into two major groups, noticeable intra marginal loops with gland marks and obscure intra
marginal loops without gland mark. The first group, are readily distinguishable from other genera while
the second group a bit difficult as it might be confused with other genera, Aporosa from the family
Euphorbiacea and Hydnocarpus from the family Sterculiaceae but the scent of dammar is always
discernable in freshly rubbed living tissue.
The parameters of leaf characteristics were also closely examined and the results revealed that some
of the characteristics were significantly important to distinguish into species level. For example, among
the fifteen of Vatica species that used for this study, V. coriacea was the only species that having
revolute margin. The characteristic parameters were set into trinomial groups; for example 0 = short, 1
= medium and 2 = long etc, details as showed in Table 1.
Table 1 Leaf characteristics parameter based on trinomial coded system (0, 1 and 2).
Petiole
Leaf blade
Leaf
Leaf
Leaf
Species
shaped Length Diam. colour Length Width margin apex
Leaf
base
Resin
gland
V. pedicellata
0
0
1
1
1
0
0
0
1
2
V. brunigii
2
0
1
1
1
1
0
0
0
1
V. oblongifolia
1
1
2
1
2
1
0
0
0
1
V. globosa
2
0
0
1
1
0
0
2
0
0
V. sarawakensis
1
1
2
0
2
2
0
0
1
2
V. compressa
2
1
1
1
1
1
0
0
1
1
V. micrantha
0
0
0
1
1
0
0
0
1
0
V. coriacea
2
0
2
0
2
2
1
1
1
2
V. badiifolia
0
1
2
0
2
2
0
0
0
2
V. albiramis
0
1
1
1
1
0
0
0
0
0
V. nitens
1
1
2
1
2
1
0
0
0
0
V. parvifolia
2
0
0
1
0
0
0
0
1
0
V. borneensis
0
1
0
1
0
0
0
0
0
0
V. brevipes
0
1
1
1
2
1
0
0
0
0
V. maingayi
0
1
1
1
1
1
0
0
0
0
The values of each parameter were then entered into PAST software for similarities and differences
analysis. The tree dendrogram than was generated to showed relationship among the species based
on similarities and differences as showed in Figure 1.
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Figure 1 The tree dendrogram on the species relationship of 15 selected Vatica species based on the
leaf morphological characteristics generated by the PAST software.
Preliminary Scanning Electron Microscopic (SEM) analysis resulted significant characteristics of leaf
micro morphological for identification of intra specific level. Type of trichomes, number of trichome’s
segment, type of stomata and the sizes of stomata were among the parameters observed during the
study (Figure 2). Two major types of trichomes were identified that presence on almost all the fifteen
selected Vatica species, stellate and peltate. The average number of trichome’s segments was 8 – 27
with average length 44.40 m - 51.64 m. Two types of stomata were identified found on the Vatica leaf
were Actinocytic and Cycloctic with average length 15.20 m - 32.85 m.
Figure 2 Micro morphological
observed on the Vatica leaf
via SEM.
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4. CONCLUSION
Some of the leaf characteristics are significantly important for identification purposes, particularly into
intra specific level. However, the results yet to be finalised as the project is still on-going.
Acknowledgement
We would like to acknowledge the Universiti Malaysia Sarawak (UNIMAS) for providing the fund
through Dana Khas Penyelidikan (DK18/2010) to support part of this research. Thanks to Forest
Department Sarawak, particularly the Herbarium of Forest Research Centre that given us permission
with care on the specimens during identification. Thanks also due to En. Safri for the SEM images,
Hidir Marzuki, Sekudang Tedung and Shafiq Sahat for their helps in the field.
References
Ashton, P.S. (2004). Dipterocarpaceae. In: Soepadmo, E., Saw, L.G. & R.C.K. Chung (eds.). Tree
Flora of Sabah and Sarawak, Vol.5: 63 – 388.
Ashton, P.S. (1982). Flora Malesiana. Series I-Spermatophyta. Martinum Nijhoff Pub. The
Netherlands.
Cao, C.-P., Gailing, O., Siregar, I., Indrioko, S. & R. Finkeldey (2006). Genetic variation at AFLPs for
the Dipterocarpaceae and its relation to molecular phylogenies and taxonomic subdivisions.
Journal of Plant Research, 119: 553 – 558.
Indrioko, S., Gailing, O. & R. Finkeldey (2006). Molecular phylogeny of Dipterocarpaeae in Indonesia
based on chloroplast DNA. Plant Systematic and Evolutions, 261: 99 – 115.
Kocsis, M., Darok, J. & A. Borhidi (2004). Comparative leaf anatomy and morphology of some
neotropic Rondeletia (Rubiaceae) species. Plant Systematic & Evolution, 248: 205 – 218.
Martinez-Cabrera, D., Terrazas, T. & H. Ochoterena (2009). Foliar and petiole anatomy of tribe
Hamelieae and other Rubiaceae. Annals of Missouri Botanic Garden, 96: 133 – 145.
Maury-Lechon, G. and Curtet, L. 1998. Biogeography and evolutionary systematics of
Dipterocarpaceae. In: Appanah S. and Turnbull J. M. (eds.). A review of Dipterocarps:
Taxonomy, Ecology and Silviculture. Centre for International Forestry Research (CIFOR),
Indonesia, pp. 5-44.
Meekiong, K., Latiff, A., Tawan, C.S. & Miraadila, M.A. (2012). Leaf micro-morphological
characteristics of selected Vatica species (Dipterocarpaceae) from Kubah National Park,
Sarawak. Paper presented for Malaysian Science and Applied Biology Seminar, Kuala
Terengganu, 1 – 3 June 2012.
Metcalfe, C. R. and Chalk, L. 1957. Anatomy of the Dicotyledons. Vol. I. Oxford University Press,
London. 220 pp.
Noraini, T. & D. Cutler (2007). Systematic significance of petiole and midrib anatomical characters in
Parashorea (Dipterocarpaceae) of Malaysia. Malaysian Applied Biology, 36(2): 47 – 55.
Pooma, R. & M. Newman (2001). Checklist of Dipterocarpaceae in Thailand. Thai Forest Bulletin
(Botany) 29: 110 – 187.
Rojo, J. P. 1987. Petiole anatomy and infrageneric interspecific relationship of Philippine Shorea
(Dipterocarpaceae). In: Kostermans A. J. G. H. (ed.). Proceedings of the Third Round Table
Conference on Dipterocarps. Papers presented at an International Conference Held at the
Mulawarman University, Indonesia. pp. 573-598.
Smitinand, T., Santisuk, T. & Phengklai, C. (1980). The Manual of Dipterocarpaceae of Mainland
South-East Asia. The Forest Herbarium, Royal Forest Department, Bangkok: 133 pp.
Solereder, H. 1908. Systematic anatomy of theDicotyledons. The Clarendon Press, Oxford.
Srinual, A. & A. Thammathaworn (2008). Leaf anatomy of Vatica L. (Dipterocarpaceae) in Thailand.
The Natural History Journal of Chulalongkorn University 8(2): 121 – 134.
nd
Symington, G.F. (2004) 2 Edition. Foresters’ εanual of Dipterocarps. Malayan Forest Records,
No.16. A joint publication by Forest Research Institute Malaysia and Malaysian Nature Society:
441 – 483.
Tewary, P. K. and Sarkar, A. K. 1985a. Leaf epidermal studies in Dipterocarpaceae-The genera Vatica
L. and Vateria L. Journal of Plant Anatomy and Morphology, 2: 67-72.
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SPECTROSCOPY PROFILING OF TRUNKING SAGO PALM (Metroxylon sagu Rottb.) USING
NUCLEAR MAGNETIC RESONANCE (NMR)
1
1
Mohd. Hasnain Hussain , Yan Wei-Jie , Zainab Ngaini
1
2
2
Department of Molecular Biology, Department of Chemistry, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
E-mail: hhasnain@frst.unimas.my
Abstract
A basic study in the variation of metabolites in trunking sago palm using photon NMR analysis method
is reported for the first time. Metabolites extracted by polar and non-polar solvents were analyzed and
1
compared by H-NMR spectra and further foolproof with FT-IR analysis. Result from nuclear magnetic
resonance, thin layer chromatography and FT-IR analysis shows insignificant variances metabolites
spectra pattern between the trunking sago palm trees. External result comparison with non-trunking
sago palm NMR spectroscopy metabolite pattern shows insignificant variances.
Keywords: sago, trunking; NMR; metabolites; FT-IR
1. INTRODUCTION
Sago palm (Metroxylon sagu Rottb.) is one of the top 5 important agricultural plants in Malaysia. In
2006, export of sago palm agricultural product exceeded RM 42.955 million and increased to
RM51.407 million at the beginning of 2007 which contributed to approximately 1.28 % and 0.99 % of
the total export value respectively. Sago palm plantations in Malaysia are mainly located in Sarawak
state where there were 55,690 hectares of sago plantation in 2006 and increased to 56,916 hectares
in 2007. These are mainly in located Dalat, Mukah giving approximately 45.95 % and 44.96 % total
land used for sago palm plantation in Sarawak in the year 2006 and 2007 respectively (Department of
Statistics Sarawak).
Sago starch, which is the main product of export in sago palm, is located at the pith in the trunk of the
sago palm which means that the economical value of sago palm is located in its trunk. In sago palm
plantation, there are sago palms which did not form trunk after the 8 year cycles and labelled as nontrunking sago palm especially at the deep peat soil areas (Singhal et al., 2008). This issue brings an
economical lost in sago palm plantation which reduce the production of sago starch per hectares of
land.
Metabolic fingerprinting is a direct comparison of metabolite composition via spectral pattern analysis
without the need for the identity of the initial components. Individual peaks showing heterogeneous
distribution are then identified to show their variances and, if necessary, be further analyzed for detail
of the variation. Analysis have been done on several plant species such as rose (Young et al., 2004),
opium (Hagel et al., 2008) and peas (Charlton et al., 2008). Metabolic profiling on the other hand
defines the separation and identification of as many extract components as possible. Manipulation of
extraction, separation, detection and comparisons technologies are important to identify the metabolite
present in the extracted samples (Griffin, 2006).
The expressions of genes in non-trunking sago palm are the main concern to overcome the problem
because the growth and development of plants are regulated by genes. Hence, comparison of gene
expression in trunking and non-trunking sago palm can determine the difference gene expression of
non-trunking sago palm which contributed to the problem stated (Krishnan et al., 2005). Direct
sequencing of the sago palm DNA is costly and do not show the expression of the gene but only show
the variation of genetic codes if any between the sago palm which may not contributing to the nontrunking problem. As the result, the analysis of plant metabolites was selected representing the
expression of gene in sago palm. This test is to determine the variation of leaves extract in the
1
trunking sago palm population using broad spectrum H-NMR analysis which confers the accuracy of
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the later test where determination of gene expression via comparison of leaves extract of non-trunking
sago palm and trunking sago palm
2. EXPERIMENTAL
2.1 Sample preparation
Crude sample was obtained from PELITA Dalat Mukah Sago Plantation in Mukah, Sarawak.
Randomization in samples collection is to reduce selection bias and improve the result value. The
plant selected were around 96 months old where the metabolites produce during the bole formation
rd
growth stage. Sample leaves taken were the 3 frond from the latest developing frond (or from the
centre/crown). Sample pinna leaf blades taken were number 5 from the tip of the frond pinnate to
eliminate the variance due to difference in development stages (Cirak et al., 2007). Sample pinnae
leaves were cleaned with 70% ethanol and stored in -20 °C freezer at the interval of 2 days after
petiole was removed from box storage at – 4 °C.
The petiole from the 5 crude leaf samples were removed, cut into a smaller fragments, separated into
5 different 50 ml polyethylene tube as crude stock and labelled. 4.0 g for organic-base extraction and
0.12 g for water-base extraction of sago palm leaf fragment was cut, weight and grind to powder using
ceramic mortar and pestle using liquid nitrogen (Sobolev et al., 2005). Three replicates of each sample
and one negative control were prepared for organic-base extraction and water-base extraction
(Sumner et al., 2007).
2.2 Metabolite extractions
2.2.1 Water-based extraction
Phosphate buffer (0.05 M sodium phosphate acid, 0.05 M EDTA, pH 6.54) was mixed with powdered
sago palm leaves. The mixture was then homogenized, supernatant containing leaf metabolites was
then filtered, transferred into a polyethylene tube and freeze-dried. Sample was then stored at
desiccator to removes remaining moisture. Three replicates and a negative control were prepared for
each sample of five. Thin layer chromatography (TLC) using hexane/ethyl acetate (1:3) was done
before NMR analysis to determine primary component in the solvent (Franz et al., 2004). Deuterium
Oxide (D2O) was used as sample solvent during NMR analysis which to reduce the noise which will
cause by the normal H2O as solvent.
2.2.2 Organic-based extraction
Powdered leaf tissue was homogenized with 0.15 M acetic acid. Fatty acids from the mixture were
removed by using hexane and vacuum filtered. The pellet was extracted using chloroform/ methanol
(Bligh and Dyer, 1959). Chloroform and distilled water were then added into the mixture, mix and
vacuum filtered. Pellet was re-extracted using the same method and the mixture was pooled.
Chloroform fraction’s supernatant was pooled by funnel separation and dried using rotovap. Sample
was then stored at desiccator to removes remaining moisture. Three replicates and a negative control
were prepared for each sample of 5. TLC was done using hexane/ethyl acetate (3:1). Deuterated
chloroform (CDCl3) (5 ml) solvent was used during sample preparation for NMR analysis to reduce
noise which cause by the normal CHCl3 as solvent.
2.3 Spectroscopy Analysis
2.3.1 NMR Analysis
The analysis was done using JEOL ECA 500 Delta2 NMR spectrometer with field strength of 11.7474
1
Tesla. Broad band probe (5 mm) tuned to detect H signal at 500.16MHz and magnetic field was
locked at D2O for water extract and CDCl3 for organic extract samples. A relaxation delay of 5 sec was
inserted to ensure quantitative data were acquired and the probe head was maintained at the
temperature of 300 K. One replicate from each sampled tree and 1 negative control in water base
1
extract and organic base extract were analysed by H-NMR.
2.3.2 Infrared Analysis
One replicate from each samples and one negative control in water base extract and organic base
extract were run using Perkin Elmer spectrum GX 1 v5.0 FT-IR spectroscopy. Blank NaCl cell were set
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as the background for each sample and control in water base extracts and organic base extracts. The
data was identify using web tool IR-wizard (http://www.science-and-fun.de/tools/) and reviewed
manually using a standard table of wave number range to functional groups and classes of
compounds absorb in the infrared. Samples variance compared manually just by the present and the
absent of the functional group of the samples.
3. RESULTS AND DISCUSSION
3.1 TLC comparisons
3.1.1 Water soluble metabolites
The result shows that the extract of the water samples do not interact with the mobile phase of and
hence there are no migration of the samples extracts shows in the TLC separations.
A more polar solvent combination such as ether/pentane, ethanol/hexane or pentane and
dichloromethane/hexane or pentane should be used as solvent in the water base extract metabolites
so that the metabolite can travel along the silica layer for separation as suggested in Franz et al.
(2004). Franz et al. (2004) also suggested that usage of different solvent combination to obtain a
complete plant extract separation is needed. Metabolites from organic solvent did not shows complete
separation in the cluster of green and brown metabolites
Figure 1 TLC of water soluble metabolites of M. sagu in D2O from the left; Control, Sample 1,Sample
2, Sample 3, Sample 4 and Sample 5.
3.1.2 Organic soluble metabolites
The result shows that there are migration of the samples extracts but not a complete separation
around the green and brown cluster shows in the TLC separations.Visible color pigment from plant
leaves can be categorized as follows respective to their migration length; Carotenes (Golden),
Pheophytin (Olive Green), Chlorophyll A (Blue Green), Chlorophyll B (Yellow Green), Lutein (Yellow),
Violaxanthin (Yellow).
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Figure 2 TLC of organic soluble metabolites of M. sagu in D2O from the left; Control, Sample
1,Sample 2, Sample 3, Sample 4 and Sample 5.
3.2 NMR analysis
3.2.1 Water soluble metabolites
All samples and the negative control shows the same 4 major peak of D 2O, EDTA, and H2O which
means that there are no variation between all the samples but since the results of this samples are
similar to the negative control, the 1H-NMR spectroscopy shows that there are no or insignificant
metabolites presents in the water extract. The two peaks (3.2 ppm and 3.6 ppm) shows by the NMR
spectra similar to the EDTA spectra in AIST Spectral Database for Organic Compounds SDBS which
are shifted downfield a bit compared to the result due to difference solvent used in the database
sample (DMSO) and the lower magnetic resonance (399.65 MHz) used. Similar shifting of H 2O
compared to the AIST database shown in the spectra supports the EDTA presenting peaks in the
spectra.
Figure 3 1H-NMR analysis of water soluble metabolites of M. sagu in D2O
3.2.2 Organic soluble metabolites
All the samples shows similar pattern of complex major peak due to the highly complex metabolites
mixture presents in the extract which means that there are no or insignificant variation between all the
samples. There are some water peak contamination in the spectra OS3R1 and OS4R1. NMR
spectrum of the negative control shows some similar peaks around the upfield with the samples
indicating there maybe presence of contamination in the control sample.
Heavy masking on the aliphatic fatty acid regions should be overcome by the fatty acid removal step
during hexane separation but the time for separation is not adequate for the total separation of the
fatty acid. It is recommended to perform the fatty acid separation for 24-48 hours in normal room
conditions for total separation. Presence of water contamination in OS3R1 and OS4R1 may due to the
error in chloroform/methanol/water layer separation. This error occur due to the entrapment of water
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bubble in the chloroform layer and hence to overcome this error, time required for complete phase
settlement or in certain cases centrifugation can be apply to enhance phase settlement.
Negative control (OS1C) showed contamination in the upfield of the spectrum but on the other hand,
there was no spot shown in TLC analysis for OS1C. This indicated that there were contaminations of
the OS1C during NMR analysis and the possible contaminations were the NMR 5 mm tube used
which was not cleaned appropriately before used. Careful washing methods of instruments are
required to reduce the error of the spectrum although the NMR spectroscopy has lower sensitivity. It is
suggested to run a blank solvent test before performing the actual test using NMR spectroscopy to
mask the effect of solvent and NMR instrument contamination.
1
Analysis based on the presences of chemical group detected using 1D H-NMR did not show reliable
results because the metabolite contained in the sample extract are complex and unknown and it is
hard to interprets the metabolite similarity among the samples based on the common chemical group
due to the highly overlapping of the chemical group from different metabolites. Nevertheless this
method can be improve to increase the reliability by using statistical software to compare the variation
of the spectrum itself using Principal Component Analysis (PCA) and Partial Least Square (PLS) on
the fixed extract weight.
Figure 4 1H-NMR analysis of organic soluble metabolites of M. sagu in CDCl3
3.3 Infrared analysis
3.3.1 Water soluble metabolites
Although there are certain differences between the peaks form the samples, but overall all the FT-IR
spectra show similarity up to 88.98% calculated using EssentialFTIR v1.50.22 software. The functional
group of hydroxyl at the range of 3564 – 3310 cm-1, and carboxylic salt at the range of 1642 – 1635
cm-1 in the FT-IR spectra may contribute by the present of EDTA in the buffer. There are other
functional groups shown in FT-IR spectra which are not detectable in NMR due to low concentration of
the respective compound in the solvent which the identity is not predictable due to the extract
complexity.
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Table 1 Comparison of FT-IR spectra with WS1R1 sample of water soluble metabolites of M. sagu
Metric
Filename
1
WS1R1.sp
0.99524
WS2R1.sp
0.98938
WS1C.sp
0.97337
WS4R1.sp
0.92248
WS3R1.sp
0.88986
WS5R1.sp
Table 2 Common functional group presents in samples of water soluble metabolites of M. sagu
Standard Range
IR Spectra Range
Chemical Group
Abbreviation
3600 – 3200
3564 – 3310
Hydroxyl
-OH
2160 – 2080
2093 – 2089
Alkynes
vC≡C
1670 – 1630
1642 – 1635
Carbonyl in 3° amides
RCOR
1400 – 1310
1406 – 1359
Carboxylic salt
-COO
-
84.4
80
70
60
2089
50
1406
40
1158
989
1079
30
20
%T
10
0
1642
3401
568
-10
-20
-30
-40
-44.2
4000.0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
370.0
cm-1
Figure 5 FT-IR analysis of water soluble metabolites of M. sagu in D2O (WS1R1)
3.3.2 Organic soluble metabolites
Although there are certain differences between the peaks form the samples, but overall the FT-IR
spectra shows similarity up to 88.65% except for sample OS5R1 calculated using EssentialFTIR
v1.50.22 software after truncated to the range of 4000 – 550 cm-1. All FT-IR spectra show the
presents 3435 – 3389 cm-1 of hydroxyl, 2955 – 2851 cm-1 of alkane and 1737 – 1733 cm-1 of
aldehydes functional group which confers the NMR analysis. Identities of other functional groups are
not predictable due to the extract complexity. Interpretation of the functional group presents in the
sample extracts is nearly impossible without any references because the samples extract is not a pure
component but a mixture of unknown components hence to predict the functional group in base on
personal opinion only without support. FT-IR analysis can be improves by the use of High
Performance Liquid Chromatography (HPLC) to separates the mixture compounds into pure
metabolite and refers with a standard.
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Appropriate equipment handling is essential to eliminates human error in the analysis. Variances
shown by the sample OS5R1 in FT-IR analysis which is highly different from the other samples are
due to the error in background scanning where the NaCl cell is not cleaned appropriately before scan
leaving the result of the reading as error.
Table 3 Comparison of FT-IR spectra with OS1R1 sample of organic soluble metabolites of M. sagu
Metric
Filename
1
OS1R1.sp
0.97572
OS3R1. sp
0.91448
OS4R1. sp
0.88648
OS2R1. sp
0.47028
OS1C. sp
0.11876
OS5R1. sp
Table 4. Common functional group presents in samples of organic soluble metabolites of M. sagu
Standard Range
IR Spectra Range
Chemical Group
Abbreviation
3600 – 3200
3435 – 3389
hydroxyl
-OH
2990 – 2850
2955 – 2851
Aliphatic alkane
-CHx
1740 – 1720
1737 – 1733
aldehydes
RCOH
101.2
95
719
557
90
1377
1737
85
1460
1161 1070
3390
80
75
2853
2954
70
65
2923
%T 60
55
50
45
40
35
30
25
21.0
4000.0
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
600
370.0
cm-1
Figure 6 FT-IR analysis of organic soluble metabolites of M. sagu in CDCl3 (OS1R1)
4. CONCLUSION
Overall the test shows that there are no significant variances in metabolites pattern of the samples
from different trunking sago palm leaf extract and hence we accept Ho. Comparison of the result with
non-trunking sago palm leaf extract shows that there are insignificant variances of metabolites pattern
1
in H-NMR between trunking and non-trunking sago palm (Amirrudin A., 2010). Further study can be
done in identifying the identity of the sample extract components and variance of metabolite contents
by using 2D NMR analysis to separates the masking and overlapping 1H-NMR spectrum for better
contrast (Lewis et al, 2007).
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Acknowledgements
The authors would like to thank Universiti Malaysia Sarawak for the facilities and funding under vote
22799, 27699 and 29999 to carry out the research.
References
Amiruddin A. (2010). Spectroscopy Profiling of Non-Trunking Sago Palm (Metroxylon sagu Rottb.)
using Nuclear Magnetic Resonance (NMR). Unpublished report. Universiti Malaysia Sarawak
Bligh, E. G. & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can. J.
Biochem. Physiol. 37:911-917
Charlton, A. J., Donarski, J. A., Harrison, M., Jones, S. A., Godward, J., Oehlschlager, S., Arques, J.
L., Ambrose, M., Cinoy, C., Mullineaux, P. M. and Domoney, C. (2008). Responses of the pea (Pisum
sativum L.) leaf metabolome to drought stress assessed by nuclear magnetic resonance
spectroscopy. Metabolomics, 4:312-327. doi:10.1007/s11306-008-0128-0
Cirak, C., Radusiene, J., Janulis, V., and Ivanauskas, L. (2007). Secondary metabolites in Hypericum
perfoliatum: variation among plant parts and phenological stages. Botanica Helvetica, 117:29 –
36. doi 10.1007/s00035-007-0777-z
Department of Statistics Sarawak, (n.d.). Estimated Area of Sago by District 2003 – 2007. Retrieved
July 27, 2009 from http://www.doa.sarawak.gov.my/statistik07_6_1.pdf
Department of Statistics Sarawak, (n.d.). Export of Sago Products 1998 – 2007. Retrieved July 27,
2009 from http://www.doa.sarawak.gov.my/statistik07_6_3_4.pdf
Department of Statistics Sarawak, (n.d.). Export Value of Agricultural Products 1998 – 2007. Retrieved
July 27, 2009 from http://www.doa.sarawak.gov.my/statistik07_1_9_10.pdf
Franz, K. J., Shea, K. M., Danheiser R. L., and Swager, T. M. (2004). Laboratory Manual.
Massachusetts Institute of Technology Department of Chemistry.
Griffin, J. L. (2006). The Cinderella story of metabolic profiling: does metabolomics get to go to the
functional genomics ball? Phil. Trans. R. Soc. B, 361:147–161. doi:10.1098/rstb.2005.1734
Hagel, J. M., Weljie, A. M., Vogel, H. J. and Facchini P. J. (2008). Quantitative 1H Nuclear Magnetic
Resonance Metabolite Profiling as a Functional Genomics Platform to Investigate Alkaloid
Biosynthesis in Opium Poppy. Plant Physiology, 147:1805–1821. doi:10.1104/pp.108.120493
Krishnan, P., Kruger, N. J., and Ratcliffe, R. G. (2005). Metabolite fingerprinting and profiling in plants
using NMR. Making Sense of the Metabolome Special Issue, Journal of Experimental Botany,
410(56): 255–265. doi:10.1093/jxb/eri010
Lewis, I. A., Schommer, S. C., Hodis, B., Robb, K. A., Tonelli, M., Westler, W. M., Sussman, M. R. and
Markley, J. L. (2007). Fast and Accurate Method for Determining Molar Concentrations of
Metabolites in Complex Solutions from Two-Dimensional 1H-13C NMR Spectra. Anal Chem,
79(24): 9385–9390. doi:10.1021/ac071583z
Singhal, R. S., Kennedy, J. F., Gopalakrishnan, S. M., Kaczmarek, A., Knill, C. J., Akmar P. F. (2008).
Industrial production, processing, and utilization of sago palm-derived products. Carbohydrate
Polymers, 72(1):1-20. doi:10.1016/j.carbpol.2007.07.043
Sobolev, A. P., Brosio, E., Gianferri, R. and Segre, A. L. (2005). Metabolic profile of lettuce leaves by
high-field NMR spectra. Magnetic Resonance in Chemistry (MRC), 43:625–638. doi:
10.1002/mrc.1618
Sumner, L. W., Amberg, A., Barrett, D., Beger, R., Beale, M. H., Daykin, C., Fan, T. W.- M., Fiehn, O.,
Goodacre, R., Griffin, J. L., Hankemeier, T., Hardy, N., Higashi, R., Kopka, J., Lindon, J. C., Lane,
A. N., Marriott, P., Nicholls, A. W., Reily, M. D., Viant, M. R. (2007). Proposed Minimum Reporting
Standards for Chemical Analysis. Metabolomics 3(3):211-221. doi: 10.1007/s11306-007-0082-2
Young, H. C.,Tapias, C. A., Hye, K. K., Lefeber, A. W. M., Erkelens, C., Verhoeven, J. Th.J., Brzin, J.,
Zel, J., and Verpoorte R. (2004). Metabolic Discrimination of Catharanthus roseus leaves infected
by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis. Plant Physiol,
135:2398–2410. doi:10.1104/pp.104.041012.
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FUNCTIONAL STUDY OF PREL2A IN ZEBRAFISH
Ai-Peng Tay* and Kui-Soon Lee*
Faculty of Resource Science & Technology, Universiti Malaysia Sarawak (MALAYSIA)
*Emails: aipeng.tay@gmail.com, kslee@frst.unimas.my
Abstract
Ena/VASP family proteins are conversed family of action regulatory proteins. It acts as important
regulator of cell migration by assembly and undergoes rearrangement during lamellipodia and
filopodia formation. Proline-rich EVH1 Ligand (PREL) family proteins are adaptor proteins consist of
Pleckstrin Homology (PH) domain and Ras-association (RA) domain. It has reported as one of the
binding partner of Ena/VASP proteins and colocalizes with them at the tips of lamellipodia and at focal
adhesions in respond to Ras activation. However, the exact mechanisms on how is the interaction
between PREL and Ena/VASP family proteins remain largely unknown. In zebrafish, we have
indentified three members of PREL family proteins (PREL1, PREL2a and PREL2b). Hence, in this
study the functional roles of PREL2a were studied through gene expression pattern in zebrafish. To
establish the expression pattern of PREL2a in developing zebrafish, total RNA was isolated from 10
embryonic stages of zebrafish which comprise of 3hpf, 8hpf, 11hpf, 18hpf, 24hpf, 36hpf, 48hpf, 72hpf,
96hpf and 120hpf. Reverse Transcriptase-PCR revealed that PREL2a was expressed along the
embryonic development in zebrafish, started from 3hpf and remained until 120hpf. The findings
provide and insight that PLEL2a plays important roles during zebrafish embryonic development.
Keywords: PREL2, Ena/VASP, Zebrafish
1. INTRODUCTION
The actin cytoskeleton dynamic represents fundamental of molecular machinery in the regulation of
cell adhesion, cell migration and polarity in respond to the extracellular signaling. Remodeling of action
cytoskeleton provides the force required for cell motility, structural changes needed for cell shape
modulation and intracellular anchoring support for adhesion. Axon guidance and T-cell polarization are
examples of process with activated in response to motility changes due to environmental signaling
(Bailly, 2004; Krause, Dent, Bear, Loureiro & Gertler, 2004).
Ena/ Vasodilator-stimulated phosphoprotein (VASP) family protein are conversed family of actin
regulatory proteins. It acts as important regulators of cell migration by assembly and undergoes
rearrangement during lamellipodin and filopodia formation (Bailly, 2004; Jenzora, Behrendt, Small,
Wehland & Stradal, 2005; Krause et al., 2003). In addition, Ena/VASP proteins family also involves in
modulate morphology and behavior of membrane protusions (Bear et al., 2002).
The study of Proline-rich Ena/VASP Ligand (PREL) becomes significantly important when Krause et
al. (2002) identified PREL2 as an Ena/VASP binding protein. PREL2 and Ena/VASP proteins
colocalize in the migration cells at the tips of lamellipodial plasma membrane and filopodia.
Interestingly, localization of PREL2 was found to be independent of Ena/VASP proteins.
Overexpression of PREL2 increases lamellipodial protrusion velocity. On the other hands, knockdown
of PREL2 impairs the formation of lamellipodia and decrease in F-actin content. These findings
suggested that the important role of PREL2 as regulator involved in action cytoskeleton regulation. In
year 2006, Chang et al. found that PREL2 has significant functions on cell signaling and direct
migration and promoting axon guidance. However, it is still a gap of knowledge on how PREL2 acts as
an actin cytoskeleton regulator. This gap has attract our interest to further study the role of PREL2 in
developmental.
In our study, zebrafish was selected as model organism to study the functional role of PREL2 in
embryonic developmental stage due to it several advantages compare to others model. At the very
beginning of this study, the functional role of PREL2 was study through the expression pattern on
zebrafish embryonic developmental stage by using reverse transcriptase polymerase chain reaction.
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2. BACKGROUND INFORMATION
2.1 The Zebrafish
In the past decade, zebrafish (Danio rerio) has emerged as an important model organism (Grunwald &
Eisen, 2002) especially in developmental biology and genetic disease study (Dodd, Curtis, Williams &
Love, 2000; Zon, 1999) because of its several advantages over others model. Firstly is because of its
relatively short generation time (2-4month) and can be easily maintained in the laboratory (Briggs,
2002; Dodd et al., 2000; Haffter & Nüsslein-Volhard, 1996). Secondly, the transparency of the embryo
and external developmental system allowing virtual visualization and manipulation of embryogenesis
and organogenesis (Driever et al., 1996; Gates et al., 1999; Fleming, 2007). Furthermore, large
number of progeny, approximately 200 embryos can be produced per mating pair per week enable
large-scale phenotypic screening. In addition, zebrafish as a vertebrate definitely represents a closer
model to human system compares to Drosophila or Caenhorhabditis elegans (Barut & Zon, 2000;
Runinstein, 2003, Zon, 1999). Also, genetic screens showed that many zebrafish mutant phenotypes
are resemble human disease states (Dodd et al., 2000).
2.2 Proline-rich EVA/VASP Ligand (PREL) 2
PREL2 or lamellipodin (Lpd) is one of the members of MRL protein family. The term lamellipodin was
named by Krause et al. (2004). The N-terminal of PREL2 comprises of 50 amino acid is highly
charged, followed by a putative coiled-coil motif, RA and PH domains. The C-terminal of PREL2 is 500
amino acids longer than PREL1. It is rich in proline with eight potential SH3 binding sites, three
potential Profilin binding sites and six putative EVH1 binding sites. Each motif of PREL2 can bind to
EVL directly, different to PREL1 which requires two motifs for Mena binding site (Jenzora et al., 2005;
Krause et al., 2004). In 2009, Lee, Lim, Puzon-McLaughlin, Shattil and Ginsberg identify a short talin
binding sequence in PREL2. Binding of talin to PREL2 and links to Rap1 membrane targeting
sequence is sufficient to recruit talin and induces integrins activation.PREL2 mediate activation of
integrin by a common scaffolding mechanism; in which contains amphipathic helices that mediate
direct binding to talin and RA domains that bind Ras superfamily GTPases (Lee et al., 2009). This
finding further confirms that PREL genes family is contributed to the actin polymerization pathway and
cell migration. The study of Lee (2008) and Tay (2010) indentified the present of two copies of PREL2
gene (named PREL2a and PREL2b) which might be due to gene duplication.
3. METHODOLOGY
3.1 Primer Design
The primer for this project was designed by Primer3 software (http://frodo.wi.mit.edu/primer3/). The
product size with the range of 500-700bp was preferred. Subsequently, Netprimer
(http://www.premierbiosoft.com/netprimer/index.html) was used to evaluate the designed primer pair
with their hairpin, palindromes, dimmers and melting temperature (Tm).
3.2 Fish Maintenance
The zebrafish used in this research were provided by Dr Lee Kui Soon under Animal Biotech
Laboratory, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak. Fish facility is
established in the lab. The zebrafish were fed three times per day and kept under room temperature
with a schedule of 14 hours light and 10 hours darkness.
3.3 Fish Breeding and Egg Collection
Prior to the breeding, the zebrafish were isolated from the aquarium and placed into mating tank.
Mating tank was prepared by covered the bottom of the tank with marbles and a piece of net on the
marbles, filled the tank with aquarium water. A pair of zebrafish (one male and one female) was
placed into the tank for overnight in condition as mentioned in Section 3.1. At least three mating tank
were prepared for each mating section. The next day, the eggs were collection by using a sieve and
washed with tap water to remove the impurity. Subsequently transferred to a petri dish containing
embryo medium (Fig. 3). Unfertilized eggs were removed from the dish. The fertilized eggs were
placed into the incubator for normal development.
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3.4 Staging and Dechorionation
The development of zebrafish embryos were observed under light microscope. The development
stage of zebrafish was indentified based on the study of Kimmel, Ballard, Kimmel, Ullmann and
Schilling (1995). The dechorionation was done before achieved the desired stage. The dechorionated
embryos were placed into petri dish containing embryo medium for normal development until reached
the desired stage subsequently transferred into a 1.5ml Eppendorf tube for RNA Isolation.
Approximately 100-150 embryos were allocated into a single tube. For the purpose of this study, 10
embryonic developmental stages were selected: 3hpf, 8hpf, 11hpf, 18hpf, 18hpf, 24hpf, 36hpf, 48hpf,
72hpf, 96hpf and 120hpf.
3.5 Total RNA Isolation
1ml of Trizol Reagent was added into the Eppendorf tube containing zebrafish embryo with desired
stage (From Section 3.3). Homogenization was done manually by using gauge and syringe.
Subsequently, the tube was centrifuged at 12,000rpm for 10minutes in 4ºC centrifuge. The
supernatant was transferred to a new tube followed by adding of 200 δ of chloroform. The tube was
left at room temperature for 15minutes on ice. Next, the tube was centrifuged at 12,000rpm for
15minutes in 4ºC centrifuge. Upper phase was transferred to a new tube and 500 δof isopropanol
was added. The tube was incubated on ice for 10minutes prior to centrifuge to pellet the RNA.
Supernatant was discard subsequently 1ml of 70% ethanol was added. The tube was centrifuged
again at 7500rpm, 5 minutes, at 4ºC centrifuge. The supernatant was discarded and the RNA was
dried on air for not more than 15minutes. Finally 10 l of DEPC water was added and the tube
containing RNA was stored at -20ºC for subsequent analysis.
3.6 Agarose Gel Electrophoresis
1 l of each RNA sample together with 5 l of loading dye pre-mix was loaded into 1% agarose gel and
electrophoresis was performed at 120volt for 30 minutes. The gel was visualized under UV
transilluminator.
3.7 RNA Quantification
The purity and concentration of extracted RNA was determined using spectrophotometer with a
dilution factor of 35. The absorbance values of 260nm, 280nm and 320nm were recorded. The
concentration of RNA was calculated as shown below:
RNA ( g/ l) = [A260 × (40 g RNA/ml)/ (1 A260 unit) × (dilution factor)] / 1000
3.8 cDNA Synthesis
cDNA synthesis was performed using SuperScriptTM III First-Strand Synthesis System (Invitrogen
brand) according to manufacturer’s technical manual. Negative control was carried out for each
sample.
3.9 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
RT-PCR was performed using Kyratec SuperCycler SC200 Thermal Cycling Engine with preparation
of master mix as shown in Table 1 and performed at condition as shown in Table 2.
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Table 1 Preparation of RT-PCR master mix
Experimental Reaction
1 rxn
10X PCR Buffer minus Mg (Final concentration: 1X)
10mM dNTP mixture (Final concentration: 0.2mM)
50 mM MgCl2 (Final concentration: 1.5 mM)
1 ε Forward Primer (Final concentrationμ 0.03 ε)
1 ε Reverse Primer (Final concentrationμ 0.03 ε))
Taq DNA Polymerase
DEPC water
Template DNA (added separately for each tubes)
2.50 l
0.50 l
0.75 l
0.75 l
0.75 l
0.50 l
18.25 l
1.00 l
Final Volume
25μl
Table 2 RT-PCR condition and cycles
Step
Reaction
Temperature
Time
Cycle
1
2
3
4
5
Initial Denaturation
Denaturation
Annealing
Extension
Final Extension
95ºC
94 ºC
61 ºC
72 ºC
72 ºC
2 minutes
30 seconds
1 minutes
1 minutes
5 minutes
1 cycle only
Repeated 35 cycles
1 cycle only
5 l of each PCR product was loaded into 1% agarose gel and electrophoresis was performed at
120volt for 30 minutes. Beta-actin was used as positive control and negative control was performed for
each sample.
4. RESULT AND DISCUSSION
4.1 Embryonic Staging of Zebrafish
The staging of zebrafish embryonic development was based on the study of Kimmel et al. (1995).
They defined zebrafish embryogenesis into seven broad periods: zygote, cleavage, blastula, gastrula,
segmentation, pharyngula and hatching periods based on morphological features. In this study, 10
embryonic stages were chosen from different periods starting from blastura period (3hpf), gastrula
period (8hpf), segmentation period (11hpf and 18hpf), pharyngula period (24hpf and 36phf), hatching
period (48hpf and 72hpf) and early larva period (96hpf and 120hpf). In 3hpf stage, it is approximately
1k of cells with 11 tiers of blastomeres. In 8hpf, 75% of epiboly was formed and dorsal side become
thicker. First somite was first observed after 10 hours post fertilization (hpf). During segmentation
period (approximately 10hpf to 24hpf, primary organogenesis starts to develop. Embryo starts to show
some movement during this stage. The tail becomes visible at 18hpf. In 24hpf, the heart becomes
visible and appear as a cone-shaped tube deep to the brain. Early pigmentation in retina and skin can
be observed after 24 hours post fertilization. In this stage, body axis straightens from its early
curvature about the yolk sac and fins begin to develop. In hatching period, the pectoral fin buds are
elongated and stretching to a height-to-width ratio of approximately 2. Embryo continues to grow at
about the same rate as earlier. At the end of hatching period, the heart is prominent with strong
beating. The blood circulation was fully developed. In early larva period, most of the morphogenesesis
has completed. The embryo in this stage is tend to swim actively and continues to grow rapidly
(Kimmel et al., 1995).
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4.2 Total RNA Isolation
Total RNA from 10 zebrafish embryonic stages (3hpf, 8hpf, 11hpf, 18hpf, 24hpf, 36hpf, 48hpf, 72hpf,
96hpf and 120hpf) were successfully extracted. The overall quality of extracted RNA is considered
good. Two clear and intact bands for each sample loaded observed from the gel. As expected, bands
represent 28S were thicker and stronger compare to 18S (As shown in Figure1). However, some
smearing was observed at the gel background. The smearing might be due to improper handling
during sample preparation. Contamination of sample is one of the causes of smearing. Also, RNA
might be degraded during extraction and gel loading steps as RNA is very unstable at room
temperature.
Figure 1 Total RNA Isolation from 10 developmental stages from zebrafish embryos: Lane 1(3hpf);
Lane 2(8hpf); Lane 3(11hpf); Lane 4(18hpf); Lane 5(24hpf); Lane 6(36hpf); Lane 7(48hpf); Lane
8(72hpf); Lane 9(96hpf); Lane 10(120hpf)
4.3 RNA Quantification
The purity and concentration of extracted RNA was determined using spectrophotometer. The
absorbance of a diluted RNA sample was measured at wavelength of 260nm and 280nm. An
absorbance of 1 unit at 260nm is equivalent to 40 g of RNA per ml and the OD at 260nm is used to
determine the RNA concentration in a solution. The ratio of A260/A280 is used to access the purity of
RNA. A pure RNA is expected to have a A260/A280 ratio closed to 2. The reading below 1.8 suggests
that the sample might be contaminated with protein. The reading obtained for 10 samples was shown
in Table 3. From the table, the concentration of extracted RNA was generally higher than 2.000 g/ l
except for 18 hpf with reading of 1.771 g/ l. The reading of A260/A280 in the range of 1.8-2.0
indicates high purity RNA had been extracted. The good quality of RNA is the key to success in
subsequent analysis.
Table 3 Spectrophotometry analysis of extracted RNA
Stage
Concentration (μg/μl)
A260/A280
A260/A230
3 hpf
8 hpf
11 hpf
18 hpf
24 hpf
36 hpf
48hpf
72 hpf
96 hpf
120 hpf
2.862
2.787
2.981
1.771
3.507
2.662
2.773
2.224
2.536
2.498
1.935
2.008
1.881
1.893
1.953
2.053
1.757
1.898
2.090
1.836
0.884
0.882
0.900
0.956
1.128
0.880
0.896
0.842
0.846
1.443
4.4 Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR)
A total of 10 single and clear brands represent 10 embryonic stages were obtained from the agarose
gel electrophoresis of PCR product. All the products size were observed allocated between 600bp and
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700bp, indicating that all the PCR products achieved the expected product size, which are 666bp.
Besides, there are no bands observed in negative control.
Figure 2 L= Ladder; Lane 1 (3hpf); Lane 2 (8hpf); Lane 3 (11hpf); Lane 4 (18hpf); Lane 5 (24hpf);
Lane 6 (36hpf); Lane 7 (48hpf); Lane 8 (72hpf); Lane 9 (96hpf); Lane 10 (120hpf)
The present of bands from 3hpf to 120hpf PCR products provide us useful information that PREL2a is
expressed along the embryonic development of zebrafish. However, PCR result is not a quantitative to
tell us how strong is the expression of the gene in certain stage.
While for the β-actin (positive control), the present of bright and clear bands for all 10 stages indicate
the strong expression of β-actin as housekeeping genes (As shown in Figure 3). All the products sizes
were between 300bp and 400bp, as expected size of 331bp.
Figure 3 L= Ladder; Lane 1 (3hpf); Lane 2 (8hpf); Lane 3 (11hpf); Lane 4 (18hpf); Lane 5 (24hpf);
Lane 6 (36hpf); Lane 7 (48hpf); Lane 8 (72hpf); Lane 9 (96hpf); Lane 10 (120hpf); Lane 11 (blank
control)
5. CONCLUSION
From the preliminary result obtained from reverse transcriptase PCR, we concluded that PREL2a was
expressed along the zebrafish embryonic developmental stages from 3hpf to 120hpf. These findings
provide information that PREL2a might be play certain important role during developmental. In coming
work, in situ hybridization will be performed to study the expression of PREL2a in vivo.
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Dodd, A., Curtis, P.M., Williams, L.C and Love, D.R. (2000). Zebrafish: bridging the gap between
development and disease. Human Molecular Genetics, 9, 2443-2449
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Fleming, A. (2007). Zebrafish as an alternative model organism for disease modeling and drug
discovery: implications for the 3Rs. National Centre for the Replacement, Refinement and
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Jenzora, A., Behrendt, B., Small, J.V., Wehland, J., Stradal, T.S. (2005). PREL1 provides a link from
Ras signaling to the action cytoskeleton via Ena/VASP proteins. Federation of European
Biochemical Societies Letters, 579, 455-463.
Krause, M., Dent, E.W., Bear, J.E., Loureiro, J.J., and Gertler, F.B. (2003). Ena/VASP proteins:
regulators of the actin cytoskeleton and cell migration. Annual Review, Cell Developmental
Biology, 19, 541-564.
Lee, H.S., Lim, C.J., Puzon-McLaughlin,W., Shattil, S.J., Ginsberg, M.H. (2009). RIAM activates
integrins by linking talin to Ras GTPase membrane-targeting sequences. Journal of Biology
Chemistry, 284, 5119-5127.
Lee, K.S. (2008). Characterization of Grb7, Prel and GYGYF family genes in zebrafish (PhD Thesis).
UK: University of Bath.
Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B. and Schilling, T.F. (1995). Stages of
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Rubinstein, A.L. (2003). Zebrafish : from disease modeling to drug discovery. Current Opinion in Drug
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Tay, A.P. (2010). Hunting for a second copy of PREL family gene in Danio rerio genome. (Final Year
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ETHNOPEDOLOGICAL KNOWLEDGE BY IBAN FARMERS OF NANGA MACHAN, KANOWIT,
SARAWAK
1*
2
1
M. E. Wasli , A. S. Bagol , H. Nahrawi & Egay, K.
3
1
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
2
Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Grand Olie Sdn Bhd
3
Brighton Square, Jalan Song, 93350 Kuching, Sarawak, Department of Sociology and Anthropology,
Faculty of Social Sciences, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
*Email: wmeffendi@frst.unimas.my
Abstract
The Iban of Sarawak still largely depend on smallholder agricultural activities as main source of
income for their livelihood. According to previous social studies on local knowledge by the Iban, they
classify their own soil based on their past experience towards the fertility or infertility of the soils. The
indigenous soil knowledge (ethnopedology) has limited information and there are requirement where
at present, people still depends on this indigenous knowledge to sustain the fertility of soil.
Ethnopedology has been defined as a hybrid discipline between the natural and social sciences that
encompasses the soil and land use knowledge systems of rural populations from the most traditional
to the modern. In ensuring that local farmers’ perception is also important in planning for future
agricultural scheme or management, the objective of this preliminary study is to clarify the correlation
between indigenous knowledge and scientific knowledge for agricultural activity based on the interview
and soil physicochemical properties analysis. Our findings showed that the farmers in the study area
still possess and practice their indigenous knowledge on soil classification for their subsistence
farming. Much of their interpretation on soil quality largely depends on their observation of topsoil and
indicators characteristics i.e. colour and texture. Indigenous knowledge is a very limited indicator to
classify soil; while scientific knowledge provides in-depth information in identifying soil characteristics.
However, both knowledge has shown major similarities and complementarities, thus would create
better soil management practice for smallholder farmers.
Keywords: ethnopedology, Sarawak, local farmers, soil classification, soil properties
1. INTRODUCTION
The relevance of traditional knowledge has established diverse aspect of natural resource
management in tropics which include the maintenance of biological diversity, biological crop pest
control strategies, soil fertility management and soil and water conservation (Lanlonde, 1993). Hence,
the local soil knowledge, soil assessment and land managing are commonly referred as
ethnopedological studies. Brady (1990) reported that farmer’s experience which based on centuries of
trial and error as well as scientific investigations of soil and their management are two basic sources
that obtained from soil knowledge. The information that gained from previous generation are passed
on through to the next generation that then refined into a system of understanding of natural resources
and relevant ecological processes (Pawluk et al., 1992).
Most of the ethnopedology studies were done by researcher from other country but the study was very
limited in Sarawak unlike China, Egypt, India and Mexico, the complex pedological wisdom were
developed more than 2000 years ago in places that are intimately related with the major centre of
plant domestication in the world (Barrera-Bassols & Zinck, 2002). Currently, in developed countries
had led many scientists and extension workers on recognizing that the rural people enclose with a rich
understanding regarding their resources (Thrupp, 1989; Warren, 1989). Studies of local environmental
knowledge are increasingly seen as a key to both the conservation of agro- and biodiversity and the
increased effectiveness of sustainable land-used.
Most of the population in Sarawak, Malaysia are largely depending on agriculture activities for their
livelihood Leigh (2002). Sarawak also encompassed of various ethnic groups whose livelihoods
largely depend on the surrounding natural resources. The Ibans constitute the largest ethnic group
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and most of them are still attached with agricultural practices such as shifting agriculture for their
subsistence or monetary income (Sutlive, 2001).
There are various reports on the knowledge by the Iban from the social and anthropology viewpoints.
However, the information on the indigenous knowledge by the Iban in understanding their surrounding
environment from the viewpoint of scientific evidence is still limited. Tanaka et al. (2007a,b) and Wasli
et al. (2009) reported on the determination of soil fertility based on the observation on indicator plants
which commonly applied in Iban communities.
Even though, the local farmer in Sarawak often referred to the extension workers and expertise from
Department of Agriculture for guidance in appropriate farming techniques but they still depending on
their previous indigenous experience in managing their soil resources. Nonetheless, at present, most
of the local farmers largely depend on fertilizer application subsidized by the local authority to ensure
the fertility of their soils towards better crop production.
In view of the fact that indigenous soil knowledge has limited information and there are requirement
where at present, people still depends on this indigenous knowledge to sustained the fertility of soil. In
ensuring that local farmers’ perception is also important in planning for future agricultural scheme or
management, the objective of this preliminary study is to clarify the correlation between indigenous
knowledge and scientific knowledge for agricultural activity based on the interview and soil
physicochemical properties analysis.
2. MATERIALS AND METHODS
2.1 Study area
This case study was conducted at Nanga εachan (N02°01’10.2, E112°00’52.6), Sibu, Sarawak. The
study area located about 100 km from Sibu town and is situated at the mouth of Rajang River. The
annual rainfall in the area is 3331 mm/year with absolute air temperature ranging from 21°C to 36°C
(Department of Statistic, 2011). The soil in the area were classified into Durin series from Saratok
Family with a very fine sandy loam and the parent materials are mixed sedimentary rocks that are
dominant by sandstone beds. Saratok family is described as a Grey-White Podzolic Soil that consists
of loam particle size class and has a residual over non-calcareous sedimentary rocks or old alluvial
deposits (Scott, 1985).
Nanga Machan encompassed of 46 Iban longhouses with estimated population of about 1500 people
whom are mainly of Iban origin. The communities in the study area were attached with agricultural
activities such as rice cultivation and cash crop farming (Rubber, pepper and cocoa) for their
livelihood.
2.2 Data collection
In this study, the data collection process was divided into two phases namely, interview session
(Phase 1) and soil sample collection (Phase 2). Interviews were conducted before the soil collection.
The location for collecting soil sample was determined by farmers. The information gained from the
interview and analyses were used for soil classification.
2.2.1 Phase 1- Interview session
In this phase, twenty-nine farmers were selected for interviewed but only eighteen respondents are
willing to help in soil sample collection. The respondents consist of different ages ranging from 30 to
above 70 years old. The questionnaires formed were to surveys on the local knowledge and types of
soils that they know. The interviewed was conducted with a single person at a time which the data
were being recorded to a semi-structured interview. Apart from that, group discussions consist of 3 to
5 people were conducted via Participatory Rural Appraisal (PRA) as proposed by Chambers (1994).
Each group discussion will discuss on a given theme such as their current agricultural practices and
their local indigenous soil classification knowledge.
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2.2.2 Phase 2- Soil sample collection
The soil sample was collected at a specific location in field that is pre-determined by the respondents
which has been interviewed. The soil samples were brought back to the Environmental Soil
Laboratory, Department of Plant Science and Environmental Ecology, Faculty of Resource Science
and Technology (FRST), Universiti Malaysia Sarawak (UNIMAS), for analysis.
2.3 Methods of selected soil analysis
The soil samples obtained from field were air-dried for one week and sieve with 2 mm mesh sieve for
soil physicochemical analysis. Soil bulk density (undisturbed soil), was determined by using the core
sampling method after drying the soil samples in an oven at 105°C to constant weight. The soil colour
was determined by referred with Munsell Soil Colour Chart that used to classified soil colour. The
Munsell Colour Chart has three simple variables that are hue, value and chroma (used to specify
colours). The Hue notation of a colour indicates its relation to the spectral colours such red, yellow,
green, blue and purple. For value, indicates the lightness of the colour; and chroma the strength, or
departure of the colour from a neutral colour of the same lightness. Soil pH was determined in water
(H2O) and measured using the glass electrode method, at the soil solution ratio of 1:5. Electrical
conductivity (EC) was measured before pH measurement using the conductivity meter (Eutech
instruments Cyberson Con II). Soil moisture content was determined by calculating the percentage of
moisture content of the air-dry soil. Soil Organic Matter (SOM) was determined using loss on ignition
method. Soil texture is the property of a soil which refers to the relative amounts of different sized
particles present in a soil. Soil texture is described using terms such sand, silt, loam, clay etc. which
indicates the relative proportions of different-sized particles present. The soil texture was determined
using feel methods.
2.4 Data analysis
The correlation of this result, were analyse using the SPSS software version 14 and interpreted by a
dendrogram in hierarchical cluster analysis using Ward’s method. The dendrogram play a vital role
which assist in listing all of the samples and specify at what level of similarity in any two clusters were
joined meanwhile Ward’s method were assist a linked cluster by minimizing the within-group sum of
squares and construct a compact cluster (Holland, 2006).
3. RESULTS AND DISCUSSION
3.1 Farmer’s perception on characterizing soil suitability for agriculture
Farmers in Nanga Machan have their own indigenous knowledge to classify, portray and characterize
types of soil based on the soils characteristics and suitability for various crops. They classifying their
soils first of all based on the soil colour, soil properties and features. As reported by Gosai et al.
(2011), soils are usually distinguished easily by farmers from the observation through physical aspect,
this aspect practices by Nyishis, one of the tribe exist in Northeast region of India which soils are
characterize based on the texture, topographic position, colour and also yield production. According to
Altieri (1λλ2) the contribution of farmers’ perception and knowledge has been highlighted by most of
the researchers while Tabor (1λλ2) stated that the studies of farmers’ knowledge and management
skills are assist the scientist to classify on the different types of soils.
Table 1 showed the summarized information on the farmer’s knowledge on soil for agricultural
purpose. From the information obtained, soil colour acted as an important indicator for farmers when
determining the suitability of the soil for farming. From the information obtained, there are five main
soil types were indicated by the respondents. These five soil types are described with their local name
which locally known as Tanah Hitam, Tanah Kuning, Tanah Pasir, Tanah Batu Lemak and Tanah
Ladu.). Soil types and their characteristics are explained as follows: For Tanah Hitam, they are black
moisture soil, non-sticky, rough and powdery texture. It contains few rocks while some said that rock is
absence in this soil. At the study area of Kanowit district, it was reported by Scott (1985) the rock that
exist in the area is referred to the sedimentary rock which dominantly by sandstones bed. Tanah hitam
has very high moisture content, higher in organic matter and contains abundance of earthworm.
According to Edwards & Baker (1992), earthworms can help to improve and recovery the soil structure
and useful for land enhancement, also in organic waste management. The earthworm locally referred
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as a ‘belut’ by farmers. As mention by farmers, Tanah hitam or Tanah chelum (Black soil) can produce
high yield of crops.
Table 1 Information on the farmers’ perception on the suitability of soil for agricultural purposes
TH
TP
TK
TBL
TL
Criteria/properties
(n = 17)
(n = 11)
(n = 16)
(n = 14)
(n = 14)
of the soil
Colour
Texture
Stickiness
Roughness
Powdery
Black (16)
White (9)
Non sticky
(13)
Rough (11)
Non sticky
(11)
Rough (11)
Powdery (16)
Powdery (11)
Yellow (14)
Red yellow (5)
White (6)
Sticky (11)
Sticky (11)
Sticky (14)
Rough (10)
Powdery
(13)
Rough (12)
Not rough (14)
Not powdery
(14)
Powdery (9)
Criteria/
TH
TP
TK
TBL
TL
properties of the
(n = 17)
(n = 11)
(n = 16)
(n = 14)
(n = 14)
soil
Rock
Few (8)
Many (7)
Few (12)
Few (9)
None (12)
Moisture
Moist (17)
Moist (6)
Moist (11)
Moist (10)
Moist (13)
Litterfall/roots
Yes (17)
No (6)
Yes (12)
Yes (7)
No (13)
Earthworm
Yes (17)
No (9)
Yes (11)
Yes (10)
No (14)
Depth
Shallow (11)
Shallow (11)
Deep (16)
Deep (12)
Deep (14)
Suitability*
Good (17)
Poor (8)
Moderate (8)
Good (8)
Poor (10)
Value in parentheses is the number of respondent that answer the given criteria, Rock: Yes refer to
the present of rock; No refer to the absent of rock, Earthworm: Yes refer to the present of earthworm;
No refer to the absent of earthworm, *Suitability of the soil for agriculture (without fertilizer application).
The soil is very shallow and easier to be seen at the soil surface. Tanah hitam has a good soil
structure that make the water easier to penetrate into the soil when rains. Tanaka et al. (2007b)
reported that, the most fertile soil referred by the Iban farmers is a black soil that usually suitable for
shifting cultivation where the soils consists of high organic matter as well as with good moisture
content. For Tanah pasir, the soil is white in colour while the soil texture is non-sticky, rough and
powdery. The soil has contains abundance of rocks and describe as moist but some of the
respondents said the soil is very dry as the water holding capacity is very low. The presence of
organic matter and earthworm in soil is not as much as Tanah hitam. It located at shallow layer of soil
and the easily penetrate with water when rains. The farmers mention that sandy soil could be a little
fertile when they mixed together with Tanah hitam and chicken dung (Tanaka et al., 2007b).
For Tanah kuning, the soils is yellow in colour, has a sticky, rough and powdery texture. The soil also
contains few rocks, has high in moisture content and high in organic matter. Based on field
observation, the abundance of earthworms can be observed in soil. Located at the subsurface or deep
layer and rainfall penetration on this type of soils is considerably good as the water penetrates into the
soil during raining day. Meanwhile for Tanah batu lemak, is referred as a red yellowish in colour and
has a sticky, rough and non-powdery texture. The soil contains few rocks while high in moisture,
organic matter and has abundance of earthworm. The soil can be found at deep layer of soil and
easily penetrates by water. Accordingly to farmers, Tanah batu lemak or known as weathered soil
were very suitable to cultivate any crops such pepper besides than Tanah hitam.
For Tanah ladu is white clayey soil, has a sticky, non-rough and non-powdery texture with the absence
of rock in soils but some of respondents said that the soil has contained few rocks. It is also have a
very high in moisture content as it is located along the riverbank. The organic matter and earthworm
are absence in this soil. The soil is located at the subsurface or deep layer and water is accumulate at
the surface layer when rains. The soils rarely being used in agriculture purposes because it known as
infertile, however, in the past, this soil are purposely used for making pot and vase.
The suitability of the soil for agriculture purpose without addition of fertilizer can be described from
fertile to less fertile: Tanah hitam, Tanah batu lemak, next is Tanah kuning and Tanah pasir while
Tanah ladu is a less fertile and very difficult to use for agriculture activities. Based on the farmer’s
knowledge, Tanah hitam is a suitable soil for cultivate vegetables, pepper, cocoa, and paddy crops.
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Next is Tanah batu lemak which suitable for cultivate pepper, rubber, ginger and paddy while Tanah
kuning is suitable for pepper and fruits cultivation. Corn, mango, watermelon, pineapple and banana,
Tanah pasir is the most suitable soil for this crops and Tanah ladu is a very difficult to be used in
agriculture activity, only paddy, oil palm and dabai fruit can cultivated in this soil.
Hence, it shows that the farmers at the study area mainly refer on tops soil surface characteristics and
used as a basic for soil description. All farmers interviewed described soils based on following
characteristics: colour, texture, rock, moisture, litterfall, earthworm, depth, water penetration and soil
fertility status. The results obtained from the farmers’ perception (interview) showed the nomenclature
given to the soils was influenced with the indigenous knowledge that inherited from one generation to
the other.
3.2 Soil physicochemical properties analysis of soil samples collected from the field that
referred by respondents for agricultural purpose
Table 2 shows the results from the laboratory analysis of the soils collected from the field of 18
respondents that involved in earlier interview session. It should be noted that the number of soil
samples collected for analysis did not involve all 18 respondents due to several reasons; during the
duration of soil samples collection 2 or more respondents refer to the soil sample collected as similar
soil based on their local knowledge i.e. 2 or more respondent refers Tanah hitam collected as similar
the soils that they possess at their farmland and, some farmers referred that the identified soils were
located at location far from the longhouses which is not accessible during the duration when the soil
sampling procedure were conducted. There are five types of soils namely Tanah hitam, Tanah pasir,
Tanah kuning, Tanah batu lemak and Tanah ladu were analyse in laboratory to determining the soil
pH, electrical conductivity, moisture content, soil organic matter and bulk density while colour, texture,
earthworm and rock are obtained from field observation. These five soils are very acidic soil with the
pH value around 5.
Table 2 Physicochemical properties of the soils in the study area
TH
TK
TBL
TP
TL
Soil properties
(n=9)
(n=10)
(n=9)
(n=7)
(n=12)
pH (H2O)
4.91 (0.38)
5.26 (0.32)
5.65 (0.78)
5.21 (0.55)
5.25 (0.32)
46.55
28.65
38.90
29.70
56.15
EC ( s/cm)
(10.60)ab
(15.17)a
(23.13)ab
(11.55)a
(28.22)b
MC (%)
3.03 (1.39)b 2.10 (1.22)ab
3.20 (1.45)b
0.94 (0.67)a
1.72 (0.70)a
SOM (%)
11.9 3 (5.88)
14.20 (7.56)
6.62 (2.54)
9.70 (6.59)
13.64 (7.57)
Bulk density
1.13 (0.14)a 1.34 (0.18)ab
1.21 (0.19)a
1.56 (0.09)b
1.18 (0.11)a
-3
(g mL )
(1) Colour
DkYB
YB
StB
DkYB
LiOB
(2) Texture
SCL
SiC
SC
SCL
SiC
(3) Earthworm
100
70
57
0
0
(4) Rock
No
No
Yes
No
No
Value in parentheses refer to the standard deviation of each properties, Value in parentheses for
colour and texture are the number of respondent that answer the given criteria, TH: Tanah Hitam; TP:
Tanah Pasir; TK: Tanah Kuning; TBL: Tanah Batu Lemak; TL: Tanah Ladu, EC; electrical conductivity,
MC; moisture content, SOM; soil organic matter, (1) observed from by Munsell Colour Chart; DkYB;
Dark Yellowish brown, YB; Yellowish brown, StB; Strong brown, LiOB; Light o;iove brown; (2)
determined from feel method; SCL; Sandy clay loam, SiC; Silty clay, SC; Sandy clay, (3) percentage
(%) of occurrence in earthworm population in each sampling points, Rock: Yes refer to the present of
rock; No refer to the absent of rock
Soil pH is often being considered as an important determinant for soil suitability and crop growth.
Nevertheless, pH also affects the nutrient availability and heavy metals in the soil, thus the leaching
through soils can change the soil condition. The farmers said that, most of the soils in the study area
are low in pH, thus referred the acidic soil as Tanah masam. Weinstock (1984) reported that, the way
of soils classified by farmers in Malaysia is similar with the western context on soil pH, commonly
based on taste which locally known as sweet (Tanah payau), neutral (Tanah tawar), and sour soil
(Tanah masam). Tanah ladu has higher electrical conductivity with value of 56.2 s/cm while Tanah
kuning is the lower electrical conductivity with value of 28.7 s/cm. Electrical conductivity indicates
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number of ions that present in the particular soil and shows the presence of salts in soil. Tanah hitam
which is 3.0 % and Tanah batu lemak with value of 3.2 % were higher moisture content whereas
Tanah pasir has the lowest moisture content which is 0.9 %. Tanah kuning contains 14.2 % of organic
matter which is the high organic matter among other four soils and commonly found associated with
litterfall and root of plants. Meanwhile the lower organic matter is Tanah batu lemak which is 6.6 %.
Furthermore, soil organic matter helps in enhancing the crop production. Higher organic content may
ascribed to the most fertile soil.
Bulk density is an important physical property that influences the penetration of root plants. Tanah
-3
pasir have a higher bulk density with the value of 1.56 g m . This may due to the lower of organic
matter content, less root growth and poor aggregation. Meanwhile, Tanah hitam possess the lowest
-3
bulk density value of 1.13 g m as compared to the other soil types. Lower level in soil bulk density
may ascribe to the higher level the organic matter content of soil (Juo & Franzluebbers, 2003). From
the field observation during soil sampling procedure, Tanah hitam and Tanah pasir are found in the
top surface of soil layer while Tanah kuning, Tanah batu lemak and Tanah ladu are found at the
subsurface soil. Usually, the depth of Tanah hitam is approximately 2 to 4 inches and consists
numerous of organic matter such as litterfall and roots. For Tanah pasir, such soil was commonly
observed at areas located along the riverbank. The study areas usually faced with flooding problem
which may affect the changes of the soil depth. From the observation using Munsell Colour Chart, the
colour of soils that establish are quite different, which described as follows: Tanah hitam and Tanah
pasir are found dark yellowish brown in colour , Tanah kuning is yellowish brown in colour, while for
Tanah batu lemak is strong brown in colour and Tanah ladu light olive brown colour.
Based on the soil texture, there are three types of texture were found, sandy clay loam, silty clay and
sandy clay. The percentage of these texture were determine using soil textural triangle. Tanah hitam
and Tanah pasir are mostly established in sandy clay loams because the soil has found larger size of
particles and gritty. Sandy clay loam is the soils that contains of 20 to 35% clay, less than 28% silt and
45% or more sand. However, Tanah hitam also found as a sandy clay texture with gritty and sticky
texture. Tanah kuning and Tanah ladu are silty clay which moderate in size and smooth texture that
comprise of 40% or more of clay and 40% or more of silt.
For Tanah batu lemak, the soil is sandy clay in texture with gritty and sticky texture and the soil
encompassed of 35% or more of clay and 45% or more of sand. The texture is use to indicate the soil
fertility status which the soils that high in silt and clay are considerably have a higher organic carbon
while soils located at lowland have the highest percentage of organic carbon (Brady, 1990). In
addition, these soils such Tanah hitam, Tanah pasir, Tanah kuning and Tanah batu lemak are under
loamy soil with medium and moderately fine texture meanwhile for Tanah ladu is under clayey soil with
fine texture. Moreover, except for the Tanah batu lemak which collected at foot hills, most of the soils
were collected nearby the riverbank and some of Tanah kuning collected at foot hills. The similarity of
soil texture between the soils affect the difficulty to differentiate texture in precisely as it was
conducting by feel method and the weather problem is one of the difficulty during conducting of this
study, which influence the soils condition. Tanah hitam has abundance of earthworm compared to
Tanah pasir and Tanah ladu, which absence of earthworm. Most of the earthworms exist in Tanah
hitam because soil provides food supply such litterfall and other organic matters. Earthworm is a good
indicator for fertile soil which can assist to fix the soil structure, so the soil would be fertile. For rocks
characteristic, it was found that none of these soil types had presence of rocks. Therefore, from all the
soils had described above, the farmer’s not just only farm in the Tanah hitam but also in other soil
such Tanah pasir, Tanah kuning and Tanah batu lemak while Tanah ladu is seldom being used for
agriculture activities.
3.3 Soil classification by the farmers for agricultural purpose based on their local knowledge
The integration of farmer’s and scientific knowledge could offer many insights about the sustainable
management of soil. As reported by Payton et al. (2003) the character of indigenous knowledge
should be consider as it is important to achieve the aims of soil research for the integration of scientific
and local knowledge. According to Bellon & Taylor (1993) the local knowledge referred by farmers and
scientific method are produced a similarity in soil quality ranking. The correlation of soil physical
properties with farmer’s soil evaluation is found more closely related than soil chemical properties
(Hirai et al., 2000; Gary & Morant, 2003). In this study, the soil classification that obtained from the
farmers knowledge with the results from the soil laboratory analysis is shown Figure 1.
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Figure 1 Soil classification based on the local knowledge & laboratory soil analysis
Overall, the indigenous and scientific soil classifications are correlated due to the similarities with
several of its criteria. In general, the farmers classified soils based on their on farm history and
experience while for scientific knowledge; the soil classification explained the scientific information
associated with the soil physicochemical properties analysis. From this relationship, the soils can be
classified into two categories, infertile and fertile soil as the soil fertility status is evaluated in
accordance with the farmer’s knowledge on the suitability of the existing soils to support crop growth
without addition of fertilizer. Infertile soil contains less organic matter comparatively to fertile soil with
the abundance of organic matter. For infertile soil, the soils are described as a dry soil with no
abundance of rock and earthworm which separated by the criteria of soil moisture, rock and
earthworm from fertile soil category.
The electrical conductivity and bulk density criteria can be separated into two groups that are one of
the groups has moderate salinity which is more than 50 s/cm with bulk density ranging from 1.0-1.2 g
-3
mL . Tanah ladu is deep clayey soils with light olive brown color and sticky texture when moist (Silty
Clay texture) is categorized as the infertile soil. The other relationship of soil moisture, rock and
earthworm criteria were linked in infertile category show low in salinity level with high bulk density, with
-3
more than 1.5 g mL . The soil was located at shallow surface of soil. Texture for Tanah pasir was
sandy soil with gritty feeling, thus known as a sandy clay loam. Based on the moisture content of
Tanah Pasir, it was found that this soil is a dry soil as the soils are rapidly decreasing of water while
low water holding capacity. The colour for Tanah pasir was dark yellowish brown.
The fertile soil was a moist soil with abundance of rock and earthworm. The soils have moderate
-3
salinity level with bulk density ranging from 1.1-1.3 g mL and located at deep layer of soils. From the
electrical conductivity and bulk density criteria, the criteria were separated into three types of soils
namely Tanah hitam, Tanah kuning and Tanah batu lemak. These soils are described as follows: For
Tanah hitam, the soil texture consists of less clay and has a gritty feeling, hence described as a sandy
clay loam. The colour for the soil was dark yellowish brown which is quite similarity with the farmers’
perception as they describe the soil as dark in colour. The dark colour show the present of organic
matter such litterfall.
For Tanah kuning, less gritty and sticky texture when moist compare to Tanah hitam, thus the soil can
be described as silty clay. The soils colour was yellowish brown soil which similar with the colour
referred by farmers. Last but not least, Tanah batu lemak could be found at the upstream of the study
area was used by the farmers for planting pepper crops. The soil has a gritty feeling with sticky texture
when moist and considerably as sandy clay. Strong brown is the colour for the soils which had a quite
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similar with soil colour referred by farmers. However, the soil textures for some soils have a significant
different due to hardly be described as the textures were determined using feel methods.
As a result, the ranking for soil fertility from most fertile to less fertile are as follows: Tanah hitam,
Tanah kuning, Tanah batu lemak, Tanah pasir and Tanah ladu. Therefore, it can be concluded that
the local farmers still largely depends on the soil indigenous knowledge which is very important for
agriculture site selection and with the suitable characteristics of soil, resulting high yield of crops.
Through the soil classification of indigenous knowledge can form basis for many management
practices to improving soil quality. In many developing countries, the local people have lead to many
researchers and extension worker about the important of indigenous knowledge (Thrupp, 1989;
Warren 1989). Thus, this knowledge provides a greater identification and present many insights about
the sustainable management of tropical soils as the farmers have been interacting with their soils for
long time (Barrios & Trejo, 2003).
Based on the study with local farmers at Nanga Machan, it is observed that their knowledge on soil is
very limited in identifying soil characteristics. According to those farmers, they identify soils based on
soil colour, texture, depth, the presence of earthworm and plant that grow on it. They also believed
that, red to yellow soil which known as Tanah tuai is less fertile compare to black soil. Other criteria
that they use as indicator are the soil type which locally known as Tanah gemuk and vegetation grows
on that area such trees and grasses. From the farmers point of view, Tanah gemuk or commonly
known as fat soil which often known as productive soil. The soil is brown in colour soil at that particular
area which already known to be area that is recently being planted by farmer and the soil is suitable to
cultivate any crops without addition of fertilizer. According to Krasilnikov & Tabor (2003) naming of
soils are based on their characteristics such as red (droughty), fat (productive) or sandy.
In addition, soil fertility level determined by crop physical appearances and health condition of the crop
has been used by farmers for decision making in selecting permanent farmlands. They also indicate
soil fertility based on their observation on the growth of the vegetation i.e. bigger sized tree is an
indication of fertile soil. Tanaka et al. (2007b) reported that Iban farmers use the traditional knowledge
to select suitable site for shifting cultivation is by looking at the size of the stem diameter of trees.
Birmingham (2003), stated that recent research in Africa and Asia (Tamang, 1993) has shown the
major similarities and complementarities between indigenous knowledge and scientific knowledge of
soils, thus through this complementary would create to the soil management (Barrera-Bassols &
Zinck, 2003). According to Buthelezi et al. (2010) that conducted in KwaZulu-Natal, South Africa, the
important of indigenous knowledge for soil classification and management from the local people and
small scale of farmer that based on soil and land characteristic are remain unknown by the scientific
community, thus by understanding the knowledge of local farmers may help the researcher to
integrate the indigenous with scientific knowledge. Therefore, the correlation of indigenous knowledge
and scientific understanding is important to be recorded as it implies to the fundamental similarities,
hence will lead to the improvement of land use for farmers. Hence, it is important that the role of this
knowledge need to be study continuously in details to develop and implement on the sustainability of
management on land.
4. CONCLUSION AND RECOMMENDATION
From the findings, this study showed that the farmers at the study area largely depends on their
knowledge about soils mainly observation on topsoil and indicators characteristics. Several criteria of
soil characteristics referred by farmers have similarity with scientific knowledge, such soil colour and
soil texture classes (sticky, sandy, etc.). There are five types of soil are found at the study area locally
known Tanah hitam, Tanah kuning, Tanah batu lemak, Tanah pasir and Tanah ladu. The soil colour
and texture is the important characteristics for farmers to evaluate the soil fertility status. In terms of its
suitability for farming, the fertility level of the soil as classified by the farmers in Nanga Machan is as
follows: TH > TBL > TK > TL & TP.
In term of soil physicochemical properties, it can be concluded that the most of the soils at the study
area are acidic soil and varied widely among classified soil types. Some physical properties of the soil
were differed among each other in terms of its texture and level bulk density level. From the soil
classification, these five types of soils are divided into two categories which are fertile and infertile soil.
Tanah hitam, Tanah kuning and Tanah batu lemak are categorise as fertile while Tanah ladu and
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Tanah pasir are infertile soils. The differences among these soil due to their criteria such moisture
content, the present of rock and earthworm, electrical conductivity, bulk density, soil organic matter,
soil depth, texture and soil colour.
Hence, it was obviously the farmers could not differentiate the soils at the first place especially when it
goes to the subsurface soil, but for surface soil, it is easier to be observed. Nevertheless, the
indigenous knowledge is important indicators for the stability and success of land management
systems. The knowledge assists for the transformation from modernization technology method into
traditional methods and help to stabilizing the environment and soil fertility. In addition, the indigenous
knowledge may influence the agriculture practise in future and it should be applied for future
generation.
Acknowledgement
The authors would like to express their gratitude to the District office of Kanowit and people of Nanga
Machan, Kanowit for their supportive assistance and co-operation during the field survey.
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Ecologist, 14 (4), 146-149.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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WATER QUALITY OF BATANG AI AND BATANG AI RESERVOIR, SARAWAK
1*
1
Ling, T.Y. , Wong, Y.M. , L. Nyanti
1
2
2
Department of Chemistry, Department of Aquatic Science,
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak (Malaysia)
*E-mail:tyling@frst.unimas.my
Abstract
Batang Ai is one of the major rivers that flows into the Batang Ai Reservoir. The reservoir was created
mainly for power generation but tourism and aquaculture activities are also carried out. However, little
is known about the water quality of the Batang Ai that flows into the reservoir. Therefore, a study was
conducted to determine the inflow water quality. Results of the study shows that during dry weather,
DO ranged from 8.00 - 9.90 mg/L with higher values near settlement. However, during wet weather,
DO dropped to 6.33 - 6.96 mg/L. During dry weather, temperature at the farthest upstream station was
o
o
25.0 C whereas at inundated stations they were 30.5 - 31.4 C. pH of the stations were 5.92 - 7.39
with higher pH values during dry weather. Turbidity ranged from 2.8 – 15.6 NTU. BOD5 of the stations
near settlement showing significantly higher values. Nitrate-nitrogen and nitrite-nitrogen
concentrations were low. During dry weather, the differences among the stations were more
pronounced than during wet weather where most of the stations did not show significant difference in
TKN, nitrate-nitrogen and nitrite-nitrogen. The water quality parameters complied with Class II of
INWQS except for BOD5 which fall in Class III and IV of INWQS.
Keywords: Dam, nutrients, reservoir inflow, biochemical oxygen demand, turbidity
1. INTRODUCTION
Batang Ai and Sg. Delok are two of the major rivers that were partially inundated in the creation of the
2
Batang Ai Reservoir. The reservoir, with a full surface area of 90 km was created mainly for power
generation. However, tourism and aquaculture activities are also carried out in the area. Fish stocking
was conducted from 1984- 1993 in order to increase fish production (Pusin, 1995). Even though the
reservoir has been in operation for about 17 years, literature on the water quality of the inflow and
outflow are limited. For the Batang Ai that flows into the reservoir, there are settlements along the river
and therefore an assessment of the water quality is important to provide the status of the water quality
and also baseline information for future reference. A water quality study of the inflow was conducted in
1989 (Pusin, 1995).
Inflow of a reservoir is important as it provides the reservoir the water not only for hydroelectric
generation but also for the aquatic life in the reservoir. The water quality of inflow will affect the cage
aquaculture in the reservoir as clean input will dilute the nutrients in the reservoir contributed by cage
aquaculture. On the other hand, poorer water quality such as high nutrients and deoxygenated inflow
will cause eutrophication which will deprive the aquatic life of dissolved oxygen. This will led to low
productivity or even death of the caged fish. Household wastewater from the settlement could
contribute to water quality deterioration as it is known to contain high organic matter and nutrients
such as nitrogen and phosphorus (Ling et al., 2010b; 2012b). Studies of the Santubong River showed
that stations near housing and construction areas have high nutrients (Ling et al., 2010a). Cage
aquaculture activities in the reservoir contribute nutrients to the water and previous studies at Batang
Ai Reservoir showed that nutrients were higher near the cage culture site than non-cage culture site
(Nyanti et al., 2012). Water quality including sulphide of the reservoir was investigated in previous
study (Ling et al. 2012). Therefore, the objective of this study was to determine the water quality of
Batang Ai as it enters the Batang Ai reservoir.
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2. MATERIALS AND METHODS
The Batang Ai is located about 235 km from Kuching City. Samplings were conducted in November
2011 and April 2012 at four stations as shown in Fig. 1. Description of the stations are given in Table
1. The coordinates of sampling sites were measured using a GPS (Garmin) and they are given in
Table 1.
Figure 1. Location of sampling stations at the study site (http://maps.google.com).
Table 1. Sampling stations selected for determination of water and sediment quality.
Station
1
2
3
4
Location
N 01° 15’ 36.1”
E 112° 02’ 33.3”
N 01° 13’ 53.4”
E 112° 00’ 40.1”
N 01° 12’ 12.4”
E 111° 56’ 57.2”
N 01° 11’ 40.8”
E 111° 55’ 32.6”
Description
Upstream of Rumah Jambu at Batang Ai
Downstream of the confluence of Sg. Delok
and Batang Ai
Upstream of the confluent of Batang Ai and Sg.
Engkari
Confluent of Batang Ai and Sg. Engkari
In-situ water quality parameters for each station were determined at the subsurface and turbidity was
determined both at the subsurface and bottom. The physical parameters such as pH, temperature,
and turbidity were measured using YSI 6600 Multiparameter Water Monitor. Dissolved oxygen (DO)
was measured using a DO meter (Hanna HI9146). Water transparency was determined by using a
Wildco® Secchi disc. Three replicates of water samples were collected from each sampling station.
These samples were kept in different HDPE and borosilicate glass bottles and stored in icebox for
transportation to the laboratory for analysis within 24 hours. Composited samples were analyzed in
triplicates. BOD5, total phosphorus (TP) and total Kjeldahl nitrogen (TKN) were analyzed according to
the Standard Methods for the Examination of Water and Wastewater (APHA, 1998). Nitrate-nitrogen
(NO3-N) and nitrite-nitrogen (NO2-N) were analyzed according to Hach (2005) after filtration using
GF/C Whatman filters (0.45 m) (Whatman International δtd, Kent, U.K.). Five-day biochemical
oxygen demand (BOD5) analysis was conducted on site with 1:1 dilution.
Twenty-five milliliters of the sample was analyzed for nitrate-nitrogen according to Cadmium
Reduction Method (Hach, 2005). Nitrite-N was analyzed according to the Diazotization Method where
10 mL of the sample was used (Hach, 2005). The concentrations of both nitrate-nitrogen and nitritenitrogen were determined by using a spectrophotometer (Hach DR 2010) at the wavelength of 507
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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nm. Total Kjeldahl Nitrogen was analyzed according to the Macro-Kjeldahl method (APHA, 1998)
where 50 mL of the digestion reagent was added to 250 mL of water sample in a beaker. Distillation
was collected in 50 mL of 2% boric acid and the concentration was determined using Nessler Method
(Hach, 2005). For total phosphorus, 50 mL of sample was digested using sulfuric acid and nitric acid.
After digestion the sample volume was adjusted to 100 mL with distilled water and it was analyzed
using the Ascorbic Acid Method (APHA, 1998). The concentration of phosphorus was determined from
a calibration curve.
Significant difference of each parameter among the stations for each trip was analyzed using one-way
analysis of variance (ANOVA) and Tukey’s test was used for pair-wise comparisons. Overall
comparisons were made using 2-way ANOVA. All data analyses were conducted using SPSS version
17.0 package.
3. RESULTS AND DISCUSSION
Table 2 show the mean values and standard deviations of temperature, pH and DO measured in the
first and second sampling trip respectively. For Trip 1, temperature of the stations ranged from 25.02
to 31.35°C whereby the lowest and highest temperature was recorded at stations 1 and 3 respectively.
For Trip 2, the mean temperature varied from 30.11 to 30.83 °C. During Trip 1, station 1 temperature
o
was about 6 C lower than the other stations due to its location upstream and it was not inundated.
However, during Trip 2, which was a wet day, the water level of the reservoir was higher and station 1
o
was also flooded and as such the temperature was only 0.2 C lower than the water temperature at
station 2. During Trip 2 stations 2 - 4 showed lower temperature than Trip 1 due to higher flow of
cooler water from upstream. Statistical analysis shows that all the stations were not significantly
different with each other (P>0.0005). Station 4 has lower temperature than station 3 for both trips
because it is location downstream of the confluence of Batang Ai and Sg. Engkari. Sg. Engkari has
bigger discharge and thus contributed higher volume of cooler water.
Table 2 Temperature, pH and dissolved oxygen (DO) at the subsurface
the two trips.
Temperature
pH
(°C)
Station
Trip 1
Trip 2
Trip 1
Trip 2
1
25.02
30.11
6.31
5.92
2
31.31
30.31
7.39
5.92
3
31.35
30.83
7.15
6.26
4
31.00
30.49
7.31
6.11
of the sampling stations for
DO
(mg/L)
Trip 1
8.00
9.90
9.40
8.70
Trip 2
6.54
6.33
6.96
6.80
During the first sampling trip the subsurface water body shows pH values ranging from 6.31 to 7.39
with station 1 showing acidic condition and the value was lower than all the other stations (Table 2).
During Trip 2, the pH values show acidic condition ranging from 5.92 to 6.26 with stations 1 and 2
showing the lowest value. The low pH values of station 1 during Trip 1 and all stations during Trip 2
are due to the water of peat swamp forest nearby which is acidic and it was observed that the water
was dark brown in colour which is typical of the peat swamp (UNDP, 2006,). Low pH range (3.03-3.84)
of peat swamp forest of Batang Igan was reported (Noraini et al., 2010). However, in Batang Ai, only
nearby station 1 was peat swamp area and thus pH values were not as low as that of Batang Igan due
to dilution from upstream. Compared to Trip 1, pH during Trip 2 were all lower than Trip 1 possibly due
to bigger influence of lower pH water from upstream in wet weather. In addition, there is some impact
of lower algal photosynthesis than Trip 1 resulting in lower pH as it was cloudy and rainy during Trip 2.
pH values fall in Class II (pH 5-7) of INWQS.
During Trip 1 the subsurface water showed concentration of DO ranging from 8.0 to 9.9 mg/L. Stations
2 and 3 have the highest DO values which were 9.9 and 9.4 mg/L respectively compared to other
stations. The lowest DO was at station 1 likely due to oxygen consumption by microorganism in the
decomposition of organic matter as the high DO corresponds to high BOD 5 (Fig. 2c). The high values
at stations 2 and 3 could be due to oxygen production through phytoplankton photosynthesis at
stations 2 and 3. During Trip 2, the concentrations of DO at the stations ranging from 5.65 to 6.96
mg/L were lower than Trip 1. This is likely due to the higher consumption of DO as organic matter as
reflected by BOD5 during Trip 2 (Fig. 2c) was higher at all stations.
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Subsurface water turbidity for Trip 1 and Trip 2 ranged from 3.2-6.1 NTU and 2.8-7.5 NTU
respectively. During Trip 1, turbidity of the subsurface water increased as we move toward the
reservoir (Fig. 2a). That shows clearer water at upstream stations 1 and 2 than stations 3 and 4.
However, during Trip 2, the trend of turbidity of subsurface water reversed with higher turbidity at the
two stations upstream where station 1 showed the highest value. This is due to the inflow of sediment
during rainfall runoff event where stations 1 and 2 had the most impact. In the bottom water, during
both trips, station 4 showed high turbidity due to its location at the confluence of two rivers (Batang Ai
and Sg. Engkari) and thus suspended sediment from turbulence resulted in high turbidity regardless of
weather condition. The bottom water of Trip 2 showed very much higher turbidity than the subsurface
water at all the stations with stations 1 and 2 showing very high values due to sediment input from
upstream and it decreased at station 3. Bottom water at station 2 showed the highest turbidity among
the four stations as it received both turbid water from station 1 upstream and from Delok River in
addition to resuspended sediment due to the turbulence at the confluence. Compared with standards
of INWQS, subsurface water of stations 1 and 2 during Trip 1 and stations 3 and 4 during Trip 2 fall in
Class I (5 NTU) whereas the other stations fall in Class II. For bottom water, turbidity at station 3
during Trip 2 complied with Class I whereas the other values complied with Class II (50 NTU).
Water transparency for Trip 1 and Trip 2 ranged from 1.09-4.00 m and 0.91-3.57 m respectively and
they show the opposite trend of turbidity as they are closely correlated with each other (Fig. 2b). For
the Trip 1, the lowest transparency value was recorded at station 2 whereas the highest was observed
at station 1. During Trip 2, the highest transparency value was measured at station 3 and this
corresponds to the lowest turbidity among the stations. During Trip 1, transparency was the highest at
station 1 and the turbidity the lowest as it is located upstream and the water was faster flowing and not
inundated when compared to other stations.
Mean BOD5 at the stations for Trip 1 and Trip 2 ranged from 3.40 to 6.00 mg/L and 4.57-6.61
respectively. BOD5 was higher at all stations at the high water level during Trip 2 (Fig. 2c). Two-way
ANOVA shows that Trip 2 mean BOD5 (5.71 mg/L) was significantly higher than that of Trip 1 (4.98
mg/L) (P=0.001). During Trip 1, BOD5 at stations 1 and 2 were significantly higher than 3 and 4
(P<0.05). During Trip 2, BOD5 at stations 1, 2 and 3 were significantly higher than station 4. Station 1
is located downstream of a longhouse where household discharge and partially treated septic tank
effluent contributed to the organic matter in the water. Household wastewater which included septic
tank effluent has been shown to contain high BOD5 ranging from 83-171 mg/L (Ling et al. 2010; 2012).
In addition, it is common for rural communities to have small scale husbandry which could also be a
non-point source of pollution. Being upstream, the river is smaller and less volume of water was
available for dilution as compared to other stations downstream. The effect of settlement was more
pronounced during Trip 2 possibly due to organic matter from the settlement entering the river through
surface runoff. This explains similar observation at station 3 where higher value was observed during
wet weather condition (Trip 2) than during dry weather condition of Trip 1 as station 3 which is located
downstream of several longhouses, a school and clinic. Compared with INWQS, BOD5 at all stations
exceeded Class II limit (3 mg/L) with stations 1 and 3 exceeding Class III (6 mg/L) during Trip 2.
For total phosphorus, during the first trip, the concentration ranged from 22.4-51.0 µg/L (Fig. 2d).
Station 4 showed the highest concentration of all the stations. Stations 1 and 2 showed higher
concentrations than station 3. Station 3 showed the lowest value of TP which corresponds to that of
BOD5 (Fig. 2c). Stations 2 and 4 showed high TP because of the higher sediment load as phosphorus
are highly associated with sediment (Lai & Lam, 2009) and those two stations are located at the
confluences, Sg. Delok - Batang Ai and Sg. Engkari - Batang Ai respectively, where turbulence
caused resuspension of the sediment as indicated by the high turbidity (Fig. 2a) and low transparency
(Fig. 2b). Ling et al. (2009) also reported that total phosphorus of sediment was strongly correlated
with silt content and organic matter of Serin River sediment. Therefore, turbulence resulted in higher
total phosphorus being detected in the water samples from those stations. During Trip 2, the
concentration ranged from 29.2-41.8 µg/L. Station 1 showed the highest TP due to the settlement
nearby which contribute phosphorus from detergents and also household waste. The trend of increase
in TP as we move from station 2 to station 3 was similar to that of BOD 5 (Fig. 2c). This indicates that
the likely source of BOD5 and TP is similar, namely the non-point source from settlement such as
waste from human and animals during wet weather condition as those waste contain phosphorus,
which is an important consituent in plants and animals (Metcalf & Eddy, 1991). The general decrease
in mean concentration during Trip 2 when compared with Trip 1 is due to dilution by rain water.
Compared with INWQS, the concentration of phosphorus complied with Class II limit of 0.2 mg/L
which is equivalent to 200 µg/L.
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(a)
(b)
(c)
(d)
Figure 2 (a) Turbidity at the subsurface (SS) and bottom (B); (b) transparency; (c) biochemical oxygen
demand (BOD5); and (d) total phosphorus (TP) of the subsurface water at the stations during the two
different trips.
For nitrogen forms, NO2-N ranged from 0.001-0.007 mg/L during Trip 1 and 0.002-0.003 mg/L during
Trip 2 (Table 3). For NO3-N concentrations, Trip 1 showed the range of 0.010-0.020 mg/L. However, in
Trip 2, all the stations have mean value of 0.010 mg/L showing the impact of dilution. Trip 1 NO 3-N
concentrations of stations 1 and 3 were the highest and higher than that of Trip 2 indicating
contributions from septic tank effluent. For NO 2-N, stations 3 and 4 were significantly higher than
stations 1 and 2 during Trip 1 but not significantly different during Trip 2. Compared with NO 2-N and
NO3-N, TKN values were the highest among the nitrogen concentrations (Table 3).
In trip 1, TKN increased significantly as we move from station 2 to station 3 showing an increase in
contributions from the settlement as we move downstream. In Trip 2, TKN of stations 1-3 were higher
than that of Trip 1 indicating that in spite of higher water level, the pollutants in runoff from the
settlement have more than offset the dilution of rain water. TKN among the stations 1-3 were not
significantly different (P>0.05) and they were significantly higher than station 4. The decrease from
station 3 to station 4 is similar to the trend of TP and BOD 5. This shows that these pollutants had the
source from settlement that produces organic waste such as sewage and animal waste. Urea and
protein are the main source of nitrogen in wastewater (Metcalf & Eddy, 1991). Compared with INWQS
limit for Class II, NO2-N and NO3-N in this study were much lower than the limits of 0.12 mg/L
(NO2=0.4 mg/L) and 1.58 mg/L (NO3=7 mg/L) respectively.
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Table 3 Values of mean and standard deviation of nitrite-nitrogen (NO2-N), nitrate-nitrogen (NO3-N)
and total Kjeldahl nitrogen (TKN) at the sampling stations for the two trips.
Station
NO2-N
(mg/L)
NO3-N
(mg/L)
Trip 1
Trip 2
Trip 1
a
a
a
1
0.002±0.001 0.002±0.001
0.020±0.000
a
a
b
2
0.001±0.000 0.003±0.000
0.010±0.000
b
a
ab
3
0.006±0.000 0.003±0.000 0.017±0.006
b
a
b
4
0.007±0.002 0.003±0.000
0.010±0.000
Mean concentrations in the same column with the same
level.
TKN
(mg/L)
Trip 2
Trip 1
Trip 2
a
a
a
0.010±0.00 0.883±0.000 1.015±0.004
a
b
a
0.010±0.00 0.772±0.000 1.010±0.002
a
c
a
0.010±0.00 0.932±0.000 1.010±0.010
a
d
b
0.010±0.00 0.913±0.004 0.729±0.034
letters are not significantly different at 5%
4. CONCLUSIONS
The trend of water quality among the stations during the dry weather was different from wet weather in
that in spite of the dilution during wet weather there were actually higher concentrations of pollutants
such as BOD5 for all stations; TKN for three stations and TP at one station due to their proximity to
human settlement. High turbidity values were observed at subsurface and bottom water of upstream
stations during wet weather and also at bottom water at the confluence regardless of weather
condition. During dry weather, the differences among the stations were more pronounced whereas
during wet weather, most of the stations do not show significant difference especially in TKN, nitratenitrogen and nitrite-nitrogen. Overall, mean concentrations of nitrite-nitrogen, nitrate-nitrogen, and TP
were higher during dry weather than wet weather.
References
APHA. (1998). Standard methods for the examination of water and wastewater. (20th ed.)
Washington, D.C.: American Public Health Association.
Hach. (2005). DR/2800 Spectrophotometer procedure manual. Hach Company, USA 1996-2000.
Lai, D. Y. F., & Lam, K. C. (2009). Phosphorus sorption by sediments in a subtropical constructed
wetland receiving stormwater runoff. Ecological Engineering, 35(5), 735-743.
Ling, T. Y., Miod, M. C., Nyanti, L., Norhadi, I., & Emang, J. J. J. (2010a). Impacts of aquaculture and
domestic wastewater on the water quality of Santubong River, Malaysia. Journal of
Environmental Science and Engineering, 4(4),11-16.
Ling, T. Y., Paka, D. D., Nyanti, L., Norhadi, I., & Emang, J. J. J. (2012a). Water quality at Batang Ai
Hydroelectric Reservoir (Sarawak, Malaysia) and implications for aquaculture. International
Journal of Applied Science and Technology, 2(6), 23-30.
Ling, T. Y., Cornellia M., & Nyanti, L. (2009). Impact of agricultural activities, motor vehicles and ewaste on sediment characteristics of the Serin River, Malaysia. Journal of Environmental Science
and Engineering, 9(3), 13-22.
Ling, T. Y., Dana, M.J., Bostam, S. & Nyanti, L. (2012b). Domestic wastewater quality and pollutant
loadings from urban housing areas. Iranica Journal of Energy & Environment 3(2): 129-133.
Ling, T. Y., Siew, T. F. & Nyanti, L. (2010b). Quantifying pollutants from household wastewater in
Kuching, Malaysia. World Applied Sciences Journal, 8(4), 449-456.
Metcalf and Eddy, (1991). Wastewater engineering: Treatment, disposal and reuse. New York:
McGraw-Hill, Inc..
Noriani, R., Gandaseca, S., Johan I., & Mohd Iqbal J. (2010). Comparative study of water quality at
different peat swamp forest of Batang Igan, Sibu Sarawak. American Journal of Environmental
Sciences, 6(5), 416-421.
Nyanti, L., Hii, K. M., Sow, A., Norhadi I. & Ling, T. Y. (2012) Impacts of aquaculture at different
depths and distances from cage culture sites in Batang Ai Hydroelectric Dam Reservoir,
.
Sarawak, Malaysia. World Applied Sciences Journal (In press)
Pusin, L. G. (1995). Cage aquaculture development in reservoirs in South-East Asia with particular
reference to Batang Ai Reservoir in Sarawak, Malaysia. (M.Sc. Thesis). Institute of Aquaculture,
University of Stirling, Scotland.
UNDP, 2006. Malaysia’s peat swamp forests – Conservation and sustainable use. United Nations
Development Program, Malaysia. Retrieved from http://www.undp.org.my/uploads/malaysia
%20peat%20swamp%20forest.pdf.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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CHARACTERIZATION OF MICROBES FROM PALM OIL MILL EFFLUENT (POME)
Chan, C.S.W.*, Lau, S.**, Husaini, A.A.S.A., Zulkharnain, A., Apun, K., Bilung, L. M. and
Vincent, M.
Department of Molecular Biology, ** Department of Chemistry,
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak, 943000 Kota Samarahan,Sarawak
*E-mail: ccsw86@hotmail.com
Abstract
Bioconversion of Palm Oil Mill Effluent (POME) to generate methane gas via anaerobic digestion
involves a consortium of microbes which are responsible in several steps of the biodegradation
process. In this study, the microbial community from a selected POME was characterized via
molecular techniques and through culture-based plating in order to determine their composition, and
subsequently understand their function in the anaerobic community. Genomic DNA of the microbial
community was extracted using direct extraction technique, followed by PCR targeting the 16S rRNA
region. Distinct fragments of approximately 1100 bp in sizes were successfully amplified using PCR
and cloned into Escherichia coli XL-1 Blue cells. Upon sequencing of the fragments, BLAST queries
identified the bacteria as Thermoanaerobacterium sp. and Ethanoligenes sp. In addition, two other
bacterial species were successfully isolated from the POME by culturing on DVS agar. The
sequencing result of these bacterial isolates showed both isolates belonged to the Bacillus genus. By
understanding the bacterial community present in the POME, this will lead to the improvement of the
anaerobic digestion process to enhance the production of biogas such as methane.
Keywords: Palm oil mill effluent, anaerobic digestion, microbial community
1. INTRODUCTION
Elaeis guineensis or more commonly known as oil palm is one of the major crops in Malaysia and
Indonesia. According to the Malaysia Palm Oil Board (MPOB) in 2008, the global production of palm
oil and plantation area has increased and Malaysia contributes to 41% of the world palm oil
production. However, rapid development of the palm oil industry has contributed to environmental
pollution due to the large quantities of wastes produced during oil extraction process. Oil extraction
process requires huge amount of water and it has been estimated that more than 50% of the water
3
ends up as POME (Ahmad et al., 2003). According to Loreatani (2006), approximately 53 million m
POME is produced every year based on production in 2005 (14.8 million tonnes). Although POME is
non-toxic, it is identified as major source of aquatic pollutions when discharged untreated into nearby
water system due to high concentration of organic matter, total solid, oil and grease, COD as well as
BOD (Ma, 2000).
Many attempts to treat POME are currently being employed worldwide. One of the treatments
introduced is the ponding system. Ponding system which is also known as waste stabilization pond
has been used in Malaysia since 1982 (Onyia et al., 2001). However, ponding systems have some
disadvantages such as the need for large areas, long hydraulic retention time (HRT), bad odour and
difficulties in maintaining liquid distribution (Onyia et al., 2001). Due to these limitations, anaerobic
treatment of POME using newer technologies such as anaerobic digesters offer a more attractive
solutions for methane gas production and clean development mechanism (CDM).
One of the key factors in determining the efficiency of anaerobic digesters is the optimal composition
of the bacterial community involved in the anaerobic degradation process, as the roles of the microbial
consortia in this process are still not completely understood. Thus, it is critical to have an accurate
understanding of the microbial population of the POME in order to provide an optimum condition for
microbial propagation and to monitor the microbial activities which could contribute to a greater
methane production. In this study, the aim of the present work was to determine the microbial
community in POME by using 16S rRNA clone library and traditional culture-based techniques.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
2. MATERIAL AND METHODS
2.1 Samples collection
Palm Oil Mill Effluent (POME) was collected from the anaerobic pond from Bau Palm Oil Mill
(BAPOM), Kuching, Sarawak. The samples were stored in sealed container immediately after
collection and preserved at 4°C in order to avoid biodegradation due to microbial activities.
2.2 Isolation of bacteria from POME
POME was cultured on DVS agar (Savant et al., 2002). The DVS medium has the following
composition (per L of distilled water): 6.0g NaCl, 0.8g CaCl 2.2H2O, 1.0g MgCl2.6H20, 10.0g Peptone,
10.0g Tryptone, 25.0g CH3CO2K, 3.0g KH2PO4, 3.0g K2HPO4, 15.0g (NH4)2SO4, 20.0g Yeast and
30.0g agar. Grown colonies were picked and purified by streaking onto agar plate. All cultures were
incubated at 50°C in an anaerobic jar.
2.3 DNA extraction and PCR amplification
Colonies from the agar plates were picked and used for colony PCR whereas direct extraction of DNA
TM
from POME was carried out using Power Soil
DNA Isolation Kit (Mo Bio Laboratories, USA),
according to the manufacture’s instruction. The DNA obtained was confirmed through electrophoresis
in 1% agarose gel. 16S rRNA from direct extraction and solid agar were amplified using the primer set
10f and 1100r (Miqueletto et al., 2011). PCR reactions were prepared in 25 l final volume containing
50-100ng of DNA, 10X Taq DNA polymerase buffer, 1.0 l of 10 mε dNTP mix (Fermentas, Canada),
2.0 l of 25 mε εgCl2, 1.0 l of each primer and 0.5 l of 5 U AmpliTaq DNA polymerase (Fermentas,
Canada). The PCR amplifications were then performed using an initial denaturation step of 5 minute at
95 °C, followed by 30 cycles of 30 seconds at 96 °C, 1 minute at 54 °C, and 1 minute at 72 °C; and a
final extension of 7 minutes at 72 °C.
2.4 Cloning 16S rRNA
PCR products were purified according to the manufacturer’s instruction (εo Bio δaboratories, USA).
Purified colony PCR products were sequenced while the purified PCR fragments from direct extraction
technique were cloned into pGEM-T Easy vector and transformed into E. coli XL-1 blue cells using the
heat shock method. White colonies were randomly selected from the agar plates and plasmids were
extracted using a plasmid extraction kit (Promega, USA). The extracted plasmids were re-amplified
through PCR reaction and sequenced.
2.5 Phylogenetic analysis
The sequences obtained were compared to known 16S rRNA sequences in GeneBank database by
using basic logical alignment tool (BLAST). Closely related sequences were aligned with PCR
sequences using the program CLUSTAL W and further edited manually. Phylogenetic tree were
constructed by neighbour-joining method provided in MEGA ver 4.0.
3. RESULTS AND DISCUSSION
16S rRNA region was successfully amplified and cloned into pGEM-T Easy vector. Out of the nine
clones screened, only four were successfully cloned with the PCR fragment size of 1100 bp as shown
in Figure 1. Upon cloning, the extracted plasmids from the successful clones were re-amplified,
purified and sequenced (Figure 2a). As for the isolates from solid agar, PCR product of 1100 bp was
successfully amplified (Figure 2b). These isolates were also cloned as well. Table 1.0 shows the result
of the DNA successfully sequenced.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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M
1
2
3
4
5
6
7
8
9
6000 bp
3000 bp
1500 bp
1100 bp
1000 bp
750 bp
Figure 1 Agarose gel electrophoresis of amplified 16S rRNA region of the isolates. Four isolates (C,
E, H and I) were identified to have the recombinant plasmid with the band size of
approximately 1100 bp. Lane M, 1kb ladder (Fermentas); Lane 1, isolate B; Lane 2, isolate C;
Lane 3, isolate E; Lane 4, isolate F; Lane 5, isolate H; Lane 6, isolate J; Lane 7, isolate K;
Lane 8, isolate L; Lane 9, isolate I.
a
M
1
2
3
4
b
M
5
6
6000 bp
3000 bp
1500 bp
1100 bp
1000 bp
6000 bp
3000 bp
1500 bp
1100 bp
1000 bp
750 bp
Figure 2 Agarose gel electrophoresis of amplified 16S rRNA region of the isolates. (a) The extracted
plasmid from 16S rRNA clone library which were successfully amplified (1100 bp). (b) PCR
products (1100 bp) of isolated bacteria from solid agar. (a) Lane M, 1kb ladder (Fermentas);
Lane 1, 2, 3 and 4, PCR products amplified by using extracted plasmid from isolates C, E, H
and I respectively. (b) Lane 5 and 6, DNA template of bacteria A and D isolated from DVS
agar.
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Table 1. Sequence homology of the isolates
Isolates
Bacteria
Gen Bank Database
A
C
D
E
H
I
Bacillus thermoamylovorans
Thermoanaerobacterium aciditolerans
Bacillus coagulans
Thermoanaerobacterium aciditolerans
Thermoanaerobacterium aciditolerans
Ethanoligenes harbinense
NR 029151
NR 042856
NR 041523
NR 042856
NR 042856
NR 042828
Sequences homology
(%)
98
97
95
97
97
90
In order to further understand the microbial community in POME, one representative from each isolate
was included in the phylogenetic tree. The sequences data set contained five sequences, including
Kluyveromyces lactis that was used as the out group (Figure 3).
Bacilli
Firmicute
s
Clostridia
Figure 3 Dendrogram of partial sequence of 16S rRNA from POME, grouped by class. The
dendrogram was constructed by the neighbour-joining method. The number at the nodes of
the tree indicates bootstrap value of each node out of 100 bootstrap resampling. The scale bar
represents 0.2 substitutions per base position.
From the phylogenetic tree, all four isolates were classified in the phylum of Firmicutes. Two of the
Firmicutes (C and I) were clustered in the class of Clostridia while isolates A and D were assigned to
the class of Bacilli. Isolate C, E, H and I were closely associated with Thermoanarobacter aciditolerans
and Ethanoligenes harbinense from the class Clostridia with 97% and 90% similarity respectively.
Thermoanaerobacter sp. and Ethanoligenes sp. had also been reported presence in POME sludge
(Khemkhao et al., 2011) and sludge of molasses wastewater from an anaerobic digester (Xing et al.,
2006). Both Thermoanaerobacter sp. and Ethanoligenes sp. are known to be associated with the
fermentation of glucose into ethanol, acetate, hydrogen and carbon dioxide (Koskinen et al., 2008;
Xing et al., 2006).
The sequences of isolates from solid agar (isolate D and A) were identified as member of the bacterial
genera Bacillus, Bacillus coagulan and Bacillus thermoamylovorans with 96% and 98% similarity
respectively. The presence of Bacillus genus bacteria in sludge and agriculture wastes were also
shown in previous findings (Ivanov et al., 2004; Vossoughi et al., 2001). It is known that Bacillus
thermoamylovorans and Bacillus coagulans are capable of producing ethanol, acetate and lactate
from glucose utilization (Tay et al., 2002). Similar results were also reported by other researchers
(Kotay and Das, 2007; Pantamas et al., 2003).
Bacillus coagulans, Bacillus thermoamylovorans, Thermoanaerobacter aciditolerans and
Ethanoligenes harbinense are thermophilic bacteria. Metabolically, they are facultative and/or strict
anaerobes and moderately acidophile (Kublanov et al., 2007; Vechi and Dargo, 2006; Combet-Blanc
et al., 1995). These characteristics enable the bacteria to survive in POME which is acidic with pH of
between 4 to 5. This is supported by previous findings regarding isolation of these bacteria from acidic
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
and/or extreme environments (Longo et al., 2010; Koskinen et al., 2008; Kublanov et al., 2007; Xing et
al., 2006).
4. CONCLUSION
The microbial community of the POME was reflected from 16S rRNA clone library and traditional
culture-based technique. Thermoanaerobacter aciditolerans and Ethanoligenes harbinense from
Clostridia class were isolated through 16S rRNA clone library while Bacillus coagulans and Bacillus
thermoamylovorans were isolated using traditional culture-based technique. High sequence
similarities (>90%) were found in both isolation methods.
Acknowledgement
This project were funded by a research grant from UNIMAS (Top-Down grant:01(TD01)/764/2010(01))
and L18403 F07 00 CMS AGROTECH.
References
Ahmad, A., Ismail S. and Bhatia S. (2003). Water Recycling from Palm Oil Mill Effluent (POME) Using
Membrane Technology. Desalination, 157, 87-95.
Combet-Blanc, Y., Ollivier, B., Streicher, C., Patel, B.K.C., Dwivedi, P.P., Pot, B., Prensier, G. and
Garcia, J.-L. (1995). Bacillus thermoamylovorans sp. nov., a Moderately Thermophilic and
Amylolytic Bacterium. International Journal of Systematic Bacteriology, 45, 9-16.
Ivanov, V.N., Wang, J.-Y., Stabnikova, O.V., Tay, S.T.-L. and Tay, J.-H. (2004) Microbiological
monitoring in the biodegradation of sewage sludge and food waste. Journal of Applied
Microbiology, 96, 641-647.
Khemkhao,M., Nuntakumjorn, B., Techkarnjanaruk, S. and Phalakornkule, C. (2011). Microbial
Diversity in Thermophilic Adaptation on Pome Treatment. KMITL Sci. Tech. J. 11(1).
Koskinen, P.E.P., Beck, S.R., O rlygsson, J., and Puhakka, J.A. (2008). Ethanol and Hydrogen
Production by Two Thermophilic, Anaerobic Bacteria Isolated From Icelandic Geothermal Areas.
Biotechnol. Bioeng, 9999,1–12.
Kotay, S.M. and Das, D. (2007). Microbial Hydrogen Production with Bacillus coagulans IIT-BT S1
isolated from anaerobic sewage sludge. Bioresource Technology, 98(6), 1183-1190.
Kublanov, I.V., Prokofeva, M.I., Kostrikina,,N.A., Kolganova, T.V., Tourova, T.P., Wiegel, J. and
Bonch-Osmolovskaya, E.A. (2007). Thermoanaerobacterium aciditolerans sp. nov., a moderate
thermoacidophile from a Kamchatka hot spring. International Journal of Systematic and
Evolutionary Microbiology, 57, 260–264.
Lorestani, A.A.Z. (2006). Biological treatment of palm oil effluent (POME) using an up-flow anaerobic
sludge fixed film (UASFF) bioreactor ,TD899. P4 L869.
Longo, M.A., Carvalho, E., Deive, F.J., and Sanroman, M.A. (2010). An Approach to The
Characterization of a Novel Termophilic Bacillus thermoamylovorans Lipase. Chemical
Engineering Transaction, 20, 145-150.
Ma, A.N. (2000). Environmental Management for the Oil Palm Industry. Palm Oil Dev., 30, 1-10.
Miqueletto, P.B., Andreote, F.D., Dias, A.C.F., Ferreira, J.C., Eugênio, S.N. and Oliveira, V.M. (2011).
Cultivation-independent methods applied to the microbial prospection of oil and gas in soil from
sedimentary basin in Brazil. AMB Express, 1(35).
M.P.O.B., 2008. Malaysian Palm Oil Board. A summary on the performance of the Malaysian oil palm
industry- 2008. http:// econ.mpob.gov.my/economy/ Performance-130109.htm.
Onyia, C.O., Uyub A.M., Akunna J.C., Norulaini N.A. and Omar A.K.M. (2001). Increasing the fertilizer
value of palm oil mill sludge: bioaugmentation in nitrification. Sludge Management Entering the
Third Millenium- Industrial, Combined, Water and Wastewater Residues, 44, 157-162.
Pantamas, P., Chaiprasert, P., and Tanticharoen, M. (2003). Anaerobic Digestion of Glucose by
Bacillus licheniformis and Bacillus coagulans at Low and High Alkalinity. Asian J. Energy
Environ., 4,1-17.
Savant, D.V., Shouche, Y.S., Prakash, S. and Ranade, D. R. (2002). Methanobrevibacter acididurans
sp. nov., a novel methanogen from a sour anaerobic digester. International Journal of
Systematic and Evolutionary Microbiology, 52, 1081–1087
Tay, S.T-L., Ivanov, V.N, Yi, S., Zhuang, W.Q., and Tay, J-H. (2002). Presence of Anaerobic
Bacteroides in Aerobically Grown Microbial Granules. Microbial Ecology, 44(3), 278-285.
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Vecchi, E.De., and Drago, L. (2006).
Lactobacillus sporogenes or Bacillus coagulans:
misidentification or mislabelling? International Journal of Probiotics and Prebiotics, 1(1), 3-10.
Vossoughi, M., Alemzadeh, I., and Salehizadeh, H. (2001). Characterization of Biopolymer Flocculant
Produced By Bacillus sp. As-101. Scientia Iranica, 8(3), 207-210.
Xing, D., Ren, N., Li, Q., Lin, M., Wang, A. and Zhao, L. (2006). Ethanoligenens harbinense gen. nov.,
sp. nov., isolated from molasses wastewater. International Journal of Systematic and
Evolutionary Microbiology, 56, 755–760.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
SIZE CONTROLLED SYNTHESIS OF STARCH NANOPARTICLES BY A MICROEMULSION
METHOD
Aressa Azman*, Chin Suk Fun and Pang Suh Cem
Department of Chemistry, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak,
94300 Kota Samarahan, Sarawak, Malaysia
E-mail: aressa88@yahoo.com
Abstract
This paper reports a simple, fast and cost effective green synthesis of starch nanoparticles through a
water-in-oil microemulsion method. Starch is a favorable precursor material for nanoparticles
synthesis as it is nontoxic, renewable, biocompatible, low cost and abundantly in nature. Synthesis of
nanoparticles by microemulsion method has the advantages of ultralow interfacial tension, large
interfacial area and thermodynamically stable. Synthesis parameters such as types of oil phases,
types of co-surfactants, different ratios of (oil:co-surfactant), volume of aqueous phase and stirring
rates were found to affect the particle size and size distribution of starch while types of surfactants and
its concentrations was found not significantly affect the size of starch particles. Our results showed
that, starch nanoparticles with average particle size of 108.6 ± 16.7 nm were synthesized by a direct
nanoprecipitation method without the microemulsion system. On the other hand, starch nanoparticles
with smaller average size of 82.5 ± 12.4 nm were obtained by using microemulsion method under
controlled conditions. The nanoparticles obtained were spherical in shape and have uniform size
particles distribution. This study showed that microemulsion system can be used to control the sizes of
starch nanoparticles.
Keywords: Starch nanoparticles, nanoprecipitation, microemulsion, particle size controls.
**Please contact the corresponding authors for further details of this paper.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
METHOD FOR IDENTIFICATION OF Aglaia spp.
Belinda Ng Ling Nah*, Mariani Omarzuki, Tiong Chia Yeo
Sarawak Biodiversity Centre, KM20, Jalan Borneo Heights, Semengoh, Locked Bag No. 3032, 93990
Kuching, Sarawak (MALAYSIA)
Email: belindang@sbc.org.my*, mariani@sbc.org.my, cyeo@sbc.org.my
Abstract
Members of the genus Aglaia are frequently reported to contain bioactive phytochemicals. The genus,
which is of the Meliaceae family, is represented by more than 100 known species of woody trees or
shrubs in the tropical rain forests. Some of these species are very similar in their morphologies and
can sometimes lead to contentions in taxonomic identification of the Aglaia species. A rapid and
definitive molecular method which can distinguish the species will be useful in efforts to extract novel
bioactive compounds from this genus. Here, we discuss the analysis of DNA sequences in the internal
transcribed spacer (ITS) sequence of the nuclear ribosomal DNA and the development of a biomarker
for the species identification. Using Aglaia stellatopilosa as a model, we identified a biomarker that
consists of a unique set of 9 nucleotides identifier (9NI). This finding was validated through sequence
comparison within the ITS regions between Aglaia stellatopilosa and other Aglaia species as well as
non-Aglaia species. Using this method, we successfully identified more than 200 samples of Aglaia
stellatopilosa which were confirmed by taxonomic identification.
Keywords: Aglaia stellatopilosa, species identification, internal transcribed spacer (ITS)
**Please contact the corresponding authors for further details of this paper.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
ESTIMATING TOURISM INCOME THROUGH ENVIRONMENTAL ACCOUNTING APPROACH FOR
PANGKOR ISLAND, PERAK
1*
1
2
3
Rosmini Ismail , Sharifah Robiah Tuan Hazam , Khalizul bin Khalid , Mohmadisa Hashim ,
1
2
2
5
4
Hartini Jaafar , Suriani Abdul Hamid , Azita Yunos , Fairisa Othman , Norasibah Abdul Jalil ,
1
1
Hazianti Abdul Halim , & Noorlela Ahmad
1
Department of Accounting & Finance, Universiti Pendidikan Sultan Idris (MALAYSIA)
Department of Management & Leadership, Universiti Pendidikan Sultan Idris (MALAYSIA)
3
Department of Geography & Environment, Universiti Pendidikan Sultan Idris (MALAYSIA)
4
Department of Economics, Universiti Pendidikan Sultan Idris (MALAYSIA)
5
Department of History, Universiti Pendidikan Sultan Idris (MALAYSIA)
2
* Email: rosmini@fpe.upsi.edu.my
Abstract
The paper reports an ongoing research with the objectives to investigate whether the management of
the hospitality service provider in Pangkor Island aware and exercise good environmental practices. If
the practices present, the study further examines whether the cost incurred in executing them are
included and inserted as individual item in the financial statement. Primary and secondary data were
then collected to estimate Pangkor Island tourism income that takes account on environmental
elements following environmental accounting approach called Environmental Accounting Sustainable
Statements (ESAS). Through this method, Pangkor Island is treated as a single entity and the income
was calculated by combining the financial outcome of these entities and deducting expenditure from
tourism activities that degrade the environment (e.g. solid waste, water quality, exhaustion of natural
resources etc.). Finally, an Environmentally Sustainable Accounting Income Statement of the ESAS
for Pangkor Island was presented in this paper.
Keywords: Tourism income, environmental accounting, hospitality, Pangkor island
1. INTRODUCTION
While contributing to the economy, tourism may also partake in the act of degrading the environment
by means of greenhouse gasses emission (Becken & Patterson, 2006), natural assets over
exploitation (Kuniyal, 2002), wildlife dependency on food resources provided by tourists (Langley,
2002), overflowing of solid wastes (Hashim, et al., 2012) and the destruction of coral reefs and its
inhabitants (Abdullah & Yasin, 2010). However, environmentally friendly and naturally sensitive
practice of tourism development and management may lessen these impacts and create more
sustainable conditions (Tuna, 2011). Sustaining the capability of tourism site is crucial due to the fact
that damaging the natural assets/environment to a point where it can no longer ‘healed’, may
obliterate the prospect of tourism for that particular site itself. On the other hand, it is unlikely to have
any sort of tourism activities without impacting the environments. Therefore, the question is, to what
extent do these activities are allowed to occur? Adopting the weak sustainability concept, it is believed
that manufactured capital can be substituted for losses in natural capital. Arguably, in the weakest
view of tourism sustainability, as long as benefit exceeds cost, the damages (limiting to recoverable
state) may be tolerated. To estimate this, benefits and costs must be quantified. Environmental
accounting approaches have been known to quantify and some monetize these environmental impact.
Examples of these are the Sustainable Assessment Model (SAM) (Bebbington & Frame, 2003) and
Environmental Engineering Group Environmental Costing (EEGECOST) (de Beer & Friend, 2006)
which concern with monetising environmental impact of a project development in order to evaluate
their project activities in the path of sustainable development.
While assessing the costs-benefits effect of tourism activities on a site, an environmental accounting
tool known as the Environmentally Sustainable Accounting Statements was developed. The main
purposes of the tool are twofold; (1) to estimates income of tourism sites by taking accounts on
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
degradation of environments caused by tourism activities, and (2) to evaluate the degree of usage of
natural assets for tourism activities.
Therefore, applying this approach on Pangkor Island, the paper described an ongoing research with
the following objectives:
(1) To examine the environmental awareness and practices of hospitality service provider (HSP)
of Pangkor Island
(2) To examine whether the cost incurred in executing these practices are included and inserted
as individual item in the financial statement
(3) To estimate tourism income (Environmentally Sustainable Accounting Income Statement)
through environmental accounting approach
However, it is important to note that objective (3) is not presented in its complete structure in the paper
due to lack of information - work in-progress.
2. THE FRAMEWORK
The Environmentally Sustainable Accounting Income Statement (known as ESAIS from this point
forward) presented in the paper is one of the three components of a tool mentioned earlier, the
Environmental Sustainable Accounting Statements (shortened to ESAS from this forth) which was
developed by Ismail, Forgie & Khalid (2012) (Please refer to Figure 1). Apart from ESAIS, the other
two components are the Environmentally Sustainable Accounting Balance Sheet (ESABS) and
Environmental Physical Unit Statement (EPUS). This paper only described the ESAIS.
ACCOUNTING TREATMENT OF
ENVIRONMENTAL EXPENDITURES
ENVIRONMENTAL COST CALCULATION
CONSTRUCTING ENVIRONMENTAL ACCOUNTS
REPORTING (DISCLOSURE) REGULATIONS
ENVIRONMENTALLY SUSTAINABLE ACCOUNTING STATEMENTS (ESAS)
Source: Ismail,R., Forgie, V., & Khalid, K. (2012), “Bridging the Environmental Accounting Gaps between Accounting and
Economics Disciplines”, American Journal of Finance and Accounting, 2,(4), pg. 303.
Figure 1. Environmental Accounting Framework
Unlike conventional accounts (business level) the ESAIS (Figure 2) incorporates environmental
elements (degradation). It still however, follows the structure of the conventional business accounts
which displays revenues and expenses items. The idea of estimating real or net income (income after
deducting environmental degradation items) has been proposed as early as in the 1λ80’s. El Serafy
and Lutz (1989), suggested that efforts should focus on sustainable income which is the true income
rather than calculating the national income (the GDP). Even though, there are several indicators,
concepts and other tools which already have been introduced to account for the environment, there is
none so far that was developed using the structure of business level account for a tourism site. The
ones that currently applied such as the Systems of Integrated Environmental and Economics
Accounting (SEEA)(United Nation, 2003), Measure of Economic Welfare (MEW) (Nordhaus & Tobin,
1973), Indicators for Sustainable Economic Welfare (ISEW)(Daly & Cobb Jr, 1994) and Genuine
Progressive Indicator (GPI) (Anielski, 2001), were all developed to estimates “sustainable” income of a
nation, region or states level. Meanwhile, other methods such as SAM, EEGECOST, and Triple
Bottom Line (TBL) were more applicable to businesses.
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TOURISM SITE XX
ENVIRONMENTALLY SUSTAINABLE ACCOUNTING INCOME STATEMENT FOR THE YEAR
ENDED 20XX
εYR `000’
REVENUE
Accommodation & Food Services
Transportations
Entertainment & Cultural Services
Travelling & Tour Services
Retail Services
Miscellaneous Services
(-)
COST OF SALES & EXPENSES
Accommodation & Food Services
Transportations
Entertainment & Cultural Services
Travelling & Tour Services
Retail Services
Miscellaneous Services
(-)
ENVIRONMENTAL EXPENSES (POLLUTANTS)
Solid Waste
Liquid Waste
Carbon Emissions
(-)
PROFIT ATTRIBUTES TO SHAREHOLDERS
DIVIDENDS
UNAPPROPRIATED
PROFIT
BROUGHT
FORWARD
UNAPPROPRIATED
PROFIT
CARRIED
FORWARD
Source: Ismail, R., Khalid, K. (2011). Taking Nature into Account: Applying Environmental Accounting Approaches in the
Tourism Sector, Hospitality and Hotel Management Symposium, Konya, Turkey
Figure 2. Environmentally Sustainable Accounting Income Statement (ESAIS)
Since the tourism sector were mostly dependent on the attraction of natural assets, it is only fitting that
a method/tool from the environmental accounting area was developed dedicate to a tourism site. Even
though ESAS/ESAIS was specifically developed for tourism sector, it still borrows concepts from
national level environmental accounting approaches namely ISEW and GPI. Since the business level
environmental accounting area does not incorporate the environmental expenditures (e.g. degradation
of the environment) directly into its Income Statement, the income calculated in the ESAIS represent
environmentally sustainable income (partly following ISEW/GPI). Several features of ESAIS can be
perceived as representing the environment more accurately for a tourism site. Through ESAIS, it can
be illustrated that the income received from tourism activities must be equally met with maintaining
and protecting the environment. If these actions are not pursued, the ESAIS will illustrate unfavourable
outcomes in regard to the total income.
3. RESEARCH DESIGN
3.1 Tourism Site
Pangkor Island is located off the coast of Perak in north-west peninsular Malaysia which has the
latitude and longitude coordinates of 4º 12’ 50” N, 100º 34’ 30” E. It has a land area of 25.85 square
kilometres. Forty minutes by ferry from Lumut (a small coastal town that links to Ipoh, or from
Sitiawan) or 10 minutes from Marina Island Jetty, As a tourism site, Pangkor is famously known its
beaches, rainforests and historical heritage (Pangkor Treaty and Dutch Fort).Besides local visitors,
Pangkor is extremely popular among travelers from Europe. There are a few small uninhabited islands
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called Pulau Giam, Pulau Mentagor and Pangkor Island Laut which are also popular among travelers.
Other small islands include Gugusan Pulau Sembilan, Pulau Tukun, Pulau Terendak, Pulau Jarak,
Pulau Dua and Pulau Batu Orang Tua which are also popular especially among scuba-divers.
Pangkor has a population of around 16,000 islanders consists of Malay, Chinese, Indian and other
races. Fishing industry is the biggest contributor to Pangkor’s economy.
3.2 Data Collection
Businesses involved in the study are only those categorized as hospitality service provider (HSP). The
HSP which engaged in tourism activities are categorized into seven (7) tourism product groups as
recommended in “Tourism Satellite Accountμ Recommended εethodological Framework’(TSARεF)”
(Commission of the European Communities, Organisation for Economic Co-operation and
Development, United Nations, & World Tourism Organization, 2001) and in accordance with the
Malaysian Standard Industrial Code (please refer to Appendix A). However, in this paper only the
accommodation category was examined.
There are 45 HSP from accommodation category operating in Pangkor Island (excluding Pangkor Laut
Resort-different Island) about 10% 4 stars, 40% 3 stars and the rest without star ranking. This study
utilizes both primary and secondary data. Instrumentations used in the study are determined upon the
type of data collected (financial, non-financial and monetary environmental information). For most
information (financial and non-financial), they are obtained from the government’s departments and
private entities through interviews. However, for those reluctant to be interviewed, their financial data,
in the form of financial statements, are purchased from the Commissions of Companies Malaysia in
Kuala Lumpur.
Data were collected during two separate periods. The first phase data collection was for three days in
September 2011 while the second for six days in November 2011. About 10 researchers and 2 field
assistants involved in the data collection process.
4. RESULTS
As mentioned earlier, the HSP examined in this paper were those from the accommodations category
such as resorts, hotels, inns and chalets of Pangkor Island. It is important to note that the 45
accommodations within this category are those legal and registered with the local authority. Most
homestays operations were not registered, therefore were excluded from this research. Due to several
constraints namely budget, time and consent, only 70% of the accommodations operator were taken
as respondents for the research. Out of 70%, 2 were 4 stars hotels, the remaining were 3 stars and
without ranking accommodations (almost the same number of respondents between the two).
Interviews were mostly with either the owner or manager or accountants of the accommodation
providers.
Objective 1: To examine the environmental awareness and practices of hospitality service
provider (HSP) of Pangkor Island
From interviews, it was found that most providers state that they are aware of the importance of
environment’s quality to their business, however only a handful conducts good environmental practice.
Many stated budget constraint as main reason for not being able to spend more on beautification and
conservation of environment. Apart from that, most perceived that it is the responsibility of the
εanjung εunicipal Council to ‘care’ for the environment and not theirs. Operators from 4 star hotels,
several from three stars and a few from without ranking accommodations which include one speciality
lodging, demonstrate good environmental practice. These include beautification and conservation
costs namely beach cleaning, tree planting and recycling. Recycling activities include separating food
leftovers into three types; dry, wet and liquid. However, one provider managed to stands out from the
rest as eco-friendly lodging. Tiger Rock Guesthouse (TRG) has won 4 awards in Best of Malaysia
Award 2011 including best eco-resort. During interview, the caretaker of this lodge has indicated that
they are very serious in promoting sustainable tourism on Pangkor Island and this can be evidently
observed in the environs of the place. Located in the middle of a jungle, using water from rivers,
committed to recycling their waste, ensuring lots of floras and faunas within the vicinity and with limited
number of rooms - warranting the privacy of their guests, TRG genuinely showed the meaning of
immersing yourself in the environment while sustaining it.
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Objective 2: To examine whether the cost incurred in executing these practices are included and
inserted as individual item in the financial statement
As mentioned in the outcome of objective 1, there are several accommodation providers of Pangkor
Island incurred costs related to beautification and conservation of environment. However, none of
them insert these costs as individual items in the financial statements or produce
environmental/sustainability report (Pangkor Laut Resort owned by YTL Corporation produced
Sustainability Report but excluded from this study). Most providers that incurred environmental costs
treated them as normal expenses.
Objective 3: To estimate tourism income (Environmentally Sustainable Accounting Income
Statement) through environmental accounting approach
ESAIS may invite criticisms due to the act of monetizing degradation where some argued that dollar
terms could not possibly capable of capturing the whole impact on the environment. However, it is
important to stress the main notion of ESAIS is to estimate ‘maintenance cost’ of tourists and illustrate
the fraction of these costs from revenue generate through tourism activities. ESAIS in Figure 3
illustrates three types of tourist maintenance costs which are solid waste, liquid waste and carbon
emissions.
PANGKOR ISLAND, PERAK
ENVIRONMENTALLY SUSTAINABILE INCOME STATEMENT FOR THE YEAR ENDED 2011
(-)
(-)
(-)
εYR `000’
33600
000
REVENUE
Accommodation & Food Services
Transportations
Entertainment & Cultural Services
Travelling & Tour Services
Retail Services
Miscellaneous Services
TOTAL REVENUES
COST OF SALES & EXPENSES
Accommodation & Food Services
33600 000
Transportations
Entertainment & Cultural Services
Travelling & Tour Services
Retail Services
Miscellaneous Services
TOTAL EXPENSES
ENVIRONMENTAL EXPENSES (POLLUTANTS)
Solid Waste*
- Direct
- Indirect
Liquid Waste*
Carbon Emissions*
TOTAL ENVIRONMENTAL EXPENSES
PROFIT ATTRIBUTES TO SHAREHOLDERS
DIVIDENDS
UNAPPROPRIATED
PROFIT
BROUGHT
FORWARD
UNAPPROPRIATED
PROFIT
CARRIED
FORWARD
20940
000
20940 000
136 320
(136 320)
12 523
680
*Estimation method is still undergoing several changes and the figure presented in the paper is purely
experimental.
Figure 3. PANGLOR ISLAND TOURISM INCOME
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The discussions in the paper focus only on solid waste. There are two types of estimation of solid
waste (1) direct – financial consequence of collecting and disposing solid waste into landfills; and (2)
indirect - estimating and converting physical unit of methane (CH4) released from landfill to financial
unit. Since the technique is currently being developed (converting physical unit to financial unit), it is
not discussed in the paper.
The waste disposal systems in Pangkor Island are mostly under the responsibility of Manjung
Municipal Council. Even though, there are payments made by some of the accommodation providers
for disposing their waste but those are mainly for transportation costs. Costs of disposal and
maintaining the landfill were borne by the Council. Information on solid waste volumes were gathered
from interviews with accommodation providers and officers at the Manjung Municipality Council. The
figure displayed in Figure three for direct costs of solid waste include waste disposals for
transportations by HSP, labours’ wages and salaries directly involved in waste disposals and other
expenses that can be directly traced to disposals of waste.
5. CONCLUSIONS
The paper discussed an ongoing research that investigates environmentally friendly practiced and
accounting treatment of expenses incurred related to beautification and conservation by hospitality
service providers (HSP) from the accommodation category. It was found that most accommodation
providers aware of the significance of the environment to their business. However, other than a
handful of these providers, most do not exercise good environmental practiced and believed that it is
within the responsibility of the local authority (MPM) to beautify and conserve the environment. The
study also found that none of the providers within the scope of the study include environmental costs
an individual item in the financial statements. Finally, a fraction of Pangkor Island ESAIS was
presented in Figure 3 that illustrates the deduction of solid waste’s direct costs from the
accommodation providers’ revenue.
Acknowledgement: This study was undertaken with financial support from the Darul Ridzuan Institute
(IDR), Perak, Malaysia.
References
[1] Abdullah, A. L., & Yasin, Z. (2010). Booming Coastal Tourism Dooming Coastal-Marine
Environment: The Case of Teluk Datai, Langkawi. In K. Din & J. Mapjabil (Eds.), Tourism
Research in Malaysia: What, which way and so what? Sintok: Universiti Utara Malaysia Press.
[2] Anielski, M. (2001). Measuring the Sustainability of Nations: The Genuine Progressive Indicator
System of Sustainable Wellbeing Accounts. Paper presented at the The Fourth Biennial
Conference of the Canadian Society for Ecological Economics: Ecological Sustainability of the
Global Market Place.
[3] Bebbington, J., & Frame, B. (2003). Moving from SDR to SAM. Chartered Accountants Journal,
82(7), 11-13.
[4] Becken, S., & Patterson, M. (2006). Measuring National Carbon Dioxide Emissions from Tourism
as a Key Step Towards Achieving Sustainable Tourism. Journal of Sustainable Tourism,
14(4), 323-338.
[5] Commission of the European Communities, Organisation for Economic Co-operation and
Development, United Nations, & World Tourism Organization (2001). Tourism Satellite
Account: Recommended Methodological Framework. New York: United Nations Publication,
OECD, WTO.
[6] Daly, H. E., & Cobb Jr, J. B. (1994). For the Common Good: Redirecting the Economy toward
Community, the Environment, and a Sustainable Future (2nd Edition ed.). Boston: Beacon
Preston.
[7] de Beer, P., & Friend, F. (2006). Environmental accounting: A management tool for enhancing
corporate environmental and economic performance. Ecological Economics, 58(3), 548-560.
[8] El Serafy, S., & Lutz, E. (1989). Environmental and Resource Accounting: An Overview.
Washington DC: The World Bank.
[9] Hashim, M., Ismail, R., Khalid, K., Jaafar, H., Abdul Halim, H., Abdul Hamid, S., et al. (2012).
Issues and Management of Solid Waste in Isolated Human Habitat: A Case of Pulau Pangkor,
Perak. Paper presented at the Human Habitat and Environmental Transformation, UKM,
Bangi, Malaysia.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
[10] Ismail, R., Forgie, V., & Khalid, K. (2012). Bridging the Environmental Accounting Gaps between
Accounting and Economics Disciplines. American Journal of Finance and Accounting, 2(4),
297-310.
[11] Kuniyal, J. C. (2002). Mountain expeditions: minimising the impact. Environmental Impact
Assessment Review, 22(6), 561.
[12] Langley, C. (2002). Development Policy for Langkawi: The Environmental and Economic
Implications of Encouraging Tourism. Unpublished Master of Science (MSc) Thesis, University
of London, London.
[13] Nordhaus, W., & Tobin, J. (1973). Is Growth Obsolete? In M. Moss (Ed.), The Measurement of
Economic and Social Performance. Princeton University, N.J: New York, National Bureau of
Economic Research; distributed by Columbia University Press.
[14] Tuna, M. (2011). Social and Environmental Impacts of Tourism Development in Turkey. In M.
Kozak & N. Kozak (Eds.), Sustainability of Tourism: Cultural and Environmental Perspectives.
Newcastle, UK: Cambridge Scholars Publishing.
[15] United Nation (2003). Handbook of National Accounting: Integrated Environmental and Economic
Accounting 2003(Final draft): United Nation, European Commission, IMF, OECD, World Bank.
Appendix A: Malaysian Standard Industrial Code (MSIC)
1
2
Product
Accommodation
Food & Beverage
Division
55
55
Group
551
552
Class
5510
5521
5522
3
Transportation
60
602
6021
6022
4
Travelling/ Travel
Agency
63
5
Cultural,
Entertainment and
Recreation
Services
92
Descriptions
Hotels
Camping sites and other provision of
accommodations
Restaurants and restaurants cum night clubs
Fast food restaurants
Cafes, snack bars
short
stay
Pubs, bars, coffee houses, cocktail lounges and karaoke
Eating and drinking places
Food stalls/hawkers
Drink stalls
Food and drink stalls/hawkers
Other scheduled passenger land transport n.e.c
Taxi, car for hire and limousine services
Other non-scheduled passenger land transport n.e.c
Passenger transportation by sea-going and coastal water
vessel
Other sea and coastal water transportation
611
6110
621
6210
622
6220
630
6304
Travel agency and tour operator services
Taxi/limosine booking service
Other activities of travel agencies and tour operators and
tourist assisstance activities n.e.c
9219
Circus, amusement park and similar attraction services
Cabarets, discotheques and kakroke lounges
Other entertainment activities n.e.c
Library and archive activities
923
9231
9232
9233
9241
9249
Passenger airline services - domestic and international
Rental services of aircraft with or without operator
Museum activities and preservation of historical buildings
Botanical and zoological gardens and nature reserves
activities
Activities of country and golf clubs
Activities of water sports and recreation
Equestrian clubs
Sports event promotions and organisations
Sports facility operations services
Other sporting services
Recreation park and beach services
Other recreational activities n.e.c
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6
Retail
52
522
5222
Retail sale of confectionery
Retail sale of beer, wine and spirits
Retail sale of tobacco, cigars, cigarettes
5232
Retail sale of articles of clothing, articles fur and clothing
accessories
Retail sale of household utensils, kitchenware, chinaware,
glassware, ornaments, etc.
Retail sale of photographic equipment
Rental of land transport equipment n.e.c
Rental of water transport equipment n.e.c
Social escort services
5233
7
Miscellaneous
Services
71
711
93
930
5238
7111
7112
9309
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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CYANOBACTERIAL COMPOSITION OF RANCHAN POOL, SERIAN, SARAWAK.
Mohd Nasarudin Harith* and Ruhana Hassan
Department of Aquatic Science, Faculty of Resource Science and Technology, Universiti Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.
*Email: hnasarudin@gmail.com
Abstract
This study serves as an update on the cyanobacterial research in Sarawak, which focusing on the
Ranchan Pool, one of the popular recreational waterfalls in Sarawak. A total of 17 species of
cyanobacteria which belong to 8 families were recorded. Overall, based on number of cyanobacterial
cells, Ranchan Pool is considered healthy except in October and December 2007. Out of 17 species
recorded, six genera namely Cylindrospermopsis, Nostoc, Lyngbya, Oscillatoria, Scytonema and
Synechococcus have potential to cause toxic effects especially for the swimmers. However, they were
no reports regarding cyanotoxin poisoning from the pool. Although no visible bloom was observed
during sampling visits, the presence of these potential toxin producers should be alerted and
monitored. It is hoped that the results could aid Sarawak state government in future monitoring of its
recreational area, besides exploring new dimensions in sustainable management of the water
resources.
Keywords: cyanobacteria, recreational area, water quality, species composition
1. INTRODUCTION
Cyanobacteria, or blue-green algae, are groups of prokaryotic photosynthetic bacteria that contain
chlorophyll a and account for up to 40% of plenary oxygen production. Among the oldest and the most
diverse lineages of bacteria, they have radiated into nearly every aquatic and terrestrial ecosystem
(Sze, 1998). However, it is their prevalence in freshwater ecosystem that caused the greatest amount
of interest and concern in recent years.
Livestock poisoning and adverse human health effects following the drinking of contaminated water
had increased the number of research related to cyanobacterial toxicity, and during the past two or
three decades, the chemical structures of a number of cyanotoxins have been identified and their
mechanisms of toxicity established (Chorus et al. 2000).
Cyanobacterial toxins occur naturally, but pollution with nutrients from agriculture and domestic
wastewater has led to increase fertilization of many water bodies (Chorus et al. 2000). This in turn
results in proliferation of cyanobacteria and thus has considerable impact upon recreational water
quality. In temperate countries, cyanobacterial dominance is most pronounced during the summer
months, when the demand for recreational water is the highest. Thus, eutrophication together with a
lack of “avoidance behaviour” by the visitors may lead to increased health risks from cyanotoxins.
Some cases of human injury through cyanotoxins were also documented. Most involved exposure to
the infected recreational waters and demonstrate that humans become ill (some serious case
recorded) through ingestion or aspiration of toxic cyanobacteria (Chorus et al. 2000). However, Yoo et
al. (1995) stated that the bulk of human illness caused by cyanobacteria probably remain
unrecognized. According to Chorus et al. (2000), human hazards of cyanobacteria occur from three
routes of exposure namely direct contact of exposed parts of the body, including the ears, eyes, mouth
and throat, accidential ingestion of water containing cells by swallowing and uptake of water
containing cells by aspiration (inhalation).
As the largest state of Malaysia, Sarawak has numerous recreational waters such as waterfalls that
are famous destinations for the public and tourists during leisure time. As recreational destinations,
waterfalls typically developed with essential services and facilities including food outlets. While most
waterfalls remain more or less in their natural state, many of the popular ones have undergone some
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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development which may include refreshment outlets of different kinds and various accommodations
(Hudson, 1998). These facilities are possible to contribute various input of substances especially
several nutrients that are required for cyanobacteria development.
Several studies reported cyanobacterial composition in selected Sarawak waterfalls and lakes.
However, the data is still very limited. It is important to assess the cyanobacterial composition in these
areas as the utilization of the water by the local community and tourists are high. Although cases
related to cyanobacterial poisoning were not yet reported, potentially toxic cyanobacteria proliferation
still needs to be addressed as they can affect humans and the local environment.
This paper serves as an update of the cyanobacterial composition research in Sarawak, with the focus
to the Ranchan Pool, one of the popular recreational areas. It is hoped that the results could aid
Sarawak state government in future monitoring of its recreational area, besides exploring new
dimensions in sustainable management of the water resources.
2. MATERIALS AND METHODS
Ranchan Recreational Park (Ranchan Pool) is a waterfall located approximately 80 km from Kuching
City. Accessible by road, this popular waterfall is well maintained recreational forest, shaded heavily
by vegetation. Amenities such as shower rooms, picnic areas, canteens and accommodation services
are available for the visitors. The lowest part of the pool (N 01° 0.8641’, E 110° 34.82λ’) was selected
for sampling during this study. Sampling was conducted monthly from February 2007 until January
2008.
Cyanobacterial samples were collected using 2 liter Van Dorn Bottle and sieved through 20 µm sieve.
Specimens retained in the sieves were kept in separate plastic bottles, preserved with δugol’s solution
and transported back to UNIMAS laboratory for species identification and enumeration.
For preserved samples, observation was carried out using the Inverted Light Microscope Olympus
M1025 – Microscope Research Fluorescence Model 1X51RFLCCD. Species identification was based
on keys by Anagnostidis and Komarek (1985; 1986; 1988; 1989; 1991); Bold and Wayne (1985); Hoek
et al. (1995); Prescott, (1954); Aishah (1996); Sze, (1998) and credible supplementary online
materials. Enumerations were carried out accordingly using hemacytometer (improved Neubeuer)
(Lawton et al. 1999). Results were expressed in cells per mililitre. The list of cyanobacteria that exists
was made available at the end of this study.
3. RESULTS AND DISCUSSION
A total of 17 species belong to 8 families were recorded (Table 1). The number of species found in this
study was higher compared to previous record by Jasmina (2008) as she reported that a total of 12
species of cyanobacteria were found in the same sampling site. This difference in cyanobacterial
composition maybe due to the duration of study as that this study covered a year sampling data
collections compared to only few sampling visits in the previous studies.
Out of 17 species recorded in Table 1, six genera namely Cylindrospermopsis, Nostoc, Lyngbya,
Oscillatoria, Scytonema and Synechococcus can be categorized as potential toxin producer as stated
by Ressom et al. (1994) and Chorus et al. (2000). Chorus et al. (2000) reported that these
cyanobacteria have potential to cause toxic effects especially for the swimmers from three routes of
exposure namely direct contact of exposed parts of the body (including the ears, eyes, mouth and
throat), accidential ingestion of water containing cells by swallowing and uptake of water containing
cells by aspiration (inhalation). For example, some species of Cylindrospermopsis may produce
cylindrospermopsin (e.g. Cylindrospermopsis raciborskii) that blocks protein synthesis which may
cause kidney and liver failure (Ohtani et al. 1992). Falconer (1996) also reported that patients
intoxicated with cylindrospermopsin via drinking in an incident in Australia escaped death only through
skilled and intensive hospital care.
However, they were no reports regarding cyanotoxin poisoning from Ranchan Pool. The isolation and
establishment of clonal cultures has not been carried out during this study to clarify the taxonomy as
well as lack of experiments to extract and verify the toxin properties for each cyanobacterial species.
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Hence, there was not enough evidence to state that waters in both areas were contaminated by
cyanotoxins. Although no visible bloom was observed during sampling visit, the presence of these
potential toxin producers should be alerted and monitored.
Table 1 Cyanobacterial taxa identified from Ranchan Pool with potential toxin producer species as
stated by Ressom et al. (1994) and Chorus et al. (2000)
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Cyanobacterial species/genera
FAMILY BORZIACEAE
Borzia sp.
FAMILY CHROOCOCCACEAE
Aphanothece microspora
Aphanothece sp.
Dactyloccopsis sp.
Chroococcus minor
Chroococcus sp.
Synechococcus sp.
FAMILY MERISMOPEDIACEAE
Aphanocapsa endophytica
Aphanocapsa sp.
Merismopedia sp.
FAMILY NOSTOCACEAE
Cylindrospermopsis sp.
Nostoc sp.
FAMILY OSCILLATORIACEAE
Lyngbya sp.
Oscillatoria sp.
FAMILY PHORMIDIACEAE
Arthrospira sp.
FAMILY PSEUDOANABAENACEAE
Pseudoanabaena sp.
FAMILY SCYTONEMATACEAE
Scytonema sp.
Potentially Toxin
Producers?
No
No
No
No
No
No
Yes
No
No
No
Yes
Yes
Yes
Yes
No
No
Yes
The variations of cyanobacteria cell density in 12 month of sampling from Ranchan Pool were
presented in Figure 1. Based on guidelines for safety practice in managing recreational water by
World Health Organization or WHO (2003), the status in terms of cyanobacteria cell density can be
considered healthy (< 20,000 cyanobacterial cells/mℓ) except in October 2007 and December 2007 as
shown as two peaks in Figure 1. Both two months showed cyanobacterial cell density of 27,000
cells/mℓ and 28,000 cells/mℓ in October 2007 and December 2007 respectively. Referring to the WHO
(2003) guidelines, this situation can be considered as low probably of adverse health effects.
However, the short-term adverse health outcomes such as skin irritations and gastrointestinal illness
might occur. Unfortunately, the cases regarding these matters were not reported, or perhaps medical
examination regards both problems as symptom of allergy and food poisoning respectively. Although
the available data does not support the peaks, it is suggested that these conditions was favorable to
cyanobacterial growth in terms of optimum temperature, light intensity and concentrations of nutrients
(Boyd & Tucker, 1998).
The cell density of cyanobacteria decrease in November 2007 and January 2008 thus showed that the
demonstration of cyanobacterial toxicity do not necessarily imply on environmental or human hazard
as long as the cells remain thinly dispersed (Chorus & Bartram, 1999). The lotic characteristic (flowing
water) of Ranchan Pool was also considered to minimize the mass development especially the
surface scum of cyanobacteria.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Cell density (Cells/mℓ)
Ranchan
Ranchan
30000
cell density
25000
20000
15000
10000
5000
0
Feb
Mar
Apr
May
June
Jul
Aug
Sept
Oct
Nov
Dec
Jan
Month
Figure 1. Variations in cyanobacterial cell density during 12 month sampling period
There were also other phytoplankton that were identified in this study. In sum, 18 genera from Division
Chlorophyta and Chrysophyta were identified (Table 3). Diatom which belongs to the Class
Bacillariophyceae in Division Chrysophyta was found to be the most diverse phytoplankton followed by
Division Chlorophyta. Recent study by Salleh et al. (2005) illustrated the same trends of phytoplankton
composition where diatoms dominate the phytoplankton flora followed by the other species. This
situation is in accordance with Sze (1998) who stated that diatoms are often major components of
planktonic communities in the freshwater and oceans. The diversity of phytoplankton of particular
water bodies can also indicate the health level of the water. According to Chakrabarty et al. (1959), if
the diversity of phytoplankton is high, the water bodies can be considered healthy. In this study, the
high species number of phytoplankton that was recorded in Ranchan Pool indicated that the water
was in good condition
Table 3: Other phytoplankton genera identified in Ranchan Pool
Division
Chlorophyta
Chrysophyta
Chlamydomonas
Cladophora
Coleochaete
Oedogonium
Staurastrum
Tetraedron
Achnanthes
Amphora
Cocconeis
Cyclotella
Diatoma
Diploneis
Eunotia
Fragilaria
Frustulia
Gomphonema
Gyrosigma
Melosira
4. CONCLUSION
A total of 17 species of cyanobacteria which belong to 8 families were recorded from this study. The
waters here can be considered healthy based on the cyanobacterial cell density safety practice
guideline by WHO. Six genera namely Cylindrospermopsis, Nostoc, Lyngbya, Oscillatoria, Scytonema.
and Synechococcus can be categorized as potential toxin producer as these cyanobacteria have
potential to cause toxic effects especially for the swimmers. However, they were no reports regarding
cyanotoxin poisoning in Ranchan Pool. Therefore, the isolation and establishment of clonal cultures
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
should be carried out to clarify the taxonomy as well as experiments to extract and verify the toxin
properties for each cyanobacterial species. The data will be useful in risk assessment of the potential
cyanotoxin contamination in water especially recreational waters in Sarawak. Although no visible
bloom was observed during sampling visit, the presence of these potential toxin producers should be
alerted and monitored.
Acknowledgment
The authors are thankful to Majlis Daerah Serian for the sampling permission. This project is partly
funded by grant FRGS/06(06)/658/2007(22) from MOHE awarded to the second author. We are
thankful to FRST staffs especially Mr. Mustafa Kamal and Mr. Zaidi Ibrahim for the unconditional help
during sampling. Thanks to UNIMAS for awarding UNIMAS Postgraduate Scholarship to the first
author and supporting in terms of transportation and lab facilities.
References
Aishah, S. (1996). Panduan Mengenali Alga Air Tawar. Kuala Lumpur. Dewan Bahasa dan Pustaka.
Anagnostidis, K. & Komarek, J. (1985). Modern approach to the classification system of Cyanophytes.
1 – Introduction. Archive fur Hydrobiology/Algological Studies, 38/39, 291 – 302.
Anagnostidis, K. & Komarek, J. (1986). Modern approach to the classification system of Cyanophytes.
2- Chroococcales. Archive fur Hydrobiology/Algological Studies, 43,157-226.
Anagnostidis, K. & Komarek, J. (1988). Modern approach to the classification system of Cyanophytes.
3- Oscillatoriales. Archive fur Hydrobiology/Algological Studies, 50/53, 327-472.
Anagnostidis, K. & Komarek, J. (1989). Modern approach to the classification system of Cyanophytes.
4- Nostocales. Archive fur Hydrobiology/Algological Studies, 56, 247-345.
Anagnostidis, K. & Komarek, J. (1991). Modern approach to the classification system of Cyanophytes.
5- Stigonematales. Algological Studies, 59,1-73.
Bold, H.C., & Wynne, J.M. (1985). Introduction to the Algae. (2nd ed). Prentice Hall. Inc.London.
Boyd, C.E. & Tucker, C.S.(1998). Pond Aquaculture Water Quality Management. Kluwer Academic
Publishers, Boston.
Chakrabarty, R.D., Roy, P & Singh, S.B. (1959). A Quantitative study of the plankton and the
physicochemical conditions of the river Jumna at Allahabad in 1954 -55. Indian Journal of
Fisheries, 6,187-203.
Chorus, I. (2000). Recreational exposure to cyanotoxins. In: Chorus, I. (Ed.). Cyanotoxins –
Occurrence, effects, controlling factors. Hiedelberg: Springer Scienctific.
Chorus, I. & Bartram, J. (1999). Toxic cyanobacteria in water: A guide to public health significance,
monitoring and management. Spon, London.
Falconer, I. (1996). Potential impact on human health of toxic cyanobacteria. Phycologia, 35, 6-11.
Hoek, C. van den., Mann, D.G. & Jahns, H.M. (1995). Algae: An Introduction to Phycology. Cambridge
University Press.
Hudson, B.J. (1998). Waterfalls resources for tourism. Annals of Tourism Research, 25, 958-973.
Jasmina, M. (2008). Water quality and phytoplankton assesment in three selected waterfalls of
Kuching and Serian District, Sarawak. Final Year Project. Universiti Malaysia Sarawak.
Unpublished.
Lawton, L., Marsalek, B., Padisak, J., Chorus, I. (1999). Chapter 12: Determination of Cyanobacteria
in the laboratory. In: Chorus, I & Bartram, J. (Eds.). Toxic Cyanobacteria in Water: A guide to
their public health consequences, monitoring and management. World Health Organization
(WHO).
Ohtani, I., Moore, R.E. & Runnegar, M.T.C. (1992). Cylindrospermopsin: a potent hepatotoxin from the
blue-green alga Cylindrospermopsis raciborskii. Journal of the American Chemical Society,
114, 7942-7944.
rd
Prescott, G. W. (1954). How to Know Freshwater Algae. 3 edition. The Pictured Key Nature Series.
United States of America.
Ressom, R., Song, F.S., Fitzgerald, J. Turczynowicz, L., Saadi, E., O., Roder, D., Maynard, T., &
Falconer, I. (1994). Health Effects of Toxic Cyanobacteria (Blue-Green Algae). National
Health & Medical Research Council NHMRC, Australia.
Salleh, A., Nadia, M.K., Hartina, M.A. & Sarini, A.W. (2005). Diversity of algae in the Hulu Selai River
and its tributaries, Endau-Rompin National Park, Johor, Malaysia. In: Mohamed, H & ZakariaIsmail, M. (Eds.). The Forests and Biodiversity of Selai Endau-Rompin, Malaysia.. Perbadanan Taman
Negara Johor. p.218.
Sze, P. (1998). Biology of the algae. 3rd ed. Mc Graw Hill.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
WHO (World Health Organization). (2003). Guidelines for Safe Recreational Water Environments, Vol.
1, Coastal and Fresh Water, World Health Organization, Geneva.
Yoo, S., Carmichael, W., Hoehn, R. & Hrudey, S. (1995). Cyanobacterial (blue-green algal) toxins: A
resource guide. American Water Works Association Research Foundation. Denver, CO.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
SHORT NOTES ON BENTHIC MICROALGAE COMPOSITION OF ASAJAYA MANGROVE, KOTA
SAMARAHAN, SARAWAK
Mohd Nasarudin Harith* and Othman Bojo
Aquatic Resource Science and Management, Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
*Emails: hmnasarudin@frst.unimas.my
Abstract
This study aimed to record the benthic microalgae assemblages’ of Asajaya mangrove area. The
sampling included sediments, rocks and roots samples from four selected stations. A total of 30
genera of microalgae were identified from all sampling stations. Qualitatively, Division Chrysophyta,
which consists of diatoms were the best represented division, as this group comprised 73% of the
microalgae composition from the samples collected. The most common genera occurred at every
sampling station were Coscinodiscus, Cyclotella, Flagilaria, Gyrosigma, Navicula, Nitzschia, Pinnularia
and Ulothrix. Out of 30 genera recorded, 17 genera were probably related to polluted water and
several of them were probably related to toxic effects. The data obtained from this study could be
useful for future microalgae monitoring especially in assessing their diversity in Sarawak aquatic
ecosystems.
Keywords: benthic microalgae, mangrove, diatom, cyanobacteria, Asajaya
1. INTRODUCTION
Benthic microalgae or microphytobenthos are microscopic, photosynthetic eukaryotic algae and
cyanobacteria that grow in marine habitats ranging from wave swept beaches to detritus-laden
backwater lagoons (Macintyre et al., 1996). These microalgae can be divided into three main groups
namely cyanobacteria (blue-green algae), diatoms (class Bacillariophyceae) and some dinoflagellates
(Underwood & Chapman, 1995).
Benthic microalgae live in the sediment of intertidal areas which include estuaries, sand flats, muddy
shores, saltmarshes and bare soft substrate (Supiah, 2004). These micro communities contribute in
biological and physiological process (Blanchard et al., 2001). Benthic microalgae serve as a main
carbon source for the higher trophic organism and macrofauna (Stocks & Grassle, 2001) and
contribute as diet for other tide zone species (Guarini et al., 2002).
In Malaysia, researches of benthic microalgae have been done in Pulau Pangkor, Perak (Cornelius,
1999), intertidal area of Kota Kinabalu, Sabah (Harun, 1990), and Batang Saribas, Sarawak (Supiah,
2004). These works has shed some lights on composition of benthic microalgae in Sarawak aquatic
ecosystems. However, these data are still very limited if comparison is made between number of
studies and data collected with the number of mangrove areas.
The information obtained can be used as baseline information for more research in the future. The
information about aquatic ecosystems is essential for references to the future management.
2. MATERIALS AND METHODS
2.1 Sampling Stations
o
o
Four stations located at Asajaya mangrove area which were Station 1 (N 01 35. 76λ’, E 110 36.
o
o
o
o
14λ’), Station 2 (N 01 35’ 4λ.5”, E 110 36’ 37.3”), Station 3 (N 01 35.757’ E 110 36.206’) and
o
o
Station 4 (N 01 35. λ63’, N 01 35.λ27’) were selected for this study. Samples were collected from
sediment, rocks and mangrove tree roots that were selected randomly. Sampling was carried out from
September 2005 to December 2005.
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2.2 Samples Collection
The sediments were collected by pushing a pistone style corer to a depth about two centimetres into
the sediments. The samples was kept in separate small size plastic storage Nasco Whirl-Pak® with
addition of some distilled water and preserved on site with δugol’s solution.
Microalgae were brushed vigorously from the surface of the rocks and roots into a small size plastic
storage Nasco Whirl-Pak® using a hard toothbrush. The toothbrush was rinsed repeatedly by distilled
water in order to transfer almost all microalgae into the plastic storage. The new toothbrushes were
used for each replicates to avoid any microalgae contamination from previous samples. Then, the
samples were preserved with δugol’s solution, labelled and later transported back to UNIMAS
Laboratory for analysis.
2.3 Microalgae Identification
Preserved samples were homogenized and 1 mℓ of the sample were placed on the Sedgewick Rafter
Counting Chamber, covered with cover slip and observed under the Nikon Eclipse 80i microscope
equipped with phase contrast and image analyzer. The benthic microalgae were identified up to genus
level. The identification was based on keys by Lokman (1991), Patrick and Reimer (1966, 1975),
Williams and Round (1987); Bold and Wynne (1985), Anagnastidis and Komarek (1985, 1986, 1988,
1989, 1991), Sze (1998), Prescott (1978); Graham and Wilcox (2000), Taylor (1987), Steidinger and
Tangen (1996) and Hendey (1964). The list of benthic microalgae that exists was made available at
the end of this study.
3. RESULTS AND DISCUSSION
A total of 30 genera of microalgae belonging to three divisions were identified from all sampling
stations with Coscinodiscus, Cyclotella, Flagilaria, Gyrosigma, Navicula, Nitzschia, Pinnularia and
Ulothrix were commonly observed (Table 1). Qualitatively, Division Chrysophyta, which consists of
diatoms from the Class Bacillariophyta was the best represented division, as this group comprised
73% of the microalgae composition from the samples collected (Figure 1). This situation is in
accordance with Sze (1998) who stated that diatoms are often reported as major components of algal
communities in the freshwater and oceans. Salleh et al. (2005) illustrated the same trends of algae
composition where diatoms dominate the algae followed by the other species.
Figure 1. Percentage of benthic microalgae identified based on division from all sampling stations.
The number of genera found in this study was higher compared to previous study by Supiah (2004)
which located in Batang Saribas, Sarawak as she reported only seven genera of microalgae were
found. The results obtained during this study also have a slight difference with Supiah (2004) with the
absence of three genera namely Okedenia, Pleurosigma and Thalassionema. However, both studies
recorded the presence of Achnanthes, Cosinodiscus, Cyclotella, Navicula and Nitzchia. These
differences were due to types of samples collected. Supiah (2004) focused on the sediment sampling
while in this study, samples from sediments, rocks and roots were collected. Moreover, the number of
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sampling sites, weather, sampling time and variation types of sampling sites were also possibly
contributed to the difference in microalgae composition.
Table 1 Microalgae identified from all sampling stations. The symbol ‘+’ represents present while
symbol ‘-‘ represents the absence of the genera.
GENERA
STATION
1
2
3
4
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Chrysophyta
Achnanthes
Amphora
Asterionella
Cocconeis
Coscinodiscus
Cyclotella
Cymatopleura
Cymbella
Diatoma
Diploneis
Eunotia
Flagilaria
Frustulia
Gomphonema
Gyrosigma
Navicula
Nitzschia
Pinnularia
Rhopalodia
Stauroneis
Synedra
Triceratium
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
23
24
25
26
27
Cyanophyta
Anabaena
Chroococcus
Lyngbya
Oscillatoria
Synechococcus
+
-
+
+
+
+
-
+
+
+
-
28
29
30
Chlorophyta
Mougeotia
Oedogonium
Ulothrix
+
+
+
+
+
+
+
Number of genera
14
22
20
25
Diatoms (Division Chrysophyta) are generally known as one of the important bioindicators for
assessing the quality of the water, particularly organic pollution (Beyene et al., 2009). According to
Bathurst et al. (2010), certain diatom genera can be used to indicate the health level of aquatic
ecosystems. Referring to Table 1, the presence of Amphora, Cocconeis, Cymbella, Diatoma,
Diploneis, Eunotia, Flagilaria, Frustulia, Gomphonema, Navicula, Nitzschia, Pinnularia, Rhopalodia,
Stauroneis and Synedra indicated that the water quality of particular area is undesirable and related to
pollution (Bathurst et al., 2010).
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Division Cyanophyta, or known as blue-green algae, comprised 17% of the microalgae composition in
the samples collected (Figure 1). Out of five genera recorded from this division, two of them namely
Anabaena and Oscillatoria can be used as bioindicator of the health status of the aquatic ecosystem.
According to Boyd and Tucker (1998), these two genera can be related to eutrophic and polluted
water. In addition, the occurrence of the genus Oscillatoria is related to phosphate and nitrate
enrichment (Ravera & Vollenweider, 1968). Anabaena and Oscillatoria were also reported as potential
toxin producer genera (Ressom et al. 1994; Skulberg et al. 1993).
However, there is not enough evidence to conclude that water in Asajaya mangrove is unsafe due to
the fact that the identification was only up to the genus level. It is possible that certain species in the
same genus especially from Division Cyanophyta, has variable capability to produce toxin. For
example, according to Sano et al. (1998), Oscillatoria agardhii was reported as toxin-producer species
whereby Oscillatoria simplicissima was claimed as non-toxic species. In this study, the isolation of
individual species and clonal culture had not been carried out. As the result, there is no detail
morphological and molecular study to clarify the taxonomy, as well as lack of experiments to analyse
toxin properties for each species. In the future, study related to taxonomy, molecular and toxin
assessments need to be done in order to clarify the health status of this aquatic ecosystem.
4. CONCLUSION
A total of 30 genera of microalgae belonging to three divisions namely Chrysophyta, Cyanophyta and
Chlorophyta were identified from all sampling stations. Genera Coscinodiscus, Cyclotella, Flagilaria,
Gyrosigma, Navicula, Nitzschia, Pinnularia and Ulothrix were commonly observed. Division
Chrysophyta, which consists of diatoms represented 73% of the microalgae composition from the
samples collected. Out of 30 genera recorded, 17 genera namely Amphora, Cocconeis, Cymbella,
Diatoma, Diploneis, Eunotia, Flagilaria, Frustulia, Gomphonema, Navicula, Nitzschia, Pinnularia,
Rhopalodia, Stauroneis Synedra, Anabaena and Oscillatoria were probably related to polluted water
and several of them were probably related to toxic effects. However, further study related to their
taxonomy, molecular and toxicity need to be carried out in order to conclude the health status of this
aquatic ecosystem. The data obtained from this study could be useful for future microalgae monitoring
especially in assessing their diversity in Sarawak aquatic ecosystems.
Acknowledgement
The authors are thankful to Universiti Malaysia Sarawak for supporting in terms of transportation and
lab facilities. The authors are also thankful to FRST staffs especially Mr. Mustafa Kamal, Mr. Zulkifli
Ahmad and Mr. Zaidi Ibrahim for the unconditional help during sampling.
References
Anagnostidis, K. & Komarek, J. (1985). Modern approach to the classification system of Cyanophytes.
1- Introduction. Algological Studies, 38/39, 291-302.
Anagnostidis, K. & Komarek, J. (1986). Modern approach to the classification system of Cyanophytes.
2- Chroococcales. Algological Studies, 43, 157-226.
Anagnostidis, K. & Komarek, J. (1988). Modern approach to the classification system of Cyanophytes.
3- Oscillatoriales. Algological Studies, 50/53, 327-472.
Anagnostidis, K. & Komarek, J. (1989). Modern approach to the classification system of Cyanophytes.
4- Nostocales. Algological Studies, 56, 247-345.
Anagnostidis, K. & Komarek, J. (1991). Modern approach to the classification system of Cyanophytes.
5- Stigonematales. Algological Studies, 59, 1-73.
Bathurst, R.R., Zori, D. & Byock, J. (2010). Diatoms as bioindicators of site use: locating turf structures
from the Viking Age. Journal of Archaeological Science, 37, 2920-2928.
Beyene, A., Addis, T., Kifle.D., Legesse,W., Kloos, H., Triest, L. (2009). Comparative study of diatoms
and macroinvertebrates as indicators of severe water pollution: case study of the Kebena and
Akaki rivers in Addis Ababa, Ethiopia. Eco. Ind, 9, 381-392.
Blanchard, G.F., Guarini, J.M., Orvain, F & Sauriau, P.G. (2001). Dynamic behaviour of benthic
microalgal biomass in intertidal mudflats. Journal of Experimental Marine Biology and Ecology,
364, 85-100.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Bold, H.C., & Wynne, J.M. (1985). Introduction to the Algae (2rd ed). Prentice Hall. Inc. London.
Boyd, C.E. & Tucker, C.S.(1998). Pond Aquaculture Water Quality Management. Kluwer Academic
Publishers, Boston.
Cornelius, E. (1999). Kajian Diatom di Perairan Pulau Pangkor. Dewan Bahasa dan Pustaka, Kuala
Lumpur.
Graham, L.E. & Wilcox, L.W. (2000). Algae. Prentice-Hall, Inc.
Guarini, J.M., Cloern, J.E., Edmunds, J. & Gros, P. (2002). Microphytobenthic potential productivity
estimated in three tidal embayments of the San Francisco Bay; A comparative study.
Estuaries, 25(3), 409-417.
Harun, W.N. (1990). Kajian ke atas Populasi Diatom Bentik di Kawasan Intertidal Kota Kinabalu.
Universiti Kebangsaan Malaysia (Kampus Sabah).
Hendey, N. I. (1964). An introductionary account of the smaller algae of British coastal water.
Otto
Koeltz Science Publishers.
Lokman, S. (1991). Diatom Air Tawar, morfologi dan Taksonomi. Dewan Bahasa dan Pustaka.
Macintyre, H.L., Geider, R.J. & Miller, D.C. (1996). Microphytobenthos: The ecological role of the
‘Secret Garden’ of unvegetated, shallow-water marine habitat. Estuaries 19, 186-201.
Patrick, R. & Reimer, C. W. (1966). The Diatoms of the United States. Vol. 1. Academy of Natural
Sciences of Philadelphia, Philadelphia.
Patrick, R & Reimer, C. W. (1975). The Diatoms of the United States. Vol. 2. Part 1. Academy of
Natural Sciences of Philadelphia, Philadelphia.
Presscott, G.W. (1978). How To Know Freshwater Algae?. WMC Brown Company Publication.
Montana. U.S.A.
Ravera, O. & Vollenweider, R.A. (1968). Oscillatoria rubescens D C as an indicator of Lago Maggiore
Eutrphication. Aquatic Science-Research Across Boundries, 30, 374-380.
Ressom, R., Song, F.S., Fitzgerald, J. Turczynowicz, L., Saadi, E., O., Roder, D., Maynard, T., &
Falconer, I. (1994). Health Effects of Toxic Cyanobacteria (Blue-Green Algae). National
Health & Medical Research Council NHMRC, Australia.
Salleh, A., Nadia, M.K., Hartina, M.A. & Sarini, A.W. (2005). Diversity of algae in the Hulu Selai River
and its tributaries, Endau-Rompin National Park, Johor, Malaysia. In: Mohamed, H & ZakariaIsmail, M. (Eds.). The Forests and Biodiversity of Selai Endau-Rompin, Malaysia.. Perbadanan
Taman Negara Johor. p.218.
Sano, T., Beattie, K.A., Codd, G.A & Kaya, K. (1998). Two (Z)-Dehydrobutyrine-Containing
Microcystins from a Hepatotoxic Bloom of Oscillatoria agardhii from Soulseat Loch, Scotland.
Journal of Natural Product, 61, 851–853
Skulberg, O.M., Carmichael, W.W., Codd, G.A. & Skulberg, R. (1993). Taxonomy Of Toxin
Cyanophyceae (Cyanobacteria). Academic Press London.
Steidinger, K.A. & Tangen, K. (1996). Dinoflagellates. In: Tomas, C.R (Ed.). Identifiying Marine
Diatoms and Dinoflagellates. Academic Press.
Stocks, K.I. & Grassle. (2001). Effects of microalgae and food limitation on the
recolonisation of benthic macrofauna into in situ saltmarsh-pond mesocosms. Marine Ecology
Progress Series, 221, 93-104.
Supiah, A. (2004). Benthic microalgae in Kampung Manggut, Batang Saribas. Final Year Project,
Universiti Malaysia Sarawak.
Sze, P. (1998). Biology of the algae. 3rd ed. Mc Graw Hill.
Taylor, F.J.R. (1987). The biology of the dinoflagellates (Vol 21). Blackwell Scientific Publication.
Underwood, A.J. & Chapman, M.G. (1995). Coastal Marine Ecology of Temperate Australia. Institute
of Marine Ecology, University of Sydney. UNSW Press.
Williams, D. M. & Round, F. E. (1987). Revision of the genus Flagilaria. Diatom Research, 2, 267-288.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
THE MANAGEMENT OF CHEPOR DEER GRAZING PADDOCKS, LENGGONG, PERAK
Siti Shuhada Mustaffa*, Siti Hajar Abdul Aziz, Norsuhana Abdul Hamid & Zuraini Zakaria
Biology Program, School of Distance Education, Universiti Sains Malaysia, 11800 Minden,
Pulau Pinang
Emails: sitishumy@gmail.com*, zuraini@usm.my
Abstract
An efficient management of Timorensis deer (Cervus timorensis) grazing ground can increase the
number of deer for the purpose of commercialization of the livestock. The effectiveness of regulating
available paddocks of deer farm is important in the management system of any farm. An achievement
in the paddock rotation system can increase the number of deer, thus helping to create a diversity in
the deer-based side products. Rotation between the paddocks should be done each week to ensure a
good quality of the grasses in the paddocks. Studies conducted in the Chepor Deer Grazing
paddocks, Lenggong, Perak reveal that all nine paddocks of total of 60 acres are grouped by deer at
all times. Rotational between the paddocks is only done once a fortnight, causing all the paddocks
having uneven grass height and a slow grass growth. This will result in a decreased food intake
caused by the decreased number and grass quality. The paddocks should be separated for the male
and female fawn when the fawn reaches three months old. Initial separation between parent deer and
fawn also allows the parent to be ready for mating. However, the separation between breed stag and
doe and fawns is not being practiced in the Chepor Deer Grazing paddocks. Such case results in a
decline in the reproductive rate of the Timorensis deer.
Keyword: Timorensis deer, management, paddock, Chepor grazing farm
1. INTRODUCTION
Breeding of deer in Kampung Chepor, Lenggong started in 2002. The 76-acre land is available for the
fourteen villagers who were to venture into the deer livestock. The pasture of Kampung is adjacent to
the main road heading to Bukit Sapi.
A deer needs three basic points in life namely food, drink and space shelter (Armstrong and Young,
2000). A study in Hutan Wanagama I, Yogyagarta showed Cervus timorensis chose to be not apart
with Sungai Oyo (Purnoma, 2010), whereas Highland Kebar in Manokwari, Indonesian Papua West,
Sungai Kasi, Sungai Api and Sungai Apriri became the natural water sources for the Timor deer
(Pattiselanno et al, 2009).
The geography and landscape of Kampung Chepor is hilly. The deer, being a wild animal loves this
kind of habitat ie.hilly areas such as the natural habitat in highland Kebar, Manokwari (Pattiselanno et
al, 2009). The land environment of Kampung Chepor which comprises various plant species as food
and deer shading place is an advantage to the grazing farms to develop the deer breeding as an
industry. From the plants, the ruminant animals receive natural mineral resources such as calcium,
potassium, iodine and zinc as additional nutrition (McDowell, 1996).
2. METHOD
In this field research, study was carried out by interview the fourteen deer farmers and staff of the
Department of Veterinary Services, Lenggong. Observations and data are garnered through the
pasture area of Kampung Chepor, Lenggong regarding the paddcok design area, paddock
management, deer production and sales, and future vision for the farmers.
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3. RESULTS AND DISCUSSION
3.1 Deer Belt
Deer Belt was inaugurated in 1993 by the Ministry of Agriculture and Agro-Based Industry. The deer
alignment covers Hulu Grik and Kuala Kangsar districts. Areas of the 'Deer Belt' in Lenggong are
Chepor, Sungai Soh A and B, Temelong, Luat and Chuas (Figure 1).
Figure 1 Deer Belt areas in Lenggong.
Only 60 acres of land were developed in the Chepor grazing grounds with a deer population of 187
consisting of 12 male parents, 75 female parents and 100 fawn (Figure 2). The targeted deer number
in Chepor grazing ground is 680, with the potential area of 76 acres to be developed into pasture. At
the moment, only the Rusa Deer species (Cervus timorensis) are bred, initially imporetd from New
Caledonia and Mauritias.
3.2 Cervus timorensis
Cervus timorensis is also commonly known as Javan Deer, Javan Rusa, Rusa Deer and Timor Deer.
They are hoofed herbivores included in the group of animal exposed to the threat of extinction
according to International Union for Conservation Nature (Stefoff, 2008; IUCN 2012). Major distribution
of Timor deer is Southeast Kalimantan (Purnoma, 2010), New Guinea (Bermica P. Bishop Museum,
1971) and New Caledonia (Garine-Wichatitsky, 2005).
Figure 2 Cervus timorensis at Chepor Grazing Paddock, Lenggong, Perak
Main nutrition of the wild Timor deer are plants and forest foliage (Stefoff, 2008; Arobaya et al, 2011) .
In Chepor, grasses such as Brachiaria decumbens (signal grass), Panicum maximum (guinea grass),
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Pennisetum purpureum (napier or Uganda grass), Brachiaria mutica (para grass) and Setaria
sphacelata (Kazungula grass) and types of legumes such as Centrosema pubescens (centro bean),
Sesbania grandiflora (West Indian pea), stylo bean, puerto bean and Phaseolus vulgaris (calypso
bean) are cultivated and serve as the deer food source (Department of Veterinary Services, 2012).
The nature of easy adaptation with environment and high disease resistance of Timor deer makes
them suit to survive in tropical climate. In Indonesia, this nocturnal species is protected under the
Indonesia Wild Animals Protection Ordinance 1931, No.134 and 226 (Stefoff, 2008; Purnoma, 2010).
This factor has motivated various research to preserve and conserve Timor deer from extinction.
A production of 62% carcass shows that Cervus timorensis is very productive to be commercialised
(Dahlan, 2009). Excellent deer breeding produces good quality of meat and this in turn determines
satisfaction to meat buyes (Challies, 1978). To generate meat that is of high quality, livestock deer
need six prime matters to be attended to : grassland and paddock, type of habitat, deer behaviour,
reproduction rate / birth, quality of health and area of wildness for the deer (Sumanto et al 2007).
Fence system for the paddocks has also to be effective, and reaches as high as 1.8-2m to avoid the
deer from springing out ( Goddart et al, 2001).
3.3 Income sources
Breeding of deer in America is an industry that has provided 7335 job opportunities, and recorded a
rapid growth with an annual total profit of USD 318.4 million (Agricultural & Food Policy Center, 2007).
In Korea, the supply of velvet from deer has become the farmers’ major source of income, whereby
the market price is 573 to 1,053 S dollars per kg (Kwak et al, 1994). In Malaysia, the minimum monthly
income for commercial farms having more than 100 deer was estimated as RM10,000.00 whilst semicommercialised farms having 50 to 99 deer recorded a minimum monthly income of RM 5,000.00
(Department of Veterinary Services, 2006). Towards 2015, RM10.47 million which is an equivalent of
402.56 sales revenue metric tonne is being targeted.
Department of Veterinary Services, Lenggong reported a decline in deer sales in Chepor grazing farm
in 2006 and 2007. As much as RM62,192.00 which represented successful 57 deer sales was achived
in 2006, and receded to RM16,099.00 (only 20 deer sold) in 2007. The sales could be improved by a
practice of sustainable use of land and an organised farm management. This is because venison
(meat of deer) has a high demand throughout the nation due to its exotic value and nutrition which is
good for our health. However, it is believed that production of meat from the pastures is unable to
accommodate demand.
Due to this, our study has proposed that Chepor Grazing Farm, Lenggong needs to be redesigned for
a more efficient and effective management. The objective is to achieve the target of making Lenggong
as a Malaysian deer belt area able to produce 4780 deer in 2015.
3.4 Current problems
The deer livestock in Chepor Grazing Farm, Lenggong is still far from achieving the deer belt target.
Various products that could be supplied from the deer livestock could not be implemented due to the
limited deer population. The farmers’ source of income is currently depending solely on venison.
Efforts need to be done to achieve the objective of the deer belt area and also the deer products. Use
of land that is effective in providing food supply (pasture) to deer, organised human resource
management and amazing pasture grounds are the main challenges to be foreseen.
In the Ninth Malaysia Plan, as much as RM4.0 million has been allocated for the deer development
programme whereby the budget distribution has injected the entrepreneur spirit in the deer farmers.
Amongst the expenditure were the costs involved in area cleaning, materials and fencing with a total
of RM8,300 per hectare or RM20,750 per acre. A diminution effort was also done for the paddock
sizes. Before the programme, Chepor Grazing Farm had six paddocks with the size of 10 acres each
(224m X 204m).
Starting 2009, the grazing ground has been modified and divided into nine paddocks with the size of
6.5 acres each (102m X 102m). Nevertheless, each paddock has an irregular ratio from the aspect of
deer age and sex. It was found that the deer population was not well distributed, which could result in
ineffective breeding. Furthermore, if parents and fawn are kept together in a paddock, inbreeding is
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likely to occur producing generations with weak disease resistance and causing death (O' Gradya et
al, 2006).
Wisconsin Department Of Natural Resources stated that any plan prepared for managing of deer
should take into consideration the balance of deer number and surrounding food in the area so that
both are directly proportional with total energy needed by the deer (Wisconsin Department of Natural
Resources, 1998). A high deer population within a paddock will affect antler growth quality. The deer
body condition, low disease resilience, thin and death are examples of effects of high deer population
in a paddock (Kerr Wildlife Management Area Research, 2000). Therefore, the deer wildness area in a
natural habitat needs to be known. This is to ensure the pasture provides a habitat that is similar to the
natural habitat of the deer. Use of satellite could facilitate the calculation area which encompasses
forests and woody plants (Wisconsin Department of Natural Resources, 1998).
The Department of Veterinary Service, Lenggong has proposed for the Chepor Grazing Farm be
limited to 20-24 deer per hectare (2.5 acreage) (Ahmad et al, 1984; Agricultural & Food Policy Center,
2007). However poor implementation occurs at most pastures in Lenggong including in Chepor.
3.5 Suggestions
Livestock development needs to consider the abilities to boost the economics effectively sustainable.
Biological factors must be added and integrated so that a safe environment is achievable (Boyazoglu,
1998).
Continuous modifications and development of paddocks should be studied to increase Cervus
timorensis population in Chepor Grazing Farm, Lenggong. The current population number
encompassing male and female parents, and male and female fawn need to be known. Overall, tag
identification is not practiced for all deer in the farm. This is a disappointment because tag
identification which is very important should be implemented for every parent admission and child birth
(Audigé et al, 1994). It facilitates total individual recorded and exact actual number. An effective
management involves data storage including health data of every individual. The grazing activity
during the first day and the last day in a paddock should also be recorded. Apart from grass height,
age and deer sex, stripping date of velvet, and other detailed information are necessarily important to
note (Audigé et al, 1994).
Figure 3 shows a paddock designed by Sumanto et al, (2007) in Kampus IPB Darmaga Bogor,
Indonesia. Isolation paddocks for male and female parents, and fawn were carried out. Paddock D1 is
for fawn aged less a year, whilst paddock D2 is for fawn aged one to two years of age. However,
Sumanto did not isolate between male and female fawn, which is essential and ideally practiced. The
separation is actually necessary to facilitate the caring activity, fattening process and and choosing the
male fawn for sales. Marital paddocks are prepared for male deer which are showing signs of mating.
Usually the sex ratio of male and female deer proposed is 1:20.
Shift system between paddocks is a good practice. A short run for deer in a paddock gives a better
effect to the deer itself and also the grass eaten (Kerr Wildlife Management Area Research, 2000) ; U.
S. Department of Agriculture, 2000; National Sustainable Agriculture Information Service, 2009 ;
Ontario Ministry of Agriculture, Food and Rural Affairs, 2012). Besides improving the pasture, shift
system is able to strengthen the soil structure, which in turn prevents landslide (U. S. Department of
Agriculture, 2000). Paddocks shift system needs to consider the soil type, fresh grass (forage),
sources of water and presence of a fence system (Ontario Ministry of Agriculture, Food and Rural
Affairs, 2012). Kerr Wildlife Management Area Research (2000) proposed that a herd of deer take
turns to three paddocks. Chepor Grazing Farm still shows some weaknesses whereby paddock shift is
carried out twice in one week and does not stick to the one week once practice as recommended by
the Department of Veterinary Service, Lenggong. Additional study is currently done to ensure the
capabiltiy of producing optimum livestock is achieved.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Figure 3 Design site paddock of Timor deer timor in Kampus IPB Darmaga Bogor (Sumanto et a.l,
2007).
4. CONCLUSION
Feasible total population of deer will increase if the paddock managing system is being practiced as
advised. The deer belt target is also achievable if the total sum of deer increases continuously.
Various programmes and activities could be implemented if the deer total population increases from
time to time. Animal tourism which involves wild and tame deer will indeed gives work opportunities to
locals. Meat which is the main source of income can be value-added through venison processing
activity in becoming various byproducts such as burger meat and venison balls. Not forgetting, the
deer skin which has the potentials in creative industries such as leather goods production. Future high
profit could also be attained by the deer farmers who endeavour into breeding programme. All these
aspects certainly bring a positive economic value.
References
Agricultural & Food Policy Center (2007). Economic impact of the Texas deer breeding industry.
Texas, United States of America. Department of Agricultural Economics.
Ahmad, M. B., Ibrahim, C. E., Hawari, H., & Mohd, D. O. (1984. ) Department of Veterinary Service,
Perak. Guide to deer farming. Unit PSM.
Arobaya, AYS., Iyai, DA., Sraun,T., & Pattiselanno, F.(2011). Forage Food of Timor Deer (Cervus
timorensis) in Manokwari, West Papua. Animal Production, 12(2):91‐95.
Bermica P.Bishop Museum (1971). Suggesteed vernacular names for New Guinea recent land
mammals.
Boyazoglu, J. (1998). Livestock farming as a factor of evironmental, social and economic stability with
special reference to research. Livestock Production Science, 57:1-14.
Challies, C. N. (1978). Assessment of the physicall well-being of red deer (Cervus elaphus L)
population in South Westland, New Zealand. University of Canterbury. Christchurch, New
Zealand.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
Dahlan, I. (2009). Characteristic and cutability of farmed rusa deer (Cervus timorensis) carcasses for
marketing of venison. Asian-Australian Journal Animal Science, 22(5):740-746.
Donoughe, J. & Wolf, M. (December, 2007). Over the limit? How can you tell if deer exceed the
carrying capacity of your habitat? With a browse impact survey. Quality Whitetails, 52-58.
Goddart, P. J.,Summers, R. W., Macdonald A. J., Murray, C., & Fawceett, A.R. (2001) Behavioural
responses of red deer to fences of five different designs. Applied Animal and Behaviour
Science, 73:289-298.
Joys, A. C., Fuller, R. J., & Dolman, P. ε. (2004). Influences of deer browsing, coppice history, and
standard trees on the growth and development of vegetation structure in coppiced woods in
lowland England. Forest Ecology and Management, 202:23–37.
Kerr Wildlife Management Area Research. (2000). White-tailed deer management in the Texas Hill
Country. Texas Parks and Wildlife Department. Texas, United Statess of America.
Kuzyk, G. W., Cool, N. L., Bork, E. W., Bampfylde, C., Franke, & Hudson, R. J. Estimating Economic
Carrying Capacity for an Ungulate Guild in Western Canada. The Open Conservation Biology
Journal, 2009(3), 24-35.
Kwak, W. S., Kim, K. H., Kim, C. W., Jeon, B. T., & Lee, S. M. (1994). Deer farming in Korea: On- farm
survey in Kyung-Kee and Chung-Buk Provinces. AJAS, 7(3):347-355.
L Audigé , Wilson P. R., & Morris, R. S. (1994). Deer-herd health and production profiling in New
Zealand. I. Study design. Vet Res, 25:126-129.
Long, Z. T., ,Pendergast, IV T. H., & Carson, W. P. (2007). The impact of deer on relationships
between tree growth and mortality in an old-growth beech-maple forest. Forest Ecology and
Management, 252:230–238.
McDowell, L. R. (1996). Feeding minerals to cattle on pasture. Animal Feed Science Technology,
60:247-27.
Morecroft, M. D., Taylor, M. E., Elwood, S. A., & Quinn, S. A. (2001). Impactsof deer herbivory on
ground vegetation at Wytham Woods, Central England. Forestry, 74(3):251-257.
National Sustainable Agriculture Information Service. (2009). Paddock design, fencing and water
systems for controlled grazing. Montana, United States of America.
O'Gradya, J., Broofeb, B. W., Reed, D. H., Ballon, J. D.,Tonkync,D.W., & Frankhama, R. (2006).
Realistic levels of inbreeding depression strongly affect extinction risk in wild populations.
Biological Conservation, 133:42-51.
Ontario Ministry of Agriculture, Food and Rural Affairs. (2012). Rotational Grazing in extensive
pastures. Ontario, Canada.
Pattiselanno, F., & Setyarini A. Y. (2009). Grazing habitat of the rusa deer (Cervus timorensis) in the
Upland Kebar Manokwari. Journal of Biological Diversity, 10(3):134-138.
Purnomo, D. W. (2010). A habitat selection model for Javan deer (Rusa timorensis) in Wanagama I
Forest, Yogyakarta. Nusantara Bioscience, 2(2):84-89.
Sumanto, εasy’ud, B., & Thohari, A. (2007). Disain penangkaran rusa timor (Cervus timorensis de
Blainville) berdasarkan sistem deer farming di Kampus IPB Darmaga Bogor. Media
Konservasi, 12(3):1-7.
U. S. Department of Agriculture. (2000). Pastures for profit: A guide to rotational grazing.
Wichatitsky, M. DE. G., Soubeyran, Y., Maillard, D., & Duncan, P. (2005). The diets of introduced rusa
deer (Cervus timorensis russa) in a native sclerophyll forest and a native rainforest of New
Caledonia. New Zealand Journal of Zoology, 32:117-126.
Wisconsin Department of Natural Resources. (1λλ8). Wisconsin’s Deer εanagement Program.
Washington, D. C.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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ANTI CANCER ACTIVITIY OF CRUDE EXTRACTS AND INDOLE ALKALOIDS FROM
Rauvolfia reflexa (Apocynaceae)
1
2
1
1
Mehran Fadaeinasab , Behnam Kamalidehghan , A.Hamid A.Hadi , Hanita Omar , Masoumeh
1
Hoseinzadeh
1. Department of Chemistry, Faculty of Science, 50603 University Malaya, Kuala Lumpur
2. Department of Pharmacy, Faculty of Medicine, 50603 University Malaya, Kuala Lumpur
Abstract
From the methanolic crude extract of Rauvolfia reflexa seven indole alkaloids were isolated namely;
(1), akuammilan (2), vomilenine (3), isoresrpiline (4), rescinnamine (5), and cantleyine (6). The
methanol crude extract showed moderate anticancer activity against MCF-7 and WRL-68 cell lines,
although the isolated compounds and dichloromethane crude extract were also tested for anticancer
activity but they didn't show significant activity against MCF-7 and WRL-86 cancer cell lines.
Keywords: alkaloids, Rauvolfia, Apocynaceae, anti cancer activity, NMR
1. INTRODUCTION
Bioactive indole alkaloids have been isolated from Rauvolfia reflexa (Apocynaceae). The chemistry of
the Rauvolfia species has been comprehensively investigated for the presence of alkaloids over a long
period of time. First part of research program was on the occurrence of alkaloids in Malaysian species
of Rauvolfia, a phytochemical analysis from bark of Rauvolfia reflexa is described. Six alkaloids were
1
isolated; the structures of all alkaloids isolated were determined by a detailed analysis of the H NMR,
13
C NMR, HMBC, HSQC, NOESY, COSY spectral data and confirmed by UV, IR and MS. Second part
of research was carried out In vitro studies of the crude extracts and pure compounds isolated from
Rauvolfia reflexa which the methanolic crude extracts exhibited considerable anticancer Activity.
2. MATERIALS AND METHODS
2.1 Cell Culture
In this study, all cells used were obtained from American Type Cell Collection (ATCC) and maintained
o
in a 37 C incubator with 5% of CO2 saturation. WRL-68 normal hepatic cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) whereas A549, non-small cell human lung
adenocarcinoma epithelial cells; MCF-7, human breast adenocarcinoma cells and PC-3, prostate
adenocarcinoma cells were maintained in Roswell Park Memorial Institution-1640 media (RPMI8
1640). Both media were supplemented with 10% of fetal bovine serum (FBS) .
2.2 Cellular Viability
The above mentioned cell lines were used to determine the inhibitory effect of dichloromethane
(CH2Cl2), methanol (MeOH) crude extracts compare with the standard compounds (G25 and
9
Thymoquinone), on cell growth using MTT assay . This calorimetric assay is based on the
conversion of yellow tetrazolium bromide (MTT) to purple formazan derivatives by mitochondrial
succinate dehydrogenase in viable cells. To measure cell viability, cells were seeded at a density of
5
o
1 × 10 cells/ml in a 96-well plate and incubated for 24 hours at 37 C with 5% of CO2. Cells were then
treated with the test agents in the next day and incubated for another 24 hours. After 24 hours, 10
µl of MTT solution at 5 mg/ml was added to each well and then the plates were incubated for
o
another 4 hours at 37 C. then, water-insoluble formazan was dissolved by adding 100 µl dimethyl
sulfoxide (DMSO) to each well. To finish, optical density (OD) was monitored at 570 nm as a
reference wavelength using. Results were expressed as a percentage of control giving percentage
cell viability after 24 hours exposure to test agents. The potency of cell growth inhibition for each
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
test agent was expressed as an inhibitory concentration 50 value (IC50). Viability was defined as
10
the ratio (expressed in percentage) of absorbance of treated cells to untreated cells .
2.3 Statistical Analysis
Each experiment was performed at least three times. Results are expressed as the mean value +
standard deviation (SD).
3. RESULT AND DISCUSSION
Plant material was collected at Kelantan, and air-dried. The methanol crude extract, from the bark,
were submitted to acid-base treatment and fractions corresponding to different pH ranges were
purified by flash-chromatography, LH-20 sephadex and preparative TLC, leading to the isolation of six
indole alkaloids (Fig. 1). Undulifoline (1) [2], akuammilan (2) [3], vomilenine (3) [4], isoresrpiline (4) [5],
rescinnamine (5) [6], and cantleyine (6) [7] were confirmed by spectroscopic analysis.
3.1 General Experimental procedures
CC was run on silica gel 60 (40-63 m). TδC was performed on aluminum and glass plates pre-coated
1
13
with silica gel 60 F254 (Merck). H NMR and C NMR and 2D NMR spectra were determined in CDCl3
(JEOL JNM-FX400), UV spectra were recorded on a Shimadzu UV-160A spectrophotometer using
MeOH as solvent. MS was obtained with Agilent 6530. The IR spectru was measured by FT-IR: pelkin-1
elmer RX 1 (fourier transform intra-red) spectrometer for frequencies 4000-400 cm
3.2 Collecting of plant material
The plant materials (barks) of Rauvolfia reflexa (KL 4900) were collected from Kelantan The botanical
identification was made by Mr.Teo Leong Eng, Faculty of Science, University of Malaya. Voucher
specimens are deposited in the Herbarium of Chemistry Department, University of Malaya.
Me
MeO2C
N
N
O
NH
N HO
Me
CO 2Me
1
2
N
MeO
MeO
Me
3
Me
O
N
H
MeO2 C
4
5
Figure 1. Indole alkaloids isolated from the bark of
Rauvolfia reflexa
6
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3.3 Extraction and Isolation
The extraction of the plant (2.0 kg) was carried out by extracted exhaustively with hexane for 48 hours
to removed non-polar organic compound, waxes and fats. Then the extract was dried on the rotary
evaporator. The plant material was dried and wetted with 10 % ammonia solution and left for overnight.
They were then re-extracted successively with dichloromethane (CH 2Cl2) and methanol (MeOH). After
removal of the solvents, the hexane crude extract (1.2 g), dichloromethane (15 g) and methanol (15 g)
were obtained.
Table 1 shows the effect of crude extracts (MeOH, CH2Cl2, G25, Thymoquinone) on two cell lines
(MCF-7 and WRL68) expressed as EC50 values in 24 hours MTT assay
Cell line
EC50 ± S.D. (µg/ml)
MeOH crude extract
CH2Cl2 crude extract
G25
Thymoquinone
MCF-7
> 200
20.10
19.65
~ 5.106
WRL-68
12.51
11.95
33.89
~ 8.517
References
Azeem S, Wasif K, Ataullah M, Ahmad I, Characterization of oxidation products of Rauvolfia alkaloids.
Pakistan Journal of Pharmaceutical Sciences 18 (2005) 33-35.
Bernas T, Dobrucki J, Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with
TMRE, JC-1, and NAO mitochondrial fluorescent probes.Cytometry (2002) 236–242.
Cesar V.F.B, Jan S, Robert V, Sandra B, Amelia T.H, Indole alkaloids from Rauvolfia sellowii.
Phytochemistry 41 (1996) 969-973.
Da silva T.M.S, Da silva B.A, Mukherjee R, The monoterpene alkaloid cantleyine from strychnos
trinervis root and its spasmolytic properties. Phtomedicine 6 (1999) 169-176.
Jennifer L.G, Doglas Y, Strain-specific differences in formation of apoptotic DNA ladders in MCF7 breast cancer cells. Cancer Letters (1999) 31-37.
Lucilia K, Raquel B, Ingrid K, Indole alkaloids from Rauvolfia bahiensis A.DC. (Apocynaceae).
Phytochemistry 60 (2002) 315-320.
Massiot G, Boumendjel A, Nuzillard J.M, le men-Olivier L, Alkaloids from Alstonia undulifolia.
Phytochemistry 31 (1992), 1078-1079.
Massiot G, Lavaud C, Vercauteren J,Le men-Olivier L, Anthony A, Rearrangement of two indole
alkaloids in trifluoroacetic acid: desformocorymine and dihydrocorymine. Helvetica Chimica
Acta 66 (1983) 2414-2430.
Mosmann T, Rapid colorimetric assay for cellular growth and survival application to proliferation and
cytotoxicity assays. Journal of Immunological Methods (1983) 55-63.
Nauvia M, Ivo J.C.V, Jan S, Leda M, Raimundo B.F, Darcyribeirine, a novel
pentacyclic alkaloid
from Rauvolfia grandiflora Mart. Tetrahedron letters 43 (2002) 1783-1787.
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IN VITRO PROPAGATION OF AQUILARIA MICROCARPA BAILL. USING SHOOT TIPS AND
LATERAL BUDS EXPLANT
Zul Helmey, M. S.,* Sepiah, M. and Sani, H.B.
Plant Tissue Culture Laboratory, Department of Plant Science and Environmental Ecology,
Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak.
*Email: zul_87fh@yahoo.com
Abstract
Aquilaria microcarpa Baill., is of economic importance source of gaharu for cosmetic and medicinal
purposes. Collection of these plants in forests has become very difficult and they are threatened with
extinction due to their indiscriminate collection and over-exploitation to meet the high demand in the
open market. The objective of this study is to develop an in vitro culture method for mass propagation
of this species. Shoot tips and lateral buds from grown seedling were used as the explants. Shoots
were cultured in modified Murashige and Skoog (MS) medium with 6-benzylaminopurine (BAP) at
various concentrations rates. The MS medium supplemented with 0.25 mg/L BAP was the highest
mean number of shoot formation for shoot tips (6.2 shoots), which is significantly higher than other
treatment. For lateral buds, MS medium supplemented with 2.0 mg/L was most effective for shoot
multiplication with 3.2 shoots, which is significantly higher than other treatment. The highest mean
shoot length for shoot tips was obtained in MS medium supplemented with 4.0 mg/L BAP (1.2 cm) and
lateral buds in MS medium supplemented with 0.25 mg/L BAP (1.8 cm). Regenerated shoots
transferred onto rooting media, they rooted within 30 days. Rooted plantlets were acclimatized in
“Jiffy-7” for four weeks before transferred in potting consisted of mix peat, soil and sand (1:1:1). The
plantlets showed 80% survival rate and good growth.
Keywords: Aquilaria microcarpa Baill., in vitro, gaharu
1. INTRODUCTION
Aquilaria species belongs to the family Thymelaeaceae, is of economic important source of non timber
forest product, gaharu or agarwood (Chakrabarty, 1994; Soehartono and Newton, 2000). There are
seven Aquilaria species have been found in Malaysia of which A. microcarpa is the most popular for
its gaharu production (Chang et al., 2002). Aquilaria species is also found in other countries, such as
in Bangladesh, Bhutan, India, Indonesia, Iran, Myanmar, Philippines, Singapore and Thailand (Oldfield
et al., 1998). In Malaysia, Aquilaria spp. have various vernacular names, however in Malay they are
known as karas, tabak, candan, kepang or depu (Lim and Noorainie, 2010).
Aquilaria species are the sources of fragrant wood that has been traded since biblical times for uses in
religious functions and for medicinal and aromatic preparations (Zich and Compton, 2001; Lim and
Noorainie, 2010). The most common forms of traded gaharu are chips, flakes and extracted oil and
powder waste (Yaacob, 1999). Chang et al., (2002) reported that prices of gaharu chips ranged from
60 sen per kg for the low and mixed grades to more than RM 2000 per kg for the high grade.
However, in 2010 a random survey in the retail local markets we found prices for the top-quality
gaharu could reach above MYR 18,000.00per kg (Lim and Noorainie, 2010).
Gaharu is not formed in each of the Aquilaria spp., but it is associated with the plant defense
mechanism due to pathological effect. Currently, it is difficult to get good quality gaharu from Aquilaria
trees that are growing in natural forest. However, the price offered in a market has stimulated illegal
and over harvesting, and as a result, Aquilaria trees are often cut down indiscriminately. Populations
of this tree have declined drastically and may lead to possible extinction in the near future if no
effective conservation effort is done. At present, all of the Aquilaria species have been included in The
World List of Threatened Tree of the Convention on International Trade in Endangered Species of
Wild Flora and Fauna (CITES) Appendix II (Lim and Noorainie, 2010). Initiatives to produce gaharu in
planted Aquilaria trees have been done especially in Indonesia, India, Cambodia, Thailand and
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Vietnam. Planting of Aquilaria species in Malaysia received attention only in the recent years. In
addition to conserving, the planting of the tree species is to ensuring sustainable supply of the
agarwood. Thus, methods for propagation need to be developed to ensure enough supply of planting
stock. In the present work, micropropagation system via tissue cultures technique such as
organogenesis was carried out to develop shoot regeneration and multiplication in A. microcarpa.
2. MATERIALS AND METHODS
The study was started with an attempt to establish axenic (contamination free) culture. The
established axenic cultures were used for subsequent experiments, induction of shoot regeneration
and multiplication. Healthy seedlings selected from cultivated A. microcarpa at UNIMAS Arboretum
were used as stock plants for explants preparation.
2.1 Induction of Multiple Shoots Formation through Direct Organogenesis
In this study, two types of explants i.e. shoot tip and lateral bud of A. malaccensis were surface
sterilized in 15% for 10 minute of Clorox, together with 0.1% Tween 20 (a wetting agent) with gentle
agitation followed rinsed three to five times (3-5 minutes each time) with sterilized distilled water. The
explants were cultured in Petri dish containing Murashige and Skoog (1962) medium without any
hormone. For shoots regeneration and multiplication, explants of A. microcarpa were cultured on full
strength MS medium with different concentrations of 6-benzylaminopurine (BAP) at 0, 0.25, 0.5, 1, 2
and 4 mg/L. The cultures were incubated at room temperature and 16 hours photoperiod on the
culture shelf until the shoots were formed. There were four replicates for each type’s explants with four
explants in each replicates. After two weeks of incubation, explants were transferred in test tubes and
bottles for further growth and shoot multiplications with the same medium and condition. The multiple
shoots were separated and subcultured to a fresh medium every four weeks using the same
conditions described above. Data on number of explants that developed shoots and number of shoots
in each Petri dish were recorded weekly after first sign of shoot formation. The mean numbers of
shoots regenerated and mean shoot length were calculated and all data were statistically analysed by
Duncan’s multiple range tests.
2.2 Rooting of Regenerated Shoots
For root initiation, the shoots regenerated from shoot tips and lateral buds were cultured on rooting
medium. For each treatment, 20 culture tubes were inoculated for individual shoot. Cultures were
maintained at 25 °C and 16 hours of photoperiod on the shelf of culture room. Number of shoots
rooted was recoded after 30 days of culture.
2.3 Hardening and Acclimatization
Rooted plantlets were washed with water in order to remove any adhering medium. Then the plantlets
were immersed in 0.1% Benomyl solution for half an hour prior planting. The plantlets were transferred
to “Jiffy-7” for four weeks before transferred in plastic pots containing autoclaved mixture of peat, soil
and sand in the ratio of 1:1:1. Hardening of potted plantlets was accomplished in room temperature by
covering them with polyethylene bags and sprayed with water to maintain high humidity. Polyethylene
bags were completely removed after 20 days. The survival rate of plantlets was recorded.
3. RESULTS AND DISCUSSION
3.1 Induction of Multiple Shoots Formation through Direct Organogenesis
Different concentration of BAP has effected on the multiplication of shoots for A. microcarpa. The
highest mean number of shoot tip explants of A. microcarpa forming the new shoots was on media
with BAP at 0.25 mg/L was 6.2 shoots compared with other concentrations (Figure 1A). The lowest
mean number of shoot tips developed the new shoots was obtained in 4.0 mg/L BAP (2.7 shoots). It
was observed that the number of shoots produced decreased as the BAP concentration in the medium
increased (Table 1). MS medium supplemented with higher concentration of BAP was also found
could induce calli surround of shoot tips explants (Figure 1B). For shoot elongation, MS containing 4.0
mg/L BAP gave the highest shoot length when compared with other concentrations (Figure 1C). Lower
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mean shoot length was obtained in MS medium containing 0.25 and 1.0 mg/L BAP. BAP alone
favored shoot multiplication, as it is considered to be one of the most useful cytokinins for achieving
the multiplication and micropropagation of plants (Joshi and Dhar, 2003).
Table 1. Effect of different concentrations of BAP on shoot multiplication from shoot tips and lateral
buds explants of A. microcarpa
BAP
Shoot tips
Lateral buds
(mg/L)
Mean no. of shoot
Mean shoot length
Mean no. of shoot
Mean shoot
regenerated
(cm)
regenerated
length (cm)
a
ab
a
a
0.25
6.2
0.5
1.0
1.8
ab
b
a
a
0.5
4.8
0.4
3.0
1.7
bc
ab
a
ab
1.0
4.2
0.5
2.8
1.2
cd
ab
a
b
2.0
2.9
0.7
3.2
0.8
d
a
a
b
4.0
2.7
1.2
1.2
0.9
a
Mean values followed by the same letters in a given column are not significantly different (P = 0.05)
according to Duncan’s test.
For lateral buds explants, the best concentrations of the cytokinins to be added in media to establish
shoot multiplication of A. microcarpa was 2.0 mg/L for BAP (3.2 shoots) (Figure 1D). Lowest mean
number of lateral buds forming shoot was obtained in MS medium containing 0.25 mg/L (1.0 shoots)
compared to the other concentrations. The shoots in MS medium supplemented with lower BAP (< 1.0
mg/L) were dumpy and twisty (Figure 1E). Different from the shoot tips explants, the highest shoots
elongation for lateral buds were observed in MS medium containing 0.25 mg/L (1.8 cm) compared to
the other concentrations (Figure 1F). Lower mean shoot length was observed in MS medium
supplemented with 2.0 mg/L BAP (0.8 cm) compared to the other concentrations. According to Joshi
and Dhar (2003), shoot elongation is retarded in cultures with increased number of shoots.
The presence of BAP to stimulate the development of new shoot formation in shoot tips and lateral
buds is well known happened in plant. Cytokinin is derivatives of adenine which promotes cell division,
regulate and development in plant tissue (Hartmann et al., 1990). BAP was reported to be more
effective for shoot multiplication in many plants. These include to regenerating shoot in Trifolium
pretense (Campbell and Tomes, 1984), Dipterocarpus intricatus (Linington and Kew, 1989), Morus
alba (Sharma and Thorpe, 1990) and olive (Ali et al., 2009). It has also been reported that high
concentration of BAP could cause stunting and or callusing of shoots (Scott et al., 1995). In this study,
shoot tips and lateral buds explants, both, were found more responsive in shoot proliferation when
appropriately balanced plant growth regulators added into the media. In addition, fast elongation of
shoots regenerated from shoot tip and lateral bud explants was also seen.
3.2 Rooting of Regenerated Shoots
When the regenerated shoots were transferred onto rooting media, they rooted within 30 days (Figure
2A). Several studies had been reported regarding the effects of plant growth regulators on rooting
induction of different plant species. Rooting in regenerated shoots of G. superba was achieved at 90%
when the excised shoots were cultured individually on root induction medium (Sayeed Hassan and
Shyamal, 2005). Fotopoulos and Sotiropoulos (2005) mention that the mineral concentration of the
culture medium affect rooting characteristics and some researchers have proposed its reduction to
half normal strength for rooting improvement.
3.3 Hardening and Acclimatization
Acclimatization is the determining key for the survival of the in vitro raised plantlets since they are not
suited for in vivo conditions (Baksha et al., 2007). The plantlets rooted on the media were used for
hardening and acclimatization. Out of 25 plants transferred to the “Jiffy-7” and potting pots, 20 plants
survived after 2 months indicating 80% survival rate (Figure 2B, Figure 2D and Figure 2F).
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
A
B
D
a
E
C
F
Figure 1 (A) Shoot multiplication from shoot tip explants in MS medium supplemented with 0.5 mg/L
BAP. (B) MS medium supplemented with higher concentration of BAP could induce calli surround of
shoot tips explants. (C) Shoot elongation from shoot tips explants in MS medium supplemented with 4.0
mg/L BAP. (D) Shoot multiplication from lateral buds explants in MS medium supplemented with
2.0mg/L BAP. (E) Dumpy and twisty shoots from lateral buds in MS medium supplemented with lower
BAP (< 1.0 mg/L). (F) Shoot elongation from lateral buds in MS medium supplemented with 0.25 mg/L
BAP. Bar: 0.5cm.
A
B
C
D
E
F
Figure 2: (A) Rooting regenerated from shoot tips and lateral buds (B) The plantlets were transferred
in “Jiffy-7” for 4 weeks. (C) New young leave and shoot formed after 4 weeks transferred in “Jiffy- 7”.
(D) The plantlets from ‘Jiffy 7’ were transferred to plastic pots containing autoclave a mixture of peat,
soil and sand in the ratio of 1:1:1. (E) and (F) Survival seedlings after transferred in polybags. Bar:
0.5cm.
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4. CONCLUSION
As conclusion, MS medium supplemented with BAP at 0.25 mg/L were effective in the induction of
higher shoot proliferation and multiplication in shoot tips and BAP at 2.0 mg/L were effective in the
induction of higher shoot proliferation and multiplication in lateral bud explants. BAP at 4.0 mg/L was
effective for shoot elongation of shoot tip and 0.25 mg/L BAP was effective for shoot elongation of
lateral bud. Shoots regenerated from shoot tips and lateral buds of A. microcarpa were rooted within
30 days after transferred in rooting media. Using shoot segments as explants, direct plantlet
regeneration of Aquilaria species can be achieved via organogenesis in short term period and the
regenerated plantlets can be used for further propagation. Thus, this study provided a very useful
method for propagation of this plant.
Acknowledgment
The authors are grateful for the financial support provided by UNIMAS, under UNIMAS Zamalah
postgraduate scholarship. We are also grateful to the staff of Department of Plant Science and
Environmental Ecology, Faculty of Resource Science and Technology, Unimas, for their technical
assistance.
References
Ali, A, Ahmad, T, Abbas, N. A. & Hafiz, I.A. (2009). Effect of different media and growth regulators on
in vitro shoot proliferation of olive cultivar ‘Moraiolo’ Pakistan. Journal of Botany, 41, 783-795.
Baksha, R., Jahan, M.A.A, Khatun, R. & Munshi, J.L. (2007). In vitro Rapid clonal propagation of
Rauvolfia serpentine (Linn.) Benth. Bangladesh J. Sci. Ind. Res., 42(1), 37-44.
Chakrabarty, K.A., Kumar, V. & Menon, V. (1994). Trade in agarwood. TRAFFIC, New Delhi, India.
Chang, Y. S., M. A. Nor Azah, A. Abu Said, E. H. Lok, S. Reader, & A. Spiers. (2002). Gaharu. FRIM
Technical Information Bulletin No. 69. Forest Research Institute of Malaysia, Kuala Lumpur,
Malaysia.
Campbell, C.T. & Tomes, D.T. (1984). Establishment and multiplication of red clover plants by in vitro
shoot tip culture. Plant Cell, Tissue and Organ Culture, 3, 49-57.
Fotopoulos, S. & Sotiropoulos, T.E. (2005). In vitro rooting of PR 204/84 rootstock (Prunus persica x
P. amaygdalus) as influenced by mineral concentration of the culture medium and exposure to
darkness for a period. Agronomy Research, 3(1), 3-8.
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Hartmann, H.T., Kester, D.E. & Davies, F.T. (1990). Plant propagation: Principal and Practice. 5 eds.
Prentice Hall, New York.
Joshi, M. & Dhar, U. (2003). In vitro propagation of Saussurea abvallata Himalaya. Plant Cell Rep., 21,
933-939.
Lim, T. W. & Noorainie, A. A. (2010). Wood for trees: A review of the agarwood (gaharu) trade in
Malaysia. TRAFFIC Southeast Asia, Petaling Jaya, Selangor, Malaysia.
Linington, I.M. & Kew, R.B.G. (1989). Micropropagation of Dipterocarpus alatus and D. intricatus. In
Vitro, 25, 22-26.
Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco
tissue culture. Physiology Plant, 15, 473–497.
Oldfield, S., Lusty, C. & MacKinven, A. (1998). The Word List of Threatened Trees. World
Conservation Press, Cambridge, UK. 650pp.
Sharma, K.K. & Thorpe, T.A. (1990). In vitro propagation of mulberry (Morus alba L.) through nodal
segments. Sci. Hort., 42, 307-320.
Scott, E.S., Rao, A.N. & Loh, C. S. (1995). Preliminary studies of micropropagation of Hopea odorata,
a dipterocarp tree. Plant Cell Tissue Organ Culture, 41, 193-196.
Soehartono, T. & Newton, A.C. (2000). “Conservation and sustainable use of tropical trees in the
genus Aquilaria. l. Status and use in Indonesia.” Biological Conservation, 96, 83-94.
Sayeed Hassan, A.K.M. & Shyamal, K. R. (2005). Micropropagation of Gloriosa superba L. through
high frequency shoot proliferation. Plant Tissue Culture, 15(1), 67-74.
Yaacob, S. (1999). Agarwood: Trade and CITES Implementation in Malaysia. Unpublished report
prepared for TRAFFIC Southeast Asia, Malaysia.
Zich, F. & Compton, J. (2001). Final Frontier: Towards sustainable management of Papua New
Guinea’s agarwood. A Traffic Oceanic Report in Conjunction with WWF South Pacific
Programme. pp. 3-4.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
THE MACROBENTHOS COMMUNITY AT SEKAMBAL RIVER ESTUARY LUNDU, SARAWAK
Harold Tinggang Ngau*, Shabdin Mohd Long, Abang Azizil Fansuri Abang Abdullah
Department of Aquatic Science, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak.
*Email: arolengang@gmail.com
Abstract
A pilot study on the community structure of macrobenthos was carried out at Sungai Sekambal,
Lundu. The main purposed of this study is to record the density and number of macrobenthos taxa
found in Sekambal River. This study consists of 7 stations, which covered from downstream to
upstream of Sekambal River. The physico-chemical parameters of water such as temperature,
turbidity, dissolved oxygen, conductivity, salinity and pH were recorded.
The triplicate of
macrobenthos sample was collected using Ekman Grab Sampler at each station. The sample was
sieved through 500 µm mesh size sieve and identified under stereomicroscope. As a result, the total of
8 taxa of macrobenthos was found in Sekambal River. These are Polycheaeta, Bivalvia, Gastropoda,
Amphipoda, Decapoda, Sipuncula, Coleoptera and Oligochaeta. The density of macrobenthos in
Sekambal River was influenced by Salinity.
Keywords: macrobenthos, taxa, density, physico-chemical parameters
1. INTRODUCTION
Benthic community is also known as benthos organisms that live on or within sediment. Benthic
community can be found in all aquatic sediment such as brackish, marine and freshwater (Morris &
Madden, 1978). Benthic community can be divided based on their sizes such as macrobenthos (larger
than 500 mm), meiobenthos (less than 500 mm; larger than 42 µm) and nannobenthos (smaller than
42 µm). Furthermore, benthos community includes crustacean, mollusks, aquatic worms and small
aquatic insect (Rak et al., 2011). Basically, the community structure of benthic community is
influenced by physico-chemical of the water (Cuomo & Zinn, 1995).
The study of macrobenthic community as biological monitoring has been widely recognized in
scientific research (Mason & Parr, 2003). The main reason of using macrobenthic community as the
bio-indicator is the sensitivity of macrobenthic to organic pollution. Furthermore, benthic community
live in the water for their whole life and easy to collect (Rak et al., 2011). Therefore, the relative
abundances of macrobenthic can be used to determine the pollution in aquatic ecosystem (Azrina et
al., 2005). In natural habitat of river, macrobenthic community typically more diverse and have high
species richness (Armitage et al.,1983). The distribution of macrobenthic depends on physical nature
of the substratum, nutrient content, oxygen content level of stability and hydrogen sulphide level
(Anbuchezian et al., 2009).
Therefore, the main objectives of this study are to record the density and the number of macrobenthos
taxa found in Sekambal River and to determine the physico-chemical factors of the water that
influence their community.
2. MATERIALS AND METHODS
Types of habitat: Basically, Sekambal River is influenced by tides twice a day. The vegetation along
the river bank is usually dominated by Rhizophora sp.
Sampling procedures: Sample of macrobenthos was collected at 7 stations using Ekman grab
sampler. Triplicate of macrobenthos samples from each station was sieved through 500 µm mesh
size. All the retained organisms on the sieves were collected and preserved in 10 % formalin solution.
Then, the sample was stained using Rose Bengal. Samples were brought back to the laboratory for
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
further identification until the lowest practical taxon under a stereomicroscope. Physico-chemical
parameters of water such as pH, salinity, temperature, turbidity, dissolved oxygen and conductivity
were recorded in-situ.
Table 1: Study Site
Stations
Coordinate
◦
1
N 01 43.995
◦
E 109 52.219
Weather Condition
Sunny
Habitat description
Sampling
station:
River
mouth
Sediment: sandy
2
N 01 43.817
◦
E 109 52.159
◦
Sunny
Sampling station: closed to
the discharged of shrimp
culture effluent.
Sediment: muddy sandy
3
N 01 43.672
◦
E 109 52.256
◦
Cloudy
Sampling station: Sebuloh
River mouth (tributary of
Sekambal River)
Sediment: muddy sandy
4
N 01 43.494
◦
E 109 52.470
◦
Cloudy
Sampling station: Dominated
by Rhizophora sp.
Sediment: fine sand plus leaf
debris
5
N 01 43.246
◦
E 109 52.521
◦
Rainy
Sampling station: dominated
by Rhizophora sp. Close to
residential area.
Sediment: muddy
6
N 01 43.124
◦
E 109 52.259
◦
Rainy
Sampling station: dominated
by Rhizophora sp.
Sediment: muddy plus leaf
debris
7
N 01 43.068
◦
E 109 52.969
◦
Cloudy
Sampling station: dominated
by Rhizophora sp. And
shallow water.
Sediment: muddy
3. RESULTS AND DISCUSSION
3.1 Macrobenthos
Based on the results, 5 classes and 3 orders of macrobenthos were recorded. The common
macrobenthos found was polychaeta which present at 7 sampling sites followed by Oligochaeta which
only absent at station 6. The less macrobenthos recorded was Coleoptera which only present at
station 7. The absence of the certain macrobenthos at sampling sites might possibly due to the
physico-chemical factors and available of habitat (Sharma & Chowdhary, 2011).
Based on the result, the highest number of taxa can be found at Station 2 followed by Station 1,
Station 3, Station 6, Station 5, Station 4 and Station 7 (Table2). Station 2 was located close to the
discharged of shrimp farm effluent. Thus, the effluent discharge from the farm has the highest
concentration of dissolved nutrient such as nitrate and phosphate (Jones, 1999). Therefore, the
availability of dissolved nutrient has increase the number of taxa in Station 2. Macrobenthic
communities regularly respond to high nutrient concentrations (Savage et al., 2002).
The density of macrobenthos was summarized in Table 2. The highest total density of macrobenthos
2
2
was at Station 7 while the lowest density was at Station 2 with 610.48 ind./m and 235.87 ind./m
respectively. Station 1 and Station 5 was recorded as the second highest of density with 513.35
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2
2
2
ind./m . The range of macrobenthos density was 235.87 ind./m – 610.48 ind./m . Density of
marcobenthos was influenced by environmental parameters (Mahapatro et al., 2011).
2
Table 2: Density (number of ind/ m ) of macrobenthos taxa in Sekambal River Estuary.
Stations
St 1
St 2
St 3
St 4
St 5
St 6
St 7
Polychaeta
152.62
69.37
97.12
138.74
263.61
27.75
41.62
Bivalvia
55.50
0.00
0.00
0.00
27.75
13.87
0.00
Oligochaeta
194.24
27.75
55.50
263.61
180.37
0.00
568.85
Gastropoda
111.00
69.37
41.62
0.00
27.75
0.00
0.00
Amphipoda
0.00
13.87
0.00
0.00
0.00
235.87
0.00
Decapoda
0.00
27.75
0.00
0.00
13.87
0.00
0.00
Sipuncula
0.00
27.75
69.37
0.00
0.00
0.00
0.00
Coleoptera
0.00
0.00
0.00
0.00
0.00
13.87
0.00
513.35
235.87
263.61
402.36
513.35
291.36
610.48
4
6
4
2
3
4
2
Taxa
Total
No. Taxa
3.2 Physico-chemical parameters of wáter
There were 6 parameters of water quality was recorded. The range of pH values was 5.48- 7.98. The
lowest value of pH was at Station 6 while the highest was at Station 3 with 5.48 and 7.98 respectively.
Based on Table 3, the range of salinity was 25 PSU-32 PSU. The lowest value for salinity was at
Station 7 with only 25 PSU. The value of Salinity was decreased at Station 7 and this might be due to
the influx of freshwater (Nuttle & Fourqurean, 2000). Moreover, location of Station 7 was closed to the
upperstream of Sekambal River.
The range of temperature at Sekambal River was 28.40◦C- 30.40◦C. According to the results
recorded, the temperature was decreased from St 1 to St 7. The temperature was influenced by the
weather condition (Table 1). The range of turbidity was 31.34 NTU- 398.33 NTU. High turbidity might
be due to the erosion of the riverbank. Based on data recorded, DO was slightly different from Station
1 to Station 7. The range of DO was 6.40 mg/l – 6.70 mg/l. The range for conductivity was 44.56 mS –
46.45 mS. Conductivity of the water was influenced by the water temperature (Roosmalen & Plomp,
1993).
Table 3: Physico-chemical parameters of water in Sekambal River Estuary.
Parameters
pH
Stations
Salinity
Temp
Turbidity
DO
Cond
(PSU)
(◦C)
(NTU)
(mg/l)
(mS)
St 1
6.46±0.03
30.00±1.00
30.40±0.44
389.33±52.58
6.50±0.10
45.36±0.01
St 2
7.93±0.04
32.33±0.58
30.27±0.06
73.33±7.64
6.53±0.06
45.36±0.01
St 3
7.98±0.01
31.00±0.00
29.97±0.06
81.67±2.52
6.70±0.20
45.68±0.01
St 4
7.80±0.01
30.67±0.58
27.43±0.06
52.67±2.08
6.60±0.10
45.87±0.03
St 5
6.39±0.01
30.00±0.00
28.27±0.06
44.08±3.15
6.40±0.10
46.30±0.06
St 6
5.48±0.03
30.00±0.00
28.40±0.00
46.08±2.45
6.50±0.10
46.45±0.07
St 7
6.13±0.01
25.67±0.58
28.40±0.00
31.34±1.79
6.60±0.10
44.56±0.05
Pearson correlation of total macrobenthos density and physico-chemical parameters in each station
showed that the total density of macrobenthos only influenced by salinity. The negative correlation
between total density and salinity showed that the total density will increase while salinity will
decreased. The results of this study showed salinity influenced the total density of macrobenthos in
Sekambal River. Thus, salinity is important in determining distribution and abundance of specific
species, and community structure of macrobenthos. Furthermore, this study showed that similar
results with study at Dead Sea Basin Region (Silberbush et al., 2005).
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The correlation between the number of macrobenthos taxa and physico-chemical parameters showed
that the number of taxa was influenced by salinity and turbidity with 0.658 and 0.600 respectively.
Thus, the number of taxa will increase with the increasing of salinity and turbidity. The number of taxa
present in the Sekambal River estuary was influenced by salinity and turbidity. The changes of salinity
are regularly considered a stress source, especially for freshwater organisms (Kokkinn & Williams,
1988). Hence, the reduction in species diversity might due to the gradients stressful environment
(Therriault & Kolasa, 2000).
Table 4: Pearson Correlation between physico-chemical parameters of the water with
and number of macrobenthos taxa.
Parameters pH
Salinity
Temperature Turbidity
Dissolved
(PSU)
(◦C)
(NTU)
Oxygen
(mg/l)
Density
-0.487
-0.791*
0.220
-0.323
-0.282
3
(ind/m )
No. Taxa
0.144
0.658*
0.112
0.600*
-0.466
(*) strong correlation at 0.05 level
total density
Conductivity
(mS)
-0.413
0.322
4. CONCLUSION
Based on the data analysis, the range of total density of macrobenthos community was 235.87 2
610.48 ind./m . In addition, a total number of 8 taxa of macrobenthos were recorded in Sekambal
River. Pearson correlation showed that total density was influenced by salinity while the number of
taxa was influenced by salinity and turbidity. The preliminary data gathered from this study will
contribute for the future biological monitoring in Sekambal River.
Acknowledgement
We would like to thanks Universiti Malaysia Sarawak for providing the facilities and logistic supports
during this study. Besides that, we would also like to thanks Ministry of Higher Education for providing
fund for this study through Fundamental Research Grant Scheme (FRGS /07(03)/786/2010(67) and
Mr Zaidi Ibrahim and Mr Nor Azlan Bujang Belly for their help during field sampling.
References
Adrian B. Jones (1999). Environmental of Aquaculture Effluents. Development of Biological Indicator
& Biological Filters. B. Sc (Hons) thesis. University of Queenland, Australia. Pp 31-35.
Anbuchezhian, R.M., Rameshkumar, G., Ravichandran, S. (2009). Macrobenthic composition and
Diversity in the Coastal Belt of Thondi, Southeast Coast of India. Global Journal of
environmental Research. (3) pp 68-69.
Armitage, P.D., Moss, D., Wright, J.F., Furse, M.T., (1983). The performance of a new biological water
quality score based on macroinvertebrates over a wide range of unpolluted running
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sites.
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Azrina, M.Z., Yap, C.K., Ismail, A.R. & Tan, S.G. (2005). Anthropogenic impacts on the distribution
and biodiversity of benthic macroinvertebrates and water quality of the Langat
River,
Peninsular Malaysia. Journal Application Environmental Management. pp2-5.
Cuomo, C. & Zinn, G.A. (1995). Benthic Invertebrates of the Lower West River. Interdisciplinary
Restoration. pp 152-161.
Kokkinn, M. J. & W. D. Williams, (1988). Adaptations to life in a hypersaline water body adaptations at
the egg and early embryonic stage of Tanytarsus barbitarsis freeman (Diptera:
Chironomidae). Aquatic Insects (10). pp 205–214.
Mahapatro, D., Panigraphy, R. C., Nalk, S., Pati, S. K. & Samal, R. N. (2011). Macrobenthos shelf
zone off Dhamara estuary, bay of Bengal. Journal of Oceanography and Marine Science.
2(2) : pp 32-42.
Mason, C.F. & Parr, B.L. (2003). Long-term trends in water quality and their impact on
macroinvertebrate assemblages in eutrophic lowland rivers. Water Research. (37) pp 2969
2979.
Morris, W.K. & Madden, M.P. (1978). Benthic Macroinvertebrate Communities Aid Water
Quality Evaluation of the Washita River. Acade Science. (58) pp 93-97.
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Nuttle, W. K.& Fourqurean, J. W. (2000). Influence of Net freshwater Supply on Salinity in Florida
Bay. Water Resources Research. 36 (7). pp 1805-1822.
Rak, A.E., Said, I., Mohamed, M. & Abas, A. (2011). Effect of Logging Activities on Water
Quality and
Benthic macroinvertebrate Assemblages of Madek River Basin, Kluang, Johor,
Malaysia.
Journal Application Environmental Management. (15). pp 337- 340.
Roosmalen, J. A. M., Huijsmans, J.P.P. & Plomp, L. (1993). Electrical conductivity in
δa1−xSrxεnO3+ . Solids State Ionic. (66). pp 279-284.
Savage, C., Elmgren, R. & Larsson, U. (2002). Effects of Sewage-derived Nutrients on an estuarine
macrobenthic community. Marine Ecology Progress Series. (243). pp 67-82.
Silberbush, A., Blaustein, L. & Margalith, Y. (2005). Influence of salinity concentration on aquatic
insect community a mesocosm experiment in the Dead Sea basin Region. Hydrobiologia.
(548). pp1-10.
Sharma, K.K. & Chowdhary, S. (2011). Macroinvetebrate assemblages as biological indicators of a
pollution in a Central Himilayan River, Tawi (J & K). International Journal of Biodiversity
Conservation 3 (5). pp 167-174.
Therriault, T. W. & J. Kolasa, (2000). Explicit links among physical stress, habitat heterogeneity and
biodiversity. Synthesising Ecology (89). pp387–391.
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DECOLOURIZATION OF SYNTHETIC DYES BY ENDOPHYTIC FUNGI ISOLATED FROM
Melastoma malabathricum
N. N. Sing*, A. A. S. A. Husaini, A. Zulkharnain and H. A. Roslan
Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia
Sarawak, 94300 Kota Samarahan, Sarawak
E-mail: melvintan5580@yahoo.com*
Abstract
Synthetic dyes are important chemical pollutants from textile and dyestuff industries. A total of 20
endophytic fungi isolated from Melastoma malabathricum (Senduduk) were examined for their ability
to decolourize three azo type dyes (Congo red, Orange G and Methyl red) and an anthraquinone type
dye (Remazol Brilliant Blue R). Initial screening on glucose minimal media agar plates amended with
200 mg/L of each respective dyes showed that only isolate MS8 was able to decolourize all the four
tested dyes. Both RBBR and Orange G dye in the agar medium were completely decolourized by
isolate MS8 in 8 days. Further quantification of dye decolourization by isolate MS8 in aqueous minimal
medium showed that isolate MS8 was able to decolourize all the tested dyes to a different extent. Dye
decolourization by isolate MS8 reached 97% for RBBR, 33% for Orange G, 48% for Congo red and
56% for Methyl red respectively, within a period of 16 days. Molecular identification of fungal isolate
MS8 using primer ITS1 and ITS4 showed that isolate MS8 shared 99% sequence similarity with
Basidiomycetes, Marasmius cladophyllus. The ability to decolourize different dyes by isolate MS8 thus
suggested a possible application of this fungus in the decolourization of dyestuff effluents.
Keywords: Decolourization, azo dyes, anthraquinone dye, endophytic fungi, Marasmius cladophyllus.
1. INTRODUCTION
5
There are more than 100,000 synthetic dyes exist commercially and over 7 × 10 tons of dyes are
produced annually worldwide (Zollinger 1991). Dyes are used throughout the world in textile, paper,
cosmetic, pharmaceutical and even in food industries. Dyes are also used as additives in petroleum
products (Husain, 2010). Synthetic dyes are extensively used particularly by the textile and dyeing
industries. In the process of dyeing, about 15-20% of the dyes used for dyeing does not bind to the
fibers and are lost in the effluent, thus causing them to be highly coloured (Husain, 2010). It has been
estimated that about 280,000 tons of textile dyes are discharged in such industrial effluents every year
worldwide (Maas & Chaudhari, 2005).
Among industrial effluents, wastewater from textile and dyestuff industries is one of the most difficult to
be treated. This is because dyes usually have synthetic and complex aromatic molecular structures
which make them more stable and difficult to degrade (Padmesh et al., 2005). Conventional
wastewater treatment plants using activated sludge treatment are unable to treat dye containing
wastewater. Up to 90% of reactive textile dyes still persist after the treatment (Pierce, 1994). Several
physical and chemical methods including membrane filtration, adsorption, ion exchange, ozonation,
flocculation-coagulation and oxidation have been used to treat dye containing wastewater. However,
due to high cost involved, disposal problems and less adaptable to a wide range of dye wastewater,
most of these methods have not been widely applied (Banat et al., 1996; Stolz, 2001). Bioremediation
of dye containing effluents using effective dye degrading microorganisms therefore is still seen as an
attractive solution due to its reputation as a low-cost, environmentally friendly and publicly acceptable
treatment technology (Banat et al., 1996).
White rot fungi have been shown to be able to degrade a wide range of organic pollutants including
synthetic dyes (Asgher et al., 2008; Erkurt et al., 2007; Kennes & Lema, 1994; Yang et al., 2009). In
recent years, many studies have focussed mostly on the use of white rot fungi to decolourize synthetic
dyes due to its ability to produce nonspecific, ligninolytic enzymes (Eichlerova et al., 2006; Lu et al.,
2007; Yang et al., 2009). Dye decolourization particularly by the white rot fungus Phanerochaete
chrysosporium has been intensively studied and the degradation pathway for sulfonated azo dye by
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this isolate has also been elucidated (Asgher et al., 2006; Goszczynski et al., 1994). There is no report
however on the use of endophytic fungi for the decolourization of dye. Most studies on endophytic
fungi have focussed on studying its relationship with its host, its diversity and also to isolate bioactive
compounds of medical value from these fungi (Debbab et al., 2011; Schulz & Boyle, 2005;
Thongsandee et al., 2012).
The main aim of our present work was therefore to examine the possible application of little studied
endophytic fungi isolated from Melastoma malabathricum to decolourize several synthetic dyes
belonging to two major dye group widely applied in the dyeing industries. All endophytes were
screened on agar medium for dye decolourization to select the best isolate for further identification
and dye decolourization studies in liquid medium.
2. MATERIALS AND METHODS
2.1 Isolation of endophytic fungi
The endophytic fungi used in this study were isolated from the stem of a healthy flowering plant M.
malabathricum. Plant samples were collected from the campus of University Malaysia Sarawak
(UNIMAS) and processed within a few hours after sample collection. The plant samples were properly
washed under running tap water to remove possible debris and air dried before cutted into 5 cm
pieces. In order to eliminate epiphytic microorganisms, all samples were surface sterilized (Schulz et
al. 1993; Thongsandee et al., 2012). The samples were immersed in 5% Clorox for 5 minutes,
followed by 70% (v/v) ethanol and rinsed twice with sterilized distilled water. The surface sterilized
samples were blot dry with sterile filter paper and then aseptically cutted into 2 cm pieces. The pieces
of stems were then transferred aseptically onto potato dextrose agar (PDA) (Merck, Germany) plates
(3 pieces per Petri plate) and incubated at room temperature for a period of 2 weeks. The plates were
observed daily and individual hyphal tips of developing fungal colonies were subcultured. Fungal
isolates were distinguished based on its colony morphology.
2.2 Dye decolourization on agar plate
The isolated endophytes were first screened for their ability to decolourize an anthraquinone dye
(Remazol Brilliant Blue R, RBBR) and 3 azo dyes (Orange G, Congo red and Methyl red) on glucose
minimal (GM) agar plates. The GM agar medium used contained (per liter): 1 g of K 2HPO4, 10 mg of
ZnSO4.7H2O, 5 mg of CuSO4.5H2O, 0.5 g of MgSO4.7H2O, 10 mg of FeSO4.7H2O, 0.5 g of KCl, 10 g
of glucose, 3 g of NaNO3 as the sole source of nitrogen and 20 g of agar. The pH of the agar medium
0
was adjusted to 5.5 before autoclaving at 121 C for 15 minutes. Dyes were added into the agar from a
stock solution to a final concentration of 200 mg/L. The agar plates were inoculated with a 5 mm agar
plug from a 7 days old fungal culture and incubated in the dark at room temperature. Plates with the
respective dye and no fungus inoculated were used as control. Each isolate was prepared in
duplicates. Plates were regularly monitored for visual disappearance of colour for a period of 16 days.
2.3 Dye decolourization in liquid medium
Based on the results obtained, the best dye decolourizing isolate was selected for further dye
decolourization in liquid medium. The same GM medium was used (excluding agar). Dyes were added
to the 20 ml GM liquid medium in 100 ml Erlenmeyer flask to a final concentration of 200 mg/L. Each
flask was inoculated with 2 pieces of 5 mm agar plugs from a 7 days old fungal culture and incubated
in the dark at room temperature under static condition. Flask with the respective dye and no fungal
inoculum was used as abiotic control. Each culture condition was prepared in triplícate, incubated for a
period of 16 days and sampled at a 4 days interval. During sampling, the whole cultures were
harvested and centrifuged at 6000 rpm for 10 minutes to separate the fungal mycelium from the
culture medium. Fungal biomass was determined by drying the fungal mycelium to a constant weight
0
at 70 C. Dye decolourization by the isolated fungus was measured by monitoring the absorbance of
each dye in the culture medium at its respective maximum absorption wavelength (595 nm for RBBR,
475 nm for Orange G, 497 nm for Congo red and 520 nm for Methyl red) using a UV-vis
spectrophotometer (Libra S12, Biochrom). Percentage decolourization was calculated according to the
following formula:
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Percentage decolourization (%) =
Ac As
100
Ac
Where Ac is the absorbance at the maximum absorption wavelength of dye in the abiotic control flask
at time t and As is the absorbance at the maximum absorption wavelength of dye in the sample flask at
time t (Ozsoy et al., 2005).
2.4 Fungal characterisation ad identification
The selected fungal isolate was identified by morphological characteristic as well as comparison of
internal transcribed spacer (ITS) sequences. The morphological appearances of the selected fungal
isolate was characterized by visual observation and by micro-morphological techniques based on
mycelium colours, growth patterns and structure of fruiting bodies. Genomic DNA of the selected
fungus was extracted according to the method of Cubero et al., (1999). Extracted fungal DNA was
then PCR amplified using a universal primer pair ITS1 and ITS4 under the following condition: initial
0
0
0
denaturation at 95 C for 5 minutes, 30 cycles of denaturation at 95 C for 1 minute, annealing at 55 C
0
0
for 1 min, extension at 72 C for 1 min and final extension at 72 C for 7 minutes. PCR products were
then purified and sequenced. Closely related sequences of the isolates were retrieved from the NCBI
GenBank database. A neighbour-joining tree (Saitou & Nei, 1987) was constructed and the distances
between sequences were calculated from the models of Jukes and Cantor (1969). Bootstrap analysis
was performed with 1000 replications to assess the confidence limits of the branching (Felsenstein,
1985).
3. RESULTS AND DISCUSSION
3.1 Isolation of endophytic fungi
A total of 20 endophytic fungi were successfully isolated from the stem of M. malabathricum. These
fungi were named according to the source from which they were isolated and followed by a number
(MS1-20 with M=Melastoma and S=Senduduk).
3.2 Dye decolourization on agar plate
Dye decolourization activity of the isolated endophytic fungi was screened using an agar plate method
with decolourization observed by the production of halo. All of the tested fungi were able to grow on
the dye containing minimal agar medium and 14 of the fungal isolates were able to decolourize at
least one of the dye (Table 1). Among the 20 tested fungi, 13 isolates were able to decolourize RBBR,
2 isolates decolourized Orange G, 13 isolates decolourized Congo red and 9 isolates decolourized
Methyl red. Only 1 isolate (MS8) was able to decolourize all the 4 different dye tested. Dye
decolourization by isolate MS8 was also the most rapid among all of the 20 fungal isolates. Both
RBBR and Orange G dye in the agar medium started to decolourize within 2 to 3 days after fungal
inoculation and were completely decolourized by isolate MS8 within a period of 8 days with no visible
traces of dye adsorption onto the fungal mycelium. Congo red and Methyl red were also partially
decolourized by isolate MS8 with the production of halo and no observable dye was adsorbed by the
fungal mycelium. Besides MS8, isolates MS4 and MS17 were also able to completely decolourized
RBBR by day 14 and Orange G by day 12 respectively with no dye adsorption to the fungal mycelium.
3.3 Dye decolourization in liquid medium
Isolate MS8 was selected for further dye decolourization in glucose minimal liquid medium due to its
ability to decolourize all the 4 different dyes on agar plate. The results obtained shows that isolate
MS8 was able to decolourize all the 4 different dyes tested to a different extent (Fig. 1). Among the 4
different dyes tested, RBBR was decolourized the fastest and to the greatest extent by isolate MS8. In
just 4 days, up to 68.4% of RBBR was completely decolourized by isolate MS8. Dye decolourization
continued to increase to 96.5 % on day 8 and further increases to 97.4% on day 16. The 3 azo dyes
were also decolourized by isolate MS8 with Methyl red being the highest, reaching 56%
decolourization by day 16. This is then followed by Congo red with 48.1% decolourization and Orange
G with 32.6% decolourization. Among the two different dye group tested, the anthraquinonic dye
RBBR was decolourized to a greater extent as compared to the 3 azo group dyes. Decolourization of
the monoazo dye Methyl red was also greater as compared to the decolourization of both the diazo
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dyes Congo red and Orange G. This shows that dyes belonging to chemically different groups are not
decolourized to the same extent and the structural differences in the dye molecule strongly affect the
decolourization process (Eichlerova et al., 2006).
Table 1 Decolourization of RBBR, Orange G, Congo red and Methyl red on agar plate by the 20
isolated endophytic fungi after 16 days
Fungal Isolates
Dye decolourization
RBBR
Orange G
Congo Red
Methyl Red
MS1
MS2
MS3
MS4
++
+
+
MS5
+
MS6
+
+
+
MS7
MS8
++
++
+
+
MS9
MS10
+
+
+
MS11
+
+
+
MS12
MS13
+
+
+
MS14
+
+
+
MS15
+
+
MS16
+
+
MS17
++
+
MS18
+
+
+
MS19
+
+
MS20
+
+
+
(-): No dye decolourization; (+): partial dye decolourization with plate showing colour fainting/ halo
production but yet with undecolourized zone after 16 days; (++): complete dye decolourization wtih
total colour disappearance without adsorption to the fungal mycelium.
8.0
Fungal Biomass (mg/mL)
100
7.0
80
6.0
5.0
60
4.0
3.0
40
2.0
20
1.0
0.0
Percentage of Dye Decolourization (%)
120
9.0
RBBR
Orange G
Congo Red
Methyl Red
No Dye
RBBR
Orange G
Congo Red
Methyl Red
0
0
4
8
Days
12
16
Figure 1 Dye decolourization and fungal biomass produced by isolate MS8 in glucose minimal liquid
medium within a period of 16 days.
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Decolourization of Orange G by isolate MS8 in liquid medium however was surprisingly lower than the
extent of Orange G decolourization achieved by the isolate on agar plate. A possible reason for this
would be that the presence of dye in liquid medium was much toxic towards the isolate as compared
to the toxicity of dye on agar medium. Further quantification of the biomass produced by isolate MS8
with the presence of the tested dye in liquid medium shows that the biomass produced was greatly
reduced as compared to the control biomass of the isolate produced in the absence of the dye (Figure
1).
3.4 Fungal characterisation ad identification
Isolate MS8 was selected for further identification based on its morphological characteristic and also
by comparison of its ITS sequences. Isolate MS8 is a fast growing fungus with mycelium that covers
the whole 90 mm Petri dish in 7 days (Fig 2a). Aerial mycelium was constantly white, flat and velvety
with no pigment production. The fungal isolates, when observed microscopically, showed the
presence of clamp connection (Fig 2b), the characteristic feature of basidiomycetous fungi (Diwaniyan
et al., 2010). The isolate however did not produce any spore like structures, which normally provide
the basis for fungal identification.PCR amplification of the ITS region of isolate MS8 using a universal
primer pair, ITS1 and ITS4, resulted in a PCR product with an approximate size of 620 bp.
Comparison of the ITS sequence with published fungal ITS sequences in the GenBank through a
standard nucleotide-nucleotide BLAST homology search shows that isolate MS8 shared 99%
sequence similarity with a Basidiomycetes, Marasmius cladophyllus. A phylogenetic tree was also
constructed to further study the phylogenetic relationship of isolate MS8 with other Marasmius species
using M. epiphyllus as the outgroup (Fig 3). The short branches and clustering of isolate MS8 together
with M. cladophyllus shows that isolate MS8 was phylogenetically very closely related to M.
cladophyllus. This is also supported by a very high bootstrap value of 100. Isolate MS8 was also
distantly related with M. rotula.
As a whole, endophytic fungus MS8 identified to be M. cladophyllus, was able to decolourize all the 4
different dyes tested especially anthraquinonic dye which was known to resist degradation due to their
fused aromatic structures (Fu & Viraraghavan, 2001). The dye decolourization ability of this isolate
was also comparable to that of white rot fungi such as I. lacteus and Thelephora sp.(Novotny et al.,
2001; Selvam et al., 2003).
(a)
(b)
Figure 2 Endophytic fungal isolate MS8. (a) Fungal colony cultured on malt extract agar for 7 days (b)
microscopic view of the isolate showing fungal hyphae with the production of a clamp connection
(arrow).
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Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
MS8
100
94 Marasmius cladophyllus (AY216475.1)
Marasmius cladophyllus (HQ248212.1)
Marasmius rotula (JN714927.1)
92
96
Marasmius oreades (EF187911.1)
100
Marasmius siccus (FJ904987.1)
Marasmius haematocephalus (EU935532.1)
Marasmius epiphyllus (JN943602.1)
0.05
Figure 3 Neighbour-joining tree from ITS sequences showing the relationship between isolate MS8
and other closely related Marasmius species retrieved from the GenBank (accession number).
Bootstrap value >70% (1000 replicates) are shown at the branches. Bar = 5 nucleotide substitutions
per 100 nucleotides.
4. CONCLUSION
Endophytic fungi isolated from the plant M. malabathricum particularly isolate MS8 were able to
decolourize both the anthraquinone and azo type synthetic dye to a different extent. This therefore
shows that besides white rot fungi, endophytic fungi were also capable of dye decolourization and
have an interesting potential to be used in the decolourization of dyestuff effluents.
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RELATIONSHIPS AMONG SEA URCHIN Diadema setosum BASED ON 16S rRNA GENE
ANALYSIS
Nursyuhaida Md Shahid* and Ruhana Hassan
Aquatic Science Department, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
Email: ms.syuhaida@gmail.com
Abstract
Study on marine invertebrates with long lived planktonic larvae have been particularly challenging due
to high dispersal abilities of the larvae and complexity on finding the barriers to gene flow in the wide
ocean which in this case. The phylogenetic relationships of a cosmopolitan species could generate
knowledge on the mode of their formation in the sea. In this study, the relationships among 30
samples of D.setosum from various locations in Sabah, have been determined using 16S rRNA gene
analysis. The sea urchin samples were collected from Mantanani Island, Kudat, Lahad Datu and
Larapan Tengah located in three different seas namely South China Sea, Sulu Sea and Celebes Sea.
Several samples from Korea and Hawaii were also used for genetic comparison purpose. Based on
this study, it is more likely that 16S rRNA gene information of approximately 600bp could not resolved
phylogenetic relationships between the populations, although various methods of phylogenetic trees
reconstruction have been applied to the data set. Due to small genetic distances between samples
involved in this study, Network analysis have been carried out to represent the potential evolutionary
path of D.setosum in Sabah waters.
Keywords: Diadema setosum, 16S rRNA gene, Phylogeny, Network analysis
** Please contact the corresponding autor for further details of this paper.
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EFFECT OF PH ON THE EFFICACY OF POTENTIAL ANTAGONISTS AGAINST PATHOGENIC
GANODERMA IN OIL PALM
Hasma bt Mat Nor*, Sepiah Muid
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak.
*Email: hasma_mn@yahoo.com
Abstract
The studies on biocontrol agent against Ganoderma pathogen in the oil palm have been carried out
progressively in Malaysia and many other countries. Several non pathogenic microbes are showed
can inhibit the growth of Ganoderma sp effectively in laboratory condition but, when those antagonists
were applied in the field condition, often it does not prove satisfactory results. The objective of this
study is to investigate the effect of pH on the efficacy of potential antagonists against Ganoderma sp.
Two Penicillium species, Penicillium citrinum and Penicillium pinophilum that were obtained from
Mycology Laboratory, FSTS were used in this study. Both species have been proved in a laboratory
observation have antagonistic effect against Ganoderma sp. (G3) that was isolated from basal trunk
disease of oil palm. Each potential antagonist was cultured in Potato Dextrose Broth (PDB) of different
pH (pH 2, pH 3, pH 4, pH 5, pH 6, pH 7, pH 8). After 7 days, the mycelia of each culture was filtered
then dried at 55 0C until constant dry weight was obtained. P. citrinum had the highest dry weight at
pH 5 while P. pinophilum at pH 6 which implies their best growth at that pH. The effect of pH on
antagonistic ability of the Penicillium against Ganoderma sp. was also studied. The fungi were grown
in the same plate containing PDA of tested pH. The radial growth of Ganoderma sp. was measured
after 7 days of incubation period. The highest percentage of reduction growth rate of Ganoderma sp.
was at pH 5(86.90%) and pH 6(86.27%). This study revealed that P. citrianum and P. pinophilum
available to control pathogenic Ganoderma under moderately acidic condition.
Keywords: Biocontrol agent, pathogenic Ganoderma, potential antagonists, pH, antagonistic effect.
** Please contact the corresponding autor for further details of this paper.
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ETHNOMEDICINAL PLANTS INDICATED FOR MANAGEMENT OF MALARIA & ITS ASSOCIATED
SYMPTOMS USED BY INDIGENOUS PEOPLE OF MALAYSIA
Ida Farah A*, Maryati Mohamad
Faculty of Science, Technology and Human Development, Universiti Tun Hussein Onn Malaysia, Batu
Pahat, Johor, Malaysia
*Email: ifahmad@yahoo.com
Abstract
Traditional medicine has been practiced for generations by the knowledge and experiences of elders
in the community. Inadequate documentation of its safety and efficacy has little effect as confidence in
traditional healers is greater. Malaria is a primarily rural problem in Malaysia addressed with
applications of traditional concoctions. Malarial symptoms are presented by high fever, dizziness,
fatigue, nausea, vomiting, muscle and joint aches and chills. Symptoms appear between 8 to 15 days
after infection by the mosquito bite. The non-Malay indigenous groups in Peninsular Malaysia, called
the Orang Asli are represented by 3 main tribal groups: Semang (Negrito), Senoi and Proto Malay; all
of which consist of 19 subgroups. Alongside other ethnic groups in Malaysia with their own unique
traditional practices, the Orang Asli’s connection to the use of their natural surrounding holds an
important role in conserving natural resources, and ultimately the precious knowledge. These
elements of traditional knowledge preservation perceived as national treasure, and potential discovery
of new or improved medicines from nature indicate the need for establishing a compilation of the
information. Through a survey interviewing practitioners and plant collection for systematic extraction
and bioactivity testing, the traditional knowledge will now have a form of scientific basis for further
investigation into potential drug discovery.
Keywords: Traditional knowledge, malaria, ethnomedicine, Orang Asli, medicinal plants
** Please contact the corresponding autor for further details of this paper.
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CHALCONES ISOLATED FROM Mitrella kentii
Ainnul Hamidah Syahadah Azizan* and A. Hamid A. Hadi
Department of Chemistry, University of Malaya, 50603 Kuala Lumpur
Email*: ainnul_azizan@yahoo.com
Abstract
Mitrella kentii is an annonaceous species of climber growing in difference area such as Malaysia
Peninsula, Sumatera, Borneo and New Guinea. The previous study on Mitrella kentii showed that this
species content difference isoquinoline alkaloids. In a continuation to our search for chemical and
biological activity of Malaysian flora, we further investigate the chemical and biological activity of other
compounds isolated from this species. This study led to isolation of chalcone derivative,
Desmosdumotin C 1, dihydrochalocone, (-)-Neolinderatin 2 and (-)-Linderatine 3. The structural
elucidation of these compounds was performed using spectroscopic techniques such as UV, IR, MS,
1D and 2D NMR.
** Please contact the corresponding autor for further details of this paper.
_________________________________________________________________________________
PHYTOCHEMICAL AND BIOLOGICAL STUDIES ON PSEUDUVARIA SPECIES FROM MALAYSIA
a*
a
a
Hairin Taha , Noraziah Nordin , A.Hamid A.Hadi , Mustafa AM
a
b
b
Department of Chemistry, Faculty of Science, University of Malaya, Department of Pharmacology,
Faculty of Medicine, University of Malaya.
Email: hairintaha167@gmail.com
Abstract
Several parts of three Pseuduvaria species from the Annonaceae family were phytochemically studied
and structurally elucidated by means of spectral analysis: Pseuduvaria rugosa, Pseuduvaria
macrophylla and Pseuduvaria monticola. The crude extracts (hexane, ethyl acetate and methanol)
yielded
oxoaporphine
and
dioxoaporphine
alkaloids
including
liriodenine,
ouregidione,
N-
methylouregidione, Pseuduvarine A and Pseuduvarine B. One derivative of tocopherol, polycerasoidol
was also isolated. Preliminary screening of the crude extracts showed promising anticancer activity
and inhibitory effects on platelet- activating factor (PAF).
** Please contact the corresponding autor for further details of this paper.
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ANTIMICROBIAL ACTIVITY AND MINIMUM INHIBITORY CONCENTRATION (MIC) OF A.
angustifolia, h. diversifolia and c. xanthorrhiza LEAVES EXTRACTS
1
1
1
2
Nurhidayah A.R. , Fatimah M.A , Asyikin O. , Salmah I , Mahmood A.A.
1
1
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, MALAYSIA
2
Institute of Biological Science, Faculty of Science, University of Malaya, MALAYSIA
E-mail: ammeen@um.edu.my
Abstract
Medicinal plants have been recognised as alternative medicines. Antimicrobial activity of three
medicinal plant extracts were investigated in vitro against nine bacterial strains (Bacillus cereus,
Staphylococcus aureus, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus
(MRSA), Escherichia coli, Pastereulla multocida, Klebsiella sp., Pseudomonas aeruginosa and
Enterobacter cloacae) using disk diffusion agar in four different concentration (50, 100, 150 and 200
mg/ml). In addition, the minimum inhibitory concentration of each extracts was determined by broth
microdilution technique. The leaves extracts of H. diversifolia and C. xanthorrhiza were inhibited the
growth of Bacillus cereus and Escherichia coli with the average of inhibition zone are 9 mm and 8 mm
respectively. The inhibition zone was shown by MRSA is ranging from 8 mm to 14 mm which are
inhibited by both A. angustifolia and H. diversifolia whereas the Staphylococcus aureus and
Staphylococcus epidermidis was only inhibited by A. angustifolia. The others bacteria (Pastereulla
multocida, Klebsiella sp., Pseudomonas aeruginosa and Enterobacter cloacae) were resistant to all
leaves extracts. The lowest minimum inhibitory concentration (MIC) was 25 mg/ml of H. diversifolia
leaves extracts against the growth of Escherichia coli and MRSA. The MIC value range from 25 mg/ml
to 200 mg/ml for all leaves extracts. The studied plant extracts have exhibit the potential properties for
preventing the bacterial growth without dependence on modern medicine. Therefore, this research
may valuable in food industry as natural preservatives, cosmetic field and medicinal therapy against
certain disease.
Keywords: Medicinal plants, antimicrobial, disk diffusion assay agar, Minimum Inhibitory Concentration
(MIC), broth microdilution, inhibition zone.
1. INTRODUCTION
Alstonia angustifolia or Pokok Pulai usually found in the Peninsular Malaysia, Singapore, Sumatra,
Bangka and Borneo. Prominent features of this plant are smooth outer bark, yellowish inner bark, no
latex, whorls leaves, up to 45 metres tall in size and small or steep buttresses with a diameter up to 70
cm (Teo, 2001). It is rich in biologically active alkaloids that are responsible as anti-amoebic agents to
treat malaria disease, which caused by Plasmodium falciparum. It also shows cytotoxic activity to
inhibit several human cancer cell lines and assist in fibrinolysis process by inhibition of enzyme known
as carboxypeptidase U (CPU) (Shin-Jowl et. al., 2010). In Malays culture, they treat remittent fever by
rubbing the A. angustifolia’s leaves on the spleen areas.
Curcuma xanthorrhhiza or Temulawak are commonly distributed in Java, Peninsular Malaysia, the
Philippines, Thailand and India. This plant grow very well in humus-rich and moist soils with the
orange-red branched rhizome, elliptical leaves shape and size up to 75cm long. The rhizome of C.
xanthorrhiza can be taken orally to treat fever and constipation as well as skin disease. In Indonesia,
this plant is really significant for women as a galactagogue and to relief uterine inflammation especially
after giving birth (Wardini et. al., 1999).
Hoya diversifolia is widespread in area of Indonesia, Malaysia, Thailand, Cambodian and Vietnam.
The fleshy leaves produce white latex which is rich in triterpenyl cinnamates components (Henri and
Bath, 1984). The tubular flowers with 5-lobed and tabular stigma appear as round or ball-like shaped
(Wiart, 2006). It is applied to relief the pain of rheumatism and as anti-nematodal agent against
Bursaphelenchus xylophilus (Alen et.al., 2000).
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Therefore, the aims of this study are to investigate the antimicrobial activity of leaves extracts of A.
angustifolia, C. xanthorrhiza and H. diversifolia on gram positive and gram negative bacteria which
may cause clinical significance diseases in humans. We also will determine the minimum inhibitory
concentration (MIC) of respective extracts towards susceptible bacterial strains.
2. MATERIALS AND METHODS
2.1 Plant specimen and preparation of extraction
Fresh A. angustifolia, H. diversifolia and C. xanthorriza leaves was obtained from Herbarium of Rimba
Ilmu, Institute of Science Biology, University of Malaya, Kuala Lumpur. The leaves was washed using
distilled water and also was dried in oven at temperature 50°C for five days. The dried leaves were
powdered using electrical blender. Hundred grams of the fine powder was soaked in 500 ml of 95%
ethanol in conical flask for 3 days. After 3 days the mixture was filtered using a fine muslin cloth
followed by filter paper (Whatman No. 1) and was distilled under reduced pressure in an Eyela rotary
evaporator (Sigma-Aldrich, USA). The dried extract was kept at -20°C in refrigerator.
2.2 Antimicrobial susceptibility testing using disk diffusion assay
The Kirby-Bauer disc diffusion assay was carried out based on recommendations given by the Clinical
Laboratory Standard Institute, CLSI (Hindler and Jorgensen, 2007; Jorgensen and Turnidge, 2007).
2.2.1 Bacterial culture
Nine different species of bacteria was used in this study which includes Escherichia coli ATCC 25922
reference strain, Staphylococcus aureus ATCC 25923 reference strains, Staphylococcus epidermidis,
Bacillus cereus, Pseudomonas aeruginosa, Klebsiella sp., Multi-methicillin Staphylococcus aureus
(MRSA), Pastereulla multocida and Enterococcus cloacae that kindly provided by Molecular
Bacteriology Laboratory, Department of Molecular Medicine, Faculty of Medicine, University of
Malaya. All bacterial was cultured and sub-cultured again for purity on Columbia Horse Blood Agar
plates (Biomedia Laboratories). Confirmation of the identity of working strains was determined using
colony morphology and Gram staining (R&M Chemicals, Essex, UK). The bacterial isolates were
maintained in Brain Heart Infusion (BHI) agar slants (BD, USA) at 4°C.
2.2.2 Preparation of disk
A stock solution of each extract was prepared by dissolving 200 mg of each extracts with 1 ml of
absolute ethanol (Hayman, UK) to produce final concentration of 200 mg/ml. The stock solution was
then diluted to concentration of 50, 100, 150 and 200 mg/ml of each extracts. 20 µl of each dilution
from respective extracts was impregnated into 6 mm in diameter sterile, blank discs. The discs were
prepared by punching Whatman No. 1 filter paper (Whatman, UK) with a punch hole and then was
autoclaved them. 5 µl of each extracts was spotted alternately on both sides of the disc and was
waited to dry before the next 5 µl spotted to ensure precise impregnation. Ethanol-loaded disc was
used as negative control. All discs were ensured to be fully dried before application on bacterial lawn.
The positive controls are vancomycin antibiotic discs (30 µg; BD, USA) for MRSA; Gentamicin
antibiotic disc (10 µg; Oxoid, UK) for B. cereus, S. aureus and S. epidermidis; Ampicillin antibiotic for
E.coli and Imipenem antibiotic for P.aeruginosa, Klebsiella sp., E. cloacae and P. multocida.
2.2.3 Preparation of bacterial inoculums
A single colony from overnight growth on blood agar was suspended in BHI broth and was incubated
until visibly turbid. Their turbidity was adjusted with spectrophotometer to within an absorbance range
of 0.08 to 0.13 at 625 nm with sterile broth, which equivalent to a 0.5 McFarland standard with a cell
count of approximately 1.5 x 108 CFU/ml (Jorgensen and Turnidge, 2007). The inoculums were used
within 15 minutes of standardization as delays may change the inoculums size.
2.2.4 Inoculation procedure
The standardized bacterial inoculums was spread over the entire surface of 90 mm Mueller Hinton
Agar (MHA; BD, USA) plates with sterile cotton swab three times, rotating the agar plate about 60
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degrees each time to ensure that the entire agar surface was covered with the inoculums. The
inoculated plates were left with the lids ajar to allow excess moisture to dry for several minutes. The
antibiotic disc was applied to the agar using sterile forceps and was pressed gently to ensure uniform
contact. The discs were placed 6 disc per plate (positive and negative control disc included)
equidistantly to avoid the overlapping of zones of inhibition. The plates were inverted and were be
incubated at 37°C overnight. A zone of inhibition was observed for visually the next day. If present, the
diameter was measured to the nearest whole millimetre with a ruler against a dark, non-reflective
background. The assay was carried out in triplicates and the mean diameter of zones was calculated.
2.3 Minimum Inhibitory Concentration
Plates showing zones of inhibition form the disc diffusion assay was further tested to determine MIC
values by broth macrodilution method as according to recommendations by CLSI (Hindler and
Jorgensen, 2007; Jorgensen and Turnidge, 2007).
2.3.1 Preparations of extract dilutions
For the ethanol extract, 400 mg of extract was dissolved in 1 ml of 0.5% carboxylmethyl cellulose
(CMC) rather than the original solvent (i.e. absolute ethanol) to obtain final concentration of 400
mg/ml. The stock solutions prepared above was serially diluted with sterile Mueller-Hinton broth (MHB;
Becton-Dickson, USA) to give concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78 and 0.39
mg/ml. The concentrations was prepared to a volume of 0.5 ml in separate microcentrifuge tubes
(Eppendorf) at double the intended concentration so that addition of equal volumes of bacterial
inoculums in later steps was result in the desired final concentration in each tube.
Inoculation procedure
Similar to the disc diffusion assay, bacteria inoculums were be prepared with Muller-Hinton broth and
8
standardized to a 0.5 McFarland standard (1.5 x 10 CFU/ml). The suspension was be diluted 1:100
6
with sterile broth to obtain a cell count of approximately 10 CFU/ml. 0.5 ml of standardized bacterial
suspension was added to the tubes containing the previously prepared 0.5 ml of diluted extracts,
5
resulting in a recommended final cell count of about 5 x 10 CFU/ml. A tube containing broth and
extract solvent (distilled water or 0.5% CMC) but without the plant extract was inoculated as the
positive control. A tube containing broth alone (uninoculated) and extract solvent was served as the
negative control. All tubes were incubated overnight at 37°C.
2.3.2 Determination of MIC values
The turbidity of the solution in each tube was observed on the next day for indication of bacterial
growth. To ensure the presence or absence of bacterial growth in the tubes, a loop of suspension in
each tube was inoculated on MHA and incubated overnight at 37°C. The plates were observed
following incubation to confirm growth or absence of bacteria. The lowest concentration of extract
dilution showing no visible growth was recorded as the MIC value. The tubes was further incubated
another 24 hours and observed after 48 hours incubation for the growth or absence of bacteria. The
MIC value after 48 hours was recorded.
3. RESULT
3.1 Antimicrobial susceptibility testing
3.1.1 Disk diffusion assay
The assay was carried out using both gram positive bacteria (Bacillus cereus, Staphylococcus aureus
ATCC 25923 reference strain, Staphylococcus epidermidis and Methicillin-resistant Staphylococcus
aureus (MRSA)) and gram negative bacteria (Pastereulla multocida, Pseudomonas aeruginosa,
Klebsiella sp., Escherichia coli ATCC 25922 reference strain and Enterococcus cloacae). From the
result of disc diffusion assay, all the tested extracts (A. angustifolia, C. xanthorrhiza and H. diversifolia)
are observed clearly possess antimicrobial properties against gram positive and negative bacteria, as
seen from the presence of zones of inhibition. The diameter of clear zones of inhibition were observed
and measured against a dark, non-reflective background. According to the interpretive standard for
disc diffusion susceptibility testing that are published by Clinical Laboratory Standards Institute (CSLI),
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all positive controls against each bacteria were fall within susceptible ranges. All negative control
showed no zones of inhibition. Fig. 1 to 4 illustrates the mean diameter of zones of inhibitions obtained
from ethanol extracts of A. angustifolia, C. xanthorrhiza and H. diversifolia respectively.
Figure 1: Mean diameter of zone of inhibitions given by ethanol extracts of A. angustifolia (As), H.
diversifolia (Hd) and C. xanthorrhiza (Cx) at concentration of 50 mg/ml.
Figure 2: Mean diameter of zone of inhibitions given by ethanol extracts of A. angustifolia (As), H.
diversifolia (Hd) and C. xanthorrhiza (Cx) at concentration of 100 mg/ml.
.
Figure 3: Mean diameter of zone of inhibitions given by ethanol extracts of A. angustifolia (As), H.
diversifolia (Hd) and C. xanthorrhiza (Cx) at concentration of 150 mg/ml.
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Figure 4: Mean diameter of zone of inhibitions given by ethanol extracts of A. angustifolia (As), H.
diversifolia (Hd) and C. xanthorrhiza (Cx) at concentration of 200 mg/ml.
The A. angustifolia leaves extract was found to show zones of inhibiton against E. coli ATCC, S.
aureus ATCC, S. epidermidis and MRSA at concentration 200, 150, 100 and 50 mg/ml, with the
ranges up to 9 mm. The leaves extract of H. diversifolia was shown highest zones of inhibition against
MRSA with mean diameter from 11.83 to 12.83 mm as well as against E. coli and B. cereus. For C.
xanthorrhiza, the extract have potential to inhibit the growth of E. coli and B. cereus in which the mean
diameter of inhibition zones are up to 7.5 mm and up to 10 mm respectively. All extracts showed no
measurable zones of inhibitions against P.aeruginosa, Klebsiella sp., P. multocida and E. cloacae.
3.2 Minimum Inhibitory Concentration (MIC)
Table 1 Minimum Inhibitory Concentration (MIC) values of Alstonia angustifolia, Hoya diversifolia and
Curcuma xanthorrhiza against susceptible bacteria strains.
MIC Value (mg/ml)
Bacteria
24 hours
48 hours
A.
angustifolia
-
H.
diversifolia
100
C.
xanthorrhiza
200
A.
angustifolia
-
H.
diversifolia
100
C.
xanthorrhiza
200
E. coli
100
25
100
50
25
100
MRSA
100
25
-
0
25
-
S. aureus
50
-
-
100
-
-
S. epidermidis
100
-
-
100
-
-
B. cereus
The tested bacterial strains that shown clearly measurable zones of inhibitions to leaves extracts of A.
Angustifolia, C. xanthorrhiza and H. diversifolia were further tested to determine their MIC values. For
A. Angustifolia, the MIC values of E. coli, S. aureus, S. epidermidis and MRSA were studied. For both
C. xanthorrhiza and H. diversifolia, determination of MIC values was focusing on B. cereus and E. coli
as well as MRSA which is for H. diversifolia only. All MIC values obtained were presented in Table 1
and the comparison by plants type and incubation period in fig. 5 and 6. The lowest MIC value
recorded was 25 mg/ml of the ethanol extract of H. diversifolia against E. coli and MRSA at 24 and 48
hours incubation. Fig. 5 compare the MIC values between the three plants extracts for 24 hours
incubation. Majority of MIC values was fall at 100 mg/ml and the highest MIC value is 200 mg/ml that
shown by B. cereus to C. xanthorrhiza leaves extracts.
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Figure 5 Effect of A. angustifolia (As), H. diversifolia (Hd) and C. xanthorrhiza (Cx) after 24 hours
incubation by determination of MIC value.
Figure 6 Effect of A. angustifolia (As), H. diversifolia (Hd) and C. xanthorrhiza (Cx) after 48 hours
incubation by determination of MIC value.
4. DISCUSSION
Recently, the people not only focusing on the modern medicine as a main medication to treat certain
diseases, but they start to find out the others alternatives which are more effective and safe by using
traditional medicine called herbs. Herbs can be obtained from any plant parts such as leaves, seeds
and flowers (Mckean and Erin, 2005). Besides use in medical treatment, these herbs also apply in
food manufacturing and preservation, pharmaceuticals, cosmetic and homeopathy (Bishnu et. al.,
2009). Thus, this herbs or natural products are widely studied to understand and explore their
mechanism as functional agents so that, we can exploit our nature for beneficial purposes.
Scientist has identified several active compounds from the natural products that are responsible for
antimicrobial activity which include simple phenols and phenolic acids, quinines, flavones and
flavonoids as well as tannins groups. These entire active compounds were functions as protector
agent to fight with aggressor agents, especially microorganisms (Silva et. al., 2010). In contrast, the
leaves extract of A. marophylla which is identical species as A. angustifolia contain alkaloid picrinine,
picralstonine, O-benzoyl vincamajine and quebrachidine but the compounds that responsible for
antimicrobial activity are β-sitosterol, ursolic acid and β-sitosterol glucoside ( Asolkar et. al., 1992;
Chattopadhyay et. al., 2001). The active compounds in species of Curcuma that responsible to the
antimicrobial properties are curcuminoids, which can be fractionated into different components
includes curcumin, demethoxycurcumin and bisdemethoxycurcumin. Curcumin are the most
significant component in biological activities (Shagufta et. al., 2012; Ong et. al., 2010). The ethanol
extraction produce huge active compound because of its polarity than the other solvents in which
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inhibition of bacterial pathogenicity in the ethanol extracts is extent to a greater degree than hexane
and ethyl acetate extracts (Ong et. al., 2010).
From the result of the disc diffusion screening, A. angustifolia, C. xanthorrhiza and H. diversifolia is
shown to clearly possess antimicrobial properties against E. coli, B. cereus, S. aureus, MRSA and S.
epidermidis, as seen from the presence of inhibition. As all plant extracts seem to give appreciable
antimicrobial activity against gram positive bacterial strains, this may indicate that the plant extract act
specifically against gram positive staphylococcal and Bacillus cell wall (Sudjana et. al., 2004). For
gram negative bacteria, only growth of E. coli are inhibited. The difference in the layer of cell wall gives
difference inhibition mechanisms. The peptidoglycan layer are much thicker in gram positive cell wall
but gram negative bacteria have extra resistance against antibiotics that cannot penetrate it, such as
antibiotics glycopeptides e.g. vancomycin. This extra resistance is referring to outer membranes that
compose lipopolysaccharides that prevent antibiotics to pass through it (Sheldon, 2007). The lack of
sensitivity of plants extracts towards inhibition of P. aeruginosa and Klebsiella sp. may due to their
protection mechanism such as inhibition of β-lactamase and bacterial topoisomerase and also removal
of toxic substances out of bacterial cell by process of efflux pump (Zuo et. al., 2008). The
effectiveness of A. angustifolia and H. diversifolia against MRSA strains used is important when one
consider the fact that most MRSA present with multiple resistance to various antibiotics.
For more accurate quantitative determination of antimicrobial strength of A. angustifolia, C.
xanthorrhiza and H. diversifolia, the MIC test was performed as compared to the disc diffusion assay.
A lower MIC value was shown the completely inhibition of bacterial growth requires the lower
concentration of extracts used or in others meaning is stronger antimicrobial strength. The lower MIC
values given by the ethanol extracts suggest that the ethanol extracts contain compound with stronger
antimicrobial strength. The patterns of inhibition for two incubation period are depending on the
efficacy of extracts. Some extracts shows similar efficacy throughout the time for example plant
extracts of H. diversifolia and C. xanthorrhiza. But plant extract of A. angustifolia indicate higher
efficacy on growth inhibition of E. coli in which the concentration of extracts was double decrease from
100 to 50 mg/ml. But efficacy of this plant extract is considered as lower when the concentration of
extract was double increase from 50 to 100 mg/ml for S. aureus. Although the concentration of crude
plants extracts are much higher than the antibiotic concentration (in µg/ml), these extracts still be
considered as having good potency (Arias et. al., 2004) and worthy of further investigation (Okusa et.
a.l, 2007).
The antimicrobial activity that are shown by plants extracts did not exceed the positive control are due
to impurity of extracts itself in which the diffusion of active compound are very poor. Thus, purification
of the active antimicrobial agents may increase it relative activity for example, give bigger zones of
inhibition or lower MIC values than obtained result (Pesewa et. al., 2008).
5. CONCLUSION
Based the result obtained, we make conclusion that all leaves extracts of medicinal plants that
involved in this study possess antimicrobial properties against tested bacterial strain especially gram
positive bacteria. The degree of sensitivity for each tested bacterial strain was varied due to
differences in tolerance mechanism of microorganisms. MIC test showed that H. diversifolia has
lowest MIC value than A. angustifolia and C. xanthorrhiza and more effective to inhibit the growth of
E.coli and MRSA. C. xanthorrhiza produce very least activities among tested leaves extracts.
ACKNOWLEDGEMENT
We are highly gratitude to the Molecular Bacteriology Laboratory, Department of Molecular Medicine,
University of Malaya for providing the facilities and specialities for this research work. This project was
supported by Postgraduate Research Fund (PPP) grant from University of Malaya (PV042/2011A).
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Arias M.F., Gomez J.D., Cudmani N.M., Vattuone M.A., & Isla M.I. (2004). Antibacterial activity of
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Ocimum sanctum, Cinnamomum zeylanicum, Xanthoxylum armatum and Origanum majorana.
Kathmandu University Journal of Science, Engineering and Technology, 5(1), 143-150.
Chattopadhyay D., Maiti K., Kundu A.P., Chakraborty M.S., Bhadra R., Mandal S.C., et al. (2001).
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Laticifers Of Hoya Diversifolia Bl. Plant Science Letters, 33, 81-91.
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Diagnostic Microbiology 3rd ed. (pp. 319-353). China: Saunders Elsevier.
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Manual of Clinical Microbiology, 9th ed. (pp. 1152-1172). Washington, DC: ASM Press.
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and antioxidant activity of Cordia gilletii De Wild (Boraginaceae). Journal of
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Ong-ard Lawhavinit, Ngampong Kongkathip, & Kongkathip, B. (2010). Antimicrobial Activity of
Curcuminoids from Curcuma longa L. on Pathogenic Bacteria of Shrimp and Chicken.
Kasetsart J. (Nat. Sci.), 44, 364-371.
Pesewu G.A., Cutler R.R., & D.P., H. (2008). Antibacterial activity of plants used in traditional
medicines of Ghana with particular reference to MRSA. Journal of Ethnopharmacology, 116,
102-111.
Shagufta Naz, Safia Jabeen, Saiqa Ilyas, Farkhanda Manzoor, Farah Aslam, & Ali, A. (2012).
Antibacterial Activity of Curcuma Longa Varieties Against Different Strains of Bacteria. Pak. J.
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(2010). Unusual indole alkaloid-pyrrole,-pyrone, and -carbamic acid adducts from Alstonia
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Backhuys Publisher.
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Zuo G.Y., Wang G.C., Zhao Y.B., Xu G.L., Hao X.Y., Han J., et al. (2008). Screening of Chinese
Medicinal plants for inhibition against clinical isolates of methicillin-resistant Staphylococcus
aureus (MRSA). Journal of Ethnopharmacology, 120, 297-290.
599
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
ANTIOXIDANT AND CYTOTOXICITY PROPERTIES OF CRUDE EXTRACT AND FRACTIONS OF
Persea declinata (BI.) KOSTERM
Putri Narrima Mohd Fauzi* and A. Hamid A. Hadi
SUCXeS, DEPARTMENT OF PHARMACOLOGY, FACULTY OF MEDICINE, UNIVERSITY OF
MALAYA, 50603 KUALA LUMPUR
Email: p_narrima@yahoo.com
Abstract
The crude extract and fractions of Persea declinata (BI.) Kosterm in the antioxidant assays proved that
the plant is as potent as compared to ascorbic acid and quercetin, in the DPPH and ORAC assay,
respectively. They were also monitored for prevention of oxidative stress in human normal hepatic
cells (HepG2) and evaluated for their cytotoxicity in the MTT assay using human breast carcinoma
cells (MCF-7), human non-small cell lung cancer cells (A549) and human normal hepatic WRL-68 cell
type. The dose responses in DPPH assay, with the positive control having an EC50 value of 3.408 ±
0.06 µg/mL, the crude extract showed EC50 value of 3.468 ± 0.54 µg/mL , whereas fraction F has the
lowest EC50 value (4.914 µg/mL), followed by C, B, A, then E and lastly D. In the ORAC assay, the
crude at a concentration of 100 and 20 g/mδ gave equivalent antioxidant activity to λ7.λ8 ± 5.6λ ε
and 68.6λ ± 0.22 ε of Trolox, respectively. Quercetin, on the other hand, at 5 µg/mL is equivalent to
59.99 ± 8.58 µM of Trolox. Fraction A showed the highest ORAC value of 241.4 ± 4.00 µM of Trolox,
followed by Fraction F, B, D, C and E, at 20 µg/mδ. As we further investigate the plant’s antioxidant
properties, we subjected the crude to an oxidative stress test using a Thermo Scientific Cellomics
ArrayScan® HCS Reader and Cellomics HCS Reagent Kit, and observed the decreased accumulation
of ethidium due to the presence of the crude extract and ascorbic acid which served as the positive
control for an antioxidant. In the cytotoxicity MTT assay, the crude showed it was toxic to all 3 cell
lines tested, being most toxic in MCF-7 cells (EC50 9.800 ± 2.21 µg/mL), however it was contradictory
for A, C and D fractions. Fraction A was most toxic to WRL-68 (EC50 33.17 ± 5.73 µg/mL), whereas
fraction B and D were most toxic to MCF-7 (EC50 31.05 ± 3.98 µg/mL) and A549 (EC50 38.55 ± 3.64
µg/mL) respectively.
** Please contact the corresponding autor for further details of this paper.
_________________________________________________________________________________
PHYTOCHEMICAL STUDIES OF CRYPTOCARYA INFECTORIA
Wan Nurul Nazneem Wan Othman, A.Hamid A.Hadi.
Chemistry Department, Faculty of Science, University of Malaya, 50603 Kuala Lumpur
Email: nazneem85@um.edu.my
Abstract
A phytochemical study on the bark of Cryptocarya infectoria, which belongs to the family of Lauraceae
afforded aporphine type of alkaloid such as Laurotetanine (1) and benzylisoquinoline type such as Nmethylisococlaurine (2) and Reticuline (3). The alkaloids were extracted from the bark by using nhexane, dichloromethane and methanol as solvents. The crude alkaloid was subjected to extensive
chromatography techniques. Structural elucidation was established through several spectroscopic
methods, such as 1D-NMR (1H, 13C, DEPT, NOE), 2D-NMR (COSY, NOESY, HMQC, HMBC), UV,
IR and MS (GCMS, LCMS and HRMS) and comparison with the published data.
Keywords: Lauraceae, Cryptocarya infectoria, aporphine, benzylisoquinoline.
** Please contact the corresponding autor for further details of this paper.
600
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
AERIAL FLOCKING VOCAL REPERTOIRE OF BLACK-NEST SWIFTLET (Aerodramus maximus)
1*
2
3
Lim Kiau Ceh , Lim Chan Koon , Hasnizam Abdul Wahid and Mustafa Abdul Rahman
1
1
2
Faculty of Resource Science and Technology, UNIMAS 94300 Kota Samarahan, Sarawak, 157
3
Lorong 4A, Off Jalan Stampin Timur, 93350 Kuching, Faculty of Applied and Creative Arts, UNIMAS,
94300 Kota Samarahan, Sarawak
Abstract
Black-nest Swiftlet (Aerodramus maximus) and White-nest Swiftlet (A. fuciphagus) are cave swiftlet
that produce edible bird’s nest. Playback of social vocalization had been widely used in the swiftlet
farming industry for the later, but no study had been conducted on the social vocalization of the
gregarious and highly vocal Black-nest Swiftlet. Our study focused on characterization and
classification of the vocalization of this species during aerial flocking, in order to produce its vocal
repertoire. Sound recordings of wild population were carried out from April 2010 to October 2011 at
two sites; at Bukit Sarang, Bintulu, and Bau, Kuching. A total of 551 calls were extracted from 46.5
hours of field recording. Sonagram characterization and analysis was done using Avisoft 5.0 software.
Classification of these calls is based on the general shape and number of elements in each call, and
the total call duration. Eight different vocal types from three categories, namely Trill Call, Rattle Call
and Chirp Call, were identified. The frequency range for each category is between 2.1-8.3kHz, 2.610.4kHz and 1.3-7.3kHz respectively. This systematic characterization of the Black-nest Swiftlet vocal
repertoire established the foundation for subsequent work to elucidate the behavior of this species by
linking each vocalization to the aerial display and flocking behavior. The ultimate outcome of this study
is to understand the meaning and functions of each call against its acoustic properties.
** Please contact the corresponding autor for further details of this paper.
_________________________________________________________________________________
HABITAT PREFRENCES OF WADERS (SCOLOPACIDAE) IN SARAWAK
1
2
Nurul Ashikeen Ab Razak , Mustafa Abdul Rahman and Andrew Alek Tuen
1
1
Institute of Biodiversity and Environmental Conservation, UNIMAS 94300 Kota Samarahan, Sarawak
2
Department of Zoology, Faculty of Resource Science and Technology, UNIMAS 94300 Kota
Samarahan, Sarawak
Abstract
Scolopacidae is one of the most diverse families in order Charadriiformes. These species usually
migrate south during winter in the temperate zone. One of the places they stopover is Borneo Island.
An assessment on habitat preferences of Scolopacidae in Sarawak was conducted using cluster
analysis that utilized Unweighted Pair Group Method with Arithmetic Mean (UPGMA). A total of 16
selected species of waders were chosen for this study which includes snipes, shanks, curlews and
small sandpipers. The result showed that two major groups can be identified through habitat
utilization. This consists of all true snipes which occupied only the inland area (clade one), whereas
the other 14 species claded together forming the second groups inhabit the coastal areas (clade two).
Morphological features of the species were described as one of the factors that lead to habitat
utilization. Thus, shorebirds do have their own preferences in choosing habitat during their stopover
migration.
** Please contact the corresponding autor for further details of this paper.
601
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
ASSIDUITY IN NEST BUILDING ACTIVITY OF THE WHITE-NEST SWIFTLET
(Aerodramus fuciphagus)
1
2
Mohamad Fizl Sidq Ramji , Lim Chan Koon and Mustafa Abdul Rahman
1
1
Department of Zoology, Faculty of Resource Science and Technology, UNIMAS 94300 Kota
2
Samarahan, Sarawak, 157 Lorong 4A, Off Jalan Stampin Timur, 93350 Kuching.
Abstract
Comparative research of the biology and breeding periodicity of the White-nest Swiftlets (Aerodramus
fuciphagus) had been done in the past decades, but not much effort is focused on the roosting and
nesting behaviour of this species despite being an economically important species widely cultivated in
the swiftlet farming industry in South-east Asia regions. Development of swiftlet houses and
advancement in video recording equipments enable behaviour research to be conducted in close
proximity with minimal interference. Daily nest building activity of a selected pair of White-nest Swiftlet
from a swiftlet house colony in Miri was observed during one month nesting period in April 2012. We
investigated the potential of sexual segregation in nesting behaviour by initial markings on the wing
tips and tail feathers on one member of the observed pair. A total of 544 hours of video monitoring
was recorded using a multifocal infrared camera system. Daily records indicated that nest building
may occur during anytime of the night but two peak activity sessions are apparent before the preemergence hours (0500-0600 hr) and immediately after the returning hours (1800-2000 hr). The
overall results revealed that both sexes participate in nest building. However, the marked individual (A)
was observed spending almost double nest building minutes compared to the unmarked partner (B).
The unequal contribution from each pair member is significant, which suggests male and female
swiftlets play different roles in nesting building. This may be an adaptive response to sustain their
multi-brooded reproductive strategy in terms of energetic. With this preliminary observation, future
work will focus along the hypothesis that it is the male that is more active in nest building because
female swiftlets need to channel substantial energy for egg production.
** Please contact the corresponding autor for further details of this paper.
_________________________________________________________________________________
BIOLOGY AND TOXICITY OF Tetraodon nigroviridis FROM SAMPADI RIVER, SARAWAK
Samsur Mohamad* and Sarmila Muthukrishnan
Deparment of Aquatic Science, Faculty of Resource Science and Technology,
94300 Kota Samarahan Sarawak, MALAYSIA
E-mail*: msamsur@frst.unimas.my
Abstract
A total of 114 individuals, spotted-green puffer were collected from Sampadi River, for biological study
(i.e. morphology, gut content analysis) and toxicity assessment. Samples were caught by using three
layer gill nets and were assessed for their standard morphometric data, gut content and toxin content.
The results showed that standard length and body weight were ranged from 7.60 to 13.00 cm, and
40.66 to 177.18 g, respectively and shows significant difference in size between male and female.
Based on the spot characteristic, seven types of spot were observed microscopically in most of the
samples. Analysis of the diet composition showed that the fish is omnivores which prey on
invertebrates such as copepod, crustacea, gastropods, polychaete, and small crabs, and leafs. The
anatomical distribution of toxicity level was tested by using mouse bioassays. Skins shows the higher
toxicity score (155.80 ± 76.17) MU/g followed by eggs, gonad, liver, and muscles. Furthermore, the
toxicity results were clarify by using TLC and the toxins extracts of all tissues exhibited same R f value
as authentic tetrodotoxin (TTX). From this finding, it suggesting that T. nigroviridis from Sampadi River
could be a potential food poison source as it contained TTX component.
Keywords: T. nigroviridis, gut content, mouse bioassay, thin layer chromatography
** Please contact the corresponding autor for further details of this paper.
602
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
ASSEMBLAGES OF FRESHWATER SNAILS IN BENUK RIVER OF PADAWAN LIMESTONE,
SARAWAK (MALAYSIA, BORNEO)
1
Mary Teh Chee Sing and Jongkar Grinang
2*
1
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan
2
Sarawak, Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak,
94300 Kota Samarahan, Sarawak
Email*: gjongkar@gmail.com
Abstract
Information on assemblages of freshwater snails is useful in understanding responses of the fauna to
environmental factors. We collected freshwater snails from 25 1-m2 plots at five stations in Benuk
River of Padawan limestone, in order to determine assemblages of the fauna in relation to habitats
(e.g., riffles, runs, pools), substrates (e.g., bedrocks, gravels, sands, leafpacks) and 10
physicochemical variables (e.g., dissolved oxygen, pH, temperature, conductivity, total dissolved solid,
calcium, magnesium, ammonium, water current, depth) using multivariate analysis of Canonical
Correspondence Analysis, Multi-response Permutation Procedure and Indicator Species Analysis. 668
individuals from five families and eight species of freshwater snails were collected from Benuk River.
The results indicated that assemblages of freshwater snails were significantly influenced by water
current and magnesium. Bedrock-riffles were dominated by large-size snails such as Brotia costula
(Rafinesque, 1833) and B. pageli (Thiele, 1908), indicating their ability to withstand strong water
current. In moderate and slow flowing water with substrates of leafpacks and gravels, the common
species were smaller-size snails of Tarebia granifera Lamark, 1822 and Thiara scabra (Muller, 1774).
The abundance of Clea nigricans A. Adams, 1855 and Paludomus lacunoides Aldrich, 1889 was low,
which only found among leafpacks at slowing flowing water. The results showed calcium was less
important in influencing assemblages of freshwater snails in limestone stream probably, in the state of
high amount in the water, the mineral might no more a limiting factor in biological activities of the
snails.
Keywords: freshwater snails; assemblages; limestone; Padawan
** Please contact the corresponding autor for further details of this paper.
_________________________________________________________________________________
DISTRIBUTION AND IDENTIFICATION KEY FOR TEN SPECIES OF THE Simulium tuberosum
SPECIES-GROUP (DIPTERA: SIMULIIDAE) IN MALAYSIA
Chee Dhang Chen , Mohd Sofian-Azirun , Zubaidah Ya’cob , Daicus Belabut , Poai Ean Tan ,
1
1
Rosli Hashim , Hiroyuki Takaoka
1*
1
1
1
2
1
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
2
Malaysia, Department of Wildlife and National Parks, Km 10, Jalan Cheras, 56100 Kuala Lumpur,
Malaysia
Email*: chen_ctbr@um.edu.my
Abstract
Black flies are one of the biting dipteran insects of medical and veterinary importance around the
world. In Malaysia, 71 species of black flies have been documented, of which ten species including
two new species recently found from Tioman Island, Pahang were placed in the tuberosum speciesgroup. Their geographical distributions and identification key are provided for the first time.
Keywords: Black flies, Similium tuberosum species-group, Tioman Island, Malaysia
** Please contact the corresponding autor for further details of this paper.
603
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
THE DIVERSITY OF THE BUTTERFLIES (INSECTA: LEPIDOPTERA) IN GENTING HIGHLANDS,
PAHANG, MALAYSIA
Karen-Chia*, H.M., Chen, C.D. and Sofian-Azirun, M.
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur,
Malaysia.
*Email: karenchia.24@gmail.com
Abstract
A study on butterfly diversity and abundance was conducted in Genting Highlands, Pahang, Malaysia.
The butterflies were collected from the sites ranged between 1,100m to 1,800m above sea level
(a.s.l). The butterflies were caught by using sweep net along the trails, roadside and different habitat
from May until December 2011. All collected specimens were preserved, identified and deposited at
Zoological Museum, Institute of Biological Sciences, Faculty of Science, University of Malaya (UMKL).
A total of 1,245 butterflies belongings to 5 families and 105 species were recorded. The dominant
butterflies obtained from Genting Highlands was belonging to the family of Nymphalidae (58.63%),
followed by Pieridae (25.46%), Lycaenidae (17.61%), Papilionidae (2.01%) and Hesperiidae (1.29%).
The Ypthima pandocus corticaria (Nymphalidae) was the most abundance and dominant species
(n=219), followed by Ypthima pandocus tahanensis (Nymphalidae; n=124) and Eurema hecabe
contubernalis (Pieridae, n=99) in Genting Highlands. The Shannon-Wiener index values (H’ = 3.5λ4)
indicates a high diversity of butterfly in Genting Highlands. Meanwhile, the eveness index (E = 0.3464)
show that Genting Highlands sites do not have an even pattern of distribution. It also found that the
number of species was reduced at the higher elevation in Genting Highlands.
** Please contact the corresponding autor for further details of this paper.
________________________________________________________________________________
PANDAN BEACH, LUNDU, SARAWAK: EPIBENTHOS, FISH AND PHYTOPLANKTON
ASSEMBLAGES
Ruhana Hassan*, Farah Akmal Idrus & Siti Akmar Khadijah Ab Rahim
Aquatic Science Department, Faculty of Resource Science and Technology, Universiti Malaysia
Sarawak, 94300 Kota Samarahan Sarawak
*Email: hruhana@frst.unimas.my
Abstract
Pandan beach, Lundu, is one of the favourite public beaches in Sarawak, comprising both white sand
and clear blue water suitable for various activities such as picnic, swimming or simply enjoying its
aesthetical beauty. Local fishermen harvest fish, crabs, prawns and jellyfish in the nearby coastal
waters, yet very little is known about the biodiversity of this area. A short study on fish, phytoplankton
th
and epibenthos assemblages was conducted on Pandan Beach and surrounding areas between 9 to
th
11 April 2012. A total of 23 individual belonging to 8 species of fish were caught using one-layer gill
nets, with Toothpony fish Gazza minuta as the most abundant fish caught. Phytoplankton study had
recorded 13 genera with marine planktonic diatom Chaetoceros as the most common genus. On the
sandy beach area, besides hermit crabs, horned-eye ghost crabs Ocypode ceratophthalmus could be
easily observed, whereas O. cordimanus was less common. Rocky shore areas are habitats for
barnacles, black mussels, rock oysters, periwinkle, limpets, sea anemone, besides some stunted
epilithic green and red algae. Results obtained during this study could only be regarded as
preliminary base line data, hence a more comprehensive study should be carried out in future in order
to get the overall picture of biodiversity of the area.
Keywords: epibenthos, fish, phytoplankton, Pandan Beach
** Please contact the corresponding autor for further details of this paper.
604
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
INSECT PEST OF Neolamarckia cadamba PLANTATION IN SARAWAK, MALAYSIA
Doreen H.S. Chai* and Marfaisal Marzuki
Sarawak Forest Tree Seed Bank, SARAWAK FORESTRY Corporation, Semengoh, KM 20, Jalan
Puncak Borneo, 93250 Kuching, Sarawak
Email*: doreen_chai@yahoo.com
Abstract
Neolamarckia cadamba, locally known as Kelampayan, is of economic importance as timber and as
source of non-timber products. Due to its growth characteristics, this species had caught the attention
of the government and private sectors for reforestation and has been introduced as a potential fast
growing tree species for forest plantation establishment in Sarawak since 2003. However, pest
problem in the established N. cadamba plantation was widespread especially for stem borers and
defoliators. Arthroschista hilaralis was found the most abundant defoliator of N. cadamba. Defoliation
of young N. cadamba seedlings stunted the growth of the trees. Factors observed triggering A.
hilaralis outbreaks in the local plantations are discussed. A study conducted to evaluate stem borer
infestation in Sarawak showed about 70% was found in several major local forest plantations. The
density of past and present attacks per tree ranged from 0.22-1.83 and 0-0.70, respectively.
Infestation was concentrated at the lower part of the trunk, less than 1 m from the ground level. The
tunnels created by the stem borer were observed to be generally about 15-30 cm deep with diameter
of around 1-2 cm. Preliminary observations suggest that intensive silvicultural practice reduced the
susceptibility of stem borer attack. Integrated pest management, approach encompassing biological,
chemical and silvicultural control, and the extent of damage relating to the wood quality, mortality and
growth performance are discussed.
Keywords: Arthroschista hilaralis, stem borer, intensive silvicultural practice
** Please contact the corresponding autor for further details of this paper.
_________________________________________________________________________________
ETHNOENTOMOLOGY AMONG SELECTED ETHNIC GROUPS IN PENINSULAR MALAYSIA
1
Ashikin Ismail N *, Maryati Mohamed
2
1
Faculty of Science, Technology and Human Development,
2
Faculty of Civil and Environmental Engineering
Universiti Tun Hussein Onn Malaysia, Batu Pahat, Johor, Malaysia.
Email: nuruleikin@yahoo.com*, maryati@uthm.edu.my
Enthnoentomology is a practice which involves insects being used for multi-purposes, mainly as food
and medicine. There are currently very few and limited work reported by Dr. Chung and his colleagues
from Sabah Forestry Department, Malaysia based on Malaysian records of ethnoentomological
studies. This study will investigate how selected indigenous ethnic groups in Peninsular Malaysia
make use of insects in their daily life. The purpose of this study is to document traditional knowledge
on the use of insects in the daily life of these ethnics and to analyse bioactive compounds from
selected insects to substantiate the traditional claims based on the potentials of these insects. Survey
will be conducted to gather information from respondents (indigenous people) on how they use insects
in their daily life, before and now. Insect samples collected would undergo laboratory analyses. It is
hoped that from the survey and laboratory work conducted, traditional claims on the potency of insects
could be substantiated through the bioactive compounds found in them. From this study, the analysis
procedures could proof to be essential for future studies and some traditional knowledge be
preserved.
Keywords: Ethnoentomology, traditional knowledge, insect, indigenous ethnics, Peninsular Malaysia
** Please contact the corresponding autor for further details of this paper.
605
4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
MORPHOLOGICAL ANALYSIS OF FAMILY ZOSTEROPIDAE IN MALAYSIA
Nor Salmizar binti Azmi* and Mustafa Abdul Rahman
Department of Zoology, Faculty of Resource Science and Technology
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak
Abstract
Family of Zosteropidae is a classic example of a great speciator which has large distributions and
consists of approximately 80 similar-looking species across the range. There are 13 genera and 86
species in this family. However, there are only five species of Zosteropidae found in Malaysia; (i) Z.
palpebrosa (oriental white-eye), (ii) Z. atricapilla (black-capped white-eye), (iii) Z. everetti (everett’s
white-eye) (iv) O. squamifrons (pygmy white-eye), and (v) C. emiliae (mountain black-eye). From all
Zosteropidae existing in Malaysia, only Z. everetti was not included in this study due to the difficulties
to capture this species. A morphological analysis of this family was carried out from 54 live samples.
Multivariate method such as Discriminant Function Analysis (DFA), Cluster Analysis and Principle
Component Analysis (PCA) were used in this study. DFA and PCA analysis were carried out in SPSS
version 18.0, while Cluster Analysis using Euclidean Distance with Unweighted Pair-Groups Method
Average (UPGMA) as the linkage method analyzed in MVSP version 3.0. A total of 10 morphological
characters; tarsus length (TR), bill length (BL), bill depth (BD), bill width (BW), head + bill (HB), wing
length (WL), wing span (WS), tail length (TA), total length (TL) and weight (WT)) were measured.
Clear division between two major species Zosterops (White-eyes) and Chlorocharis (blackeyes) was
confirmed in those three analyses. Besides, component matrix score in PCA had proven that
characteristic which highly contributed to the Factor 1 was HB (0.986). Meanwhile, BW was identified
as a character which contributed to the Factor 2. While in DFA, high value of standardized
discriminant function coefficients suggested that TA (0.462) contributed to determine score in Function
1. For Function 2, TR scored the highest character loading with the value 0.843. Therefore, this study
proves that; (i) species at higher altitude usually larger than species found in lowland. The selective
advantage of variation in size between highland species and lowland species is presumed to be
related with temperature differences., (ii) variation in bill characters (bill length, bill width, bill depth and
head+bill length) of avian is probably related to their feeding ecology.
Keywords: morphological, Zosteropidae, multivariate, variation, Discriminant function analysis (DFA).
** Please contact the corresponding autor for further details of this paper.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
BIOMASS AND CARBON SEQUESTRATION OF SELECTED WILD GROWN HERBACEOUS
SPECIES IN UNIMAS CAMPUS
Sharifah Mazenah, W.Y.*, I.B. Ipor., C.S. Tawan, Ismail J., and Siti Kamilah, M.
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak.
*Email: wysmazenah@gmail.com
Abstract
Ten selected of selected wild grown herbaceous species were selected to estimate the amount of
carbon stocks sequestered and to determine the relationship between biomass and Leaf Area Index
(LAI) in UNIMAS campus. Ten quadrates of 1m x 1m were plotted for sampling for each species and
harvested for 5 months. LAI was measured by using Portable Area Meter for every sampling. Biomass
o
was calculated after 7 days prior to oven dried in 60 C and carbon analysis were determined by using
CHN analyzer. Biomass of Nephrolepis bisserata (Sw.) Schott, Ischaemum magnum Rendle and
Imperata cylindrical (L.) Raeuschel were obviously decreased where the other species showed in
gradually decreased. However, Merremia peltata (L.) Merr. contained the highest amount of carbon
stock that is 37.43% of carbon content while Monochoria vaginalis (Burm. F.) Presl contained the
lowest percentage of 31.12%. There was a linear relationship between LAI and biomass with
2
respective r values where the reduction in LAI caused limitation in carbon absorption by leaves.
Keywords: biomass, carbon stock, carbon sequestration, LAI, herbaceous species
1. INTRODUCTION
Currently, Carbon dioxide (CO2) in the atmosphere is increasing continuously as what has been
documented in global environmental change research (Roshetko et al., 2002). It results the increasing
of CO2 concentration thus affects in primary productivity by leaf-level gas exchange. Biomass and
carbon are mainly naturally stored in the forest and be a significant part of the global carbon cycle
(Manhas et al., 2006). The study of tropical forest dynamic and structure must be carried out to ensure
the potential of carbon either it lose or store in the ecosystem as well as for a better understanding on
how forest can recover from disturbance (Vieira et al., 2004). It has been proposed that carbon
sequestration act as an effective mitigation option to manage the carbon in the atmosphere because
of the positive effect of the environmental conservation and soil fertility is combined (Smith, 2007).
Hence, to convert the excess of CO2 in the atmosphere, it needs more photosynthesis actions as
different species of herbaceous plants stored different amounts of carbon storage such as tree
contains more carbon than herbaceous. In tropical country, herbaceous species might contain
different amount of carbon as compared to temperate species and undoubtedly, the new adjusted
formula will be more acceptable (Kenzo et al., 2009). This study is intended to enable the comparative
study of biomass allocation and carbon sequestration of selected wild grown herbaceous species in
UNIMAS campus and to determine the relationship between biomass and LAI of the selected species.
2. MATERIAL AND METHOD
The study was carried out in several areas around UNIMAS campus. Areas were selected based on
bushy and dominant growth of the particular selected species in opened areas and distant from any
possible disturbance. Ten quadrates of 1m x 1m plot were established for each species where plants
within the quadrate were severed at ground level to ensure that each sample was cut at almost same
height. All plant materials were oven-dried to obtain their dry biomass to be used to estimate the
carbon stock that was based on method described by Pearson et al. (2005) where the carbon stock is
50% of the biomass. Direct method was used to estimate the value of LAI by measurement of leaf
area by using Portable Area Meter except for mimosa pudica that could not be determined due to its
habit. For the carbon analysis measurement, the amounts of carbon in each of ten species for first and
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fifth time harvests were determined by CHN Analyzer. The data obtained were analyzed using oneway ANOVA, Tukey test and regression analysis.
3. RESULT AND DISCUSSION
The biomass of ten selected species resulted different patterns of biomass reduction as stated in
Table 1. Paspalum conjugatum, Merremia peltata and Ipomea aquatica were declined gradually,
compared to Nephrolepis bisserata and Ischaemum magnum showed sudden decrease in their
biomass. The other remain species Mimosa pudica, Axonopus compressus, Monochoria vaginalis and
Colocasia esculenta were bring obvious reduction. The patterns resulted from the carbon stock
assessment were similar to those biomass assessment as the values of carbon stock were estimated
based on Pearson et al (2005) and Merremia peltata showed the highest amount of carbon stock
compared to other nine species. The outcomes were due to factors such as climate, availability of
space, nutrients, growth density and others that encourage and promote their rapid growth. The areas
with less shade will contribute to increase of ground vegetation biomass and their density (Sigurdsson
et al., 2005). However, constant declining in biomass is significant since a part of those captured
carbon in plant biomass is carried separately to roots and continually enters organic and inorganic
carbon soil where they can be stored there for the next thousand years of period (Jansson et al.,
2010).
LAI values were gradually reduced as in Table 2 except for certain species with obvious increasing in
value throughout harvesting period such as Axonopus compressus, Merremia peltata, Monochoria
vaginalis and Colocasia esculenta. As the LAI values reduced, the ability in capturing carbon was also
reduced. The linear relationship between biomass and LAI of nine selected species proved that LAI
2
values were depends on biomass value. The r values were ranged between 0.938 and 1.00 where
Imperata cylindrical give the highest value whereas Paspalum conjugatum give the lowest value. This
result of regression analysis indicates that linear models gave best fit for regression of biomass will
also increase LAI value as well. Study of aboveground tree biomass and LAI also had indicated that
both two parameters have strong positive correlation in terms of correlation patterns as their r value is
0.99 (Heiskanen, 2006).
The result of carbon sequestration of ten selected herbaceous species showed in Table 3 that
Merremia peltata had highest carbon content with 37.80% at first harvest and 37.05% at fifth harvest
while Monochoria vaginalis contained lowest percentage of carbon 30.92% and 31.31% for first and
fifth harvest. Carbon was estimating from the carbon content of dry biomass at 50% constant by
weight and practiced by most researchers while apart from the researchers 45% by weight was used.
However, to measure it directly carbon is measured directly by burning in CHN analyzer (Losi et al.,
2003). Biomass and carbon stock are important parameters in reducing CO 2 within atmosphere and
consequently essential in combating global warming.
Data analysis of one-way ANOVA by using Tukey test with significant levelν α=0.05 indicates that
there were significant differences of carbon sequestration among ten species selected based on their
mean difference values.
4. CONCLUSION
Ten selected herbaceous species in UNIMAS campus were studied where the amount of carbon stock
was estimated by using Pearson et al. (2005) conversion factor and by using CHN Analyzer. Merremia
peltata was contained highest amount of carbon stock besides of highest percentage of carbon
content if compared to other selected species. The linear relationship was determined between two
variables of LAI and biomass for each species. Biomass and carbon stock was act as important
parameters in reducing CO2 in the atmosphere and consequently essential in combating global
warming and as compared to the woody plants their role in carbon sequestration of herbaceous
species was not clear discovered thus really need further research that works on herbaceous species.
Table 1. Average biomass and carbon stock of ten selected herbaceous species.
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Plant species
Paspalum
conjugatum
Mimosa pudica
Axonopus
compressus
Merremia peltata
Nephrolepis
bisserata
Monochoria
vaginalis
Colocasia
esculenta
Ipomea aquatica
Ischaemum
magnum
Imperata cylindrica
Harvest
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Average biomass
(g)
208
126
82
78
78
234
154
112
132
146
156
188
112
118
110
488
180
190
120
128
552
106
78
48
52
136
110
189
105
70
458
118
130
160
110
164
126
92
90
50
1618
44
62
42
36
2289
150
86
70
66
609
C stock
(g)
104
63
41
39
39
117
77
56
66
73
78
94
56
59
55
244
90
95
60
64
276
53
39
24
26
68
55
94.5
52.5
35
229
59
65
80
55
82
63
46
45
25
809
22
31
21
18
1144.5
75
43
35
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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2
2
2
Table 2. Average value of leaf area (cm ) and LAI (cm / cm ) of nine selected species
according to five time harvests.
Species
Paspalum conjugatum
Axonopus compressus
Nephrolepis bisserata
Merremia peltata
Monochoria vaginalis
Colocasia esculenta
Ipomea aquatica
Ischaemum magnum
Imperata cylindrica
Harvest
Leaf area (cm²)
LAI (cm²/cm²)
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
11516.80
9583.30
6236.70
5932.40
5932.40
34465.90
37290.00
22216.00
23405.60
21819.10
12856.00
3172.60
2333.40
1432.80
1552.20
42111.00
15100.80
15939.70
10068.30
10738.50
16840.70
14055.40
24150.70
13417.00
8944.60
46545.50
18087.20
19926.40
24525.30
16860.90
33547.20
26873.20
19622.40
19195.60
10663.60
75921.60
5207.80
7338.20
4971.20
4261.40
272396.60
15968.50
9155.70
7452.30
7027.10
1.15
0.96
0.63
0.60
0.59
3.45
3.73
2.22
2.34
2.18
1.29
0.32
0.23
0.14
0.16
4.21
1.51
1.60
1.01
1.07
1.68
1.41
2.42
1.34
0.90
4.65
1.81
1.99
2.45
1.69
3.35
2.69
1.96
1.92
1.07
7.59
0.52
0.73
0.50
0.43
27.24
1.60
0.92
0.75
0.70
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Table 3. Average value of carbon content in ten selected species
according to first and final harvest.
Plant species
Paspalum conjugatum
Mimosa pudica
Axonopus compressus
Nephrolepis biserrata
Merremia peltata
Monochoria vaginalis
Colocasia esculenta
Ipomea aquatica
Ischaemum magnum
Imperata cylindrica
Harvest
Carbon (%)
H1
H2
H1
H2
H1
H2
H1
H2
H1
H2
H1
H2
H1
H2
H1
H2
H1
H2
H1
H2
34.99
33.84
36.54
36.37
33.09
34.45
33.28
33.34
37.80
37.05
30.92
31.31
33.53
31.89
33.21
33.30
36.04
35.27
34.54
34.74
References
Heiskanen, J. (2006). Estimating aboveground tree biomass and leaf area index in a mountain birch
forest using ASTER satellite data. International Journal of Remote Sensing, 27(3), 1135-1158.
Jansson, C., Wullschleger, S., Kalluri, U. C. & Tuskan, G.A. (2010). Phytosequestration: carbon
biosequestration by plants and the prospects of genetic engineering. Bioscience, 60(9), 685696.
Manhas, R.K., Negi, J.D.S., Kumar, R. & Chauhan, P.S. (2006). Temporal assessment of growing
stock, biomass and carbon stock of Indian forest. Climatic Change, 74, 191-221.
Pearson, T.R.H., Brown, S. & Ravindranath, N.H. (2005). Integrating carbon benefit estimates into
GEF projects. Capacity development and adaptation group guidelines, United Nations
Development Programme Global Environment Facility, New York, USA.
Roshetko, J.M., Delaney, M., Hairiah, K. & Purnomosidhi, P. (2002). Carbon stocks in Indonesian
homegarden systems: Can smallholder systems be targeted for increased carbon storage.
American Journal of Alternative Agriculture, 17(2), 1-11.
Sigurdsson, B.D., Magnusson, B., Elmarsdottir, A. & Bjarnadottir, B. (2005). Biomass and composition
of understory vegetation and the forest floor carbon stock across Siberian larch and mountain
birch chronosequences in Iceland. Ann. Forest Science, 62, 881-888.
Smith, A. M., Nadeau, C., Freemantle, J., Wehn, H., Teillet, P. M., Kehler, I. et al. (2005). Leaf area
th
index from CHRIS satellite data and applications in plant yield estimation. 26 Canadian
Symposium on Remote Sensing, June 14-16, 2005. Wolfville., Nova Scotia.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
Sustaining Tropical Natural Resources Through Innovations, Technologies and Practices.
ECOPHYSIOLOGY OF Ischaemum magnum Rendle: THE EFFECT OF SHADING ON THE
ALLOCATION OF NUTRIENTS
I.B. Ipor, C.S. Tawan and Nurafiza, A.*
Department of Plant Science and Environmental Ecology, Faculty of Resource Science and
Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak
*Email: feezaaman@gmail.com
Abstract
Greenhouse study was conducted to determine the effect of shading at 0%, 50% and 75% light intensity
on biomass and nutrient allocations in Ischaemum magnum Rendle. Shading significantly affected the
vegetative growth, dry-matter production, leaf area and biomass partitioning of I. magnum. Maximum
2
shading (75%) gave rise to the highest values of leaf weight ratio (0.53g), specific leaf area (404.1 cm
-1
-2
-1
g ), dry matter production (8.48g), net assimilation rate (511.0 mg cm day ) and leaf area duration
-2
(8544.9 cm day). Vegetative and reproductive parts from three shade levels were analysed for nitrogen
(N), phosphorus (P) and potassium (K). The amount of a particular element allocated to a specific
vegetative part was significantly differed between different shade levels whereas generally, most of
nutrient (N, P and K were abundant at root and leaf either of 50% or 75% shade. The different
distribution of nutrients at various vegetative parts was closely related to the patterns of biomass
allocation.
Keywords: ecophysiology, Ischaemum magnum Rendle., light intensity, biomass, nutrient allocation.
1. INTRODUCTION
Ischaemum magnum Rendle is a perennial tussock grass with stout culms and occurs widely within the
geographical areas of 20° N latitude and 5°s such as Peninsular Malaysia, Singapore, Borneo, Thailand
and Burma (Gilliland et al., 1971). It is an opportunistic and aggressive colonizer which abundantly
grown in open or disturbed habitats under full sunlight or under shade. It grows profusely in most
agricultural areas, road verges and particularly well adapted to wet sites.
I. magnum is not well recognised as a forage grass for grazing but its young vegetative parts are readily
eaten by free-ranging cattle and goats (Ng, 1992). It has been long recognised as a serious weed due
to its extensive formation of phalanx tillers with high survival although the palatability decline towards
maturity. Biomass was allocated differently according on the resources: highly light availability results in
a higher root/shoot ratio whereas high nutrient content results in vice versa (Cronin & Lodge, 2003).
The need to combine carbon with other elements creates a functional equilibrium between the shoot
and root (Brouwer, 1962; Raper et al., 1978; Hunt & Nicholls, 1986) which can be embodied in a
quotient of the biomass in each fraction. The allocation within and between both vegetative and
reproductive organ is doubtless dissimilar to that of mineral nutrient (Lovett Doust, 1981; Abrahamson &
Caswell, 1982; Fitter & Setters, 1988). If shading can alter the root-shoot equilibrium and biomass
partitioning, they may also modulate the total nutrient uptake and the nutrient allocation between organs.
In the greenhouse experiment, we report and discus nutrient partitioning in vegetative and reproductive
organs of I. magnum in different light intensities. The effect of shading on the partitioning of biomass is
also included.
2. MATERIALS AND METHODS
The uniformly seedlings at three leaf stage were transplanted into the 10 cm diameter plastic pots, filled
with soil mixture (3 : 1 : 1). Three levels of irradiance were achieved using artificial shade cloth placed 2
m above the pots. These shade cloths giving the three levels of radiation, H = l00%, 1=56% and L =
33% and it can be considered neutral because they did not modify the spectra) composition of the
transmitted radiation.
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The plants were then kept in separate light regimes at 0%, 50% and 75% shade. Special black nettings
were used to cover the cages (2m X 5m) to obtain the designated shade levels. The light intensity of the
shading regimes were checked with Skye light meter (Skye Intrument Limited, UK). The plants were
watered daily.
Four pots of plants from each light regime were assessed weekly to determine their leaf number, tillers
and height of mother plants. The other group of four plants from each light regime was harvested for
growth analysis. The plants were harvested at 49 and 56 days after transplanting. The soil from pots
was completely removed. The roots, leaves, branches and reproductive parts were separated. The leaf
area of each plant was determined using leaf area meter (V-Delta Devices, Inc.) and the dry weight of
the plant parts was determined after 3 days oven-dry at 60°c. The calculations of root weight ratio
(RWR), leaf weight ratio (LWR), shoot weight ratio (SWR), specific leaf are (SLA), dry matter production
(DMP), leaf area duration (LAD) and net assimilation rate (NAR) were based on technique developed by
Patterson and Flint (1983).
In another experiment, four plants from each light regime were harvested at 28 days after transplanting
for nutrient analysis. The plant parts were separated into leaf, branch, flower and root parts and air dried
before being used for further analysis. The soil was also air dried.
The determination of nitrogen (N) in both plant tissues and soil was carried out by following semi-micro
Kjeldhal method (Sirim, 1980). Dry ashing technique was employed for determination of both
phosphorus (P) and potassium (K) (Chapman and Pratt, 1961). The concentrate of N, P and K were
determined using Technicon autoanalyzer. All experiments were conducted in completely randomised
block arrangement with four replicates for each light regime. Data was subjected to variance analysis
and comparison of means was done with LSD.
3. RESULTS AND DISCUSSION
The height of plants was significantly increased as the shade level increased. The tallest plants were
found at 75% shading (Table 1). Similar trend of growth pattern was observed by Ipor (1992) on Mikania
micrantha. Shading had significantly decreased the number of leaves. The least number of leaves was
found at 75% shade level. The leaf number at 75% shade level was 84 as compare with 334 leaves
from 0% shade level. The leaves from higher levels of shading were generally longer than those at
lower level of shade. The same trend of result observed on the number of tillers. The number of tillers at
0% shade was more than two times of those from 75% shade level. This scenario also found in
Panicum maximum var. trichoglume grown under 40% light transmission whereas 27% increase in
shoot length (Wong & Wilson, 1980).
Leaves produced under the shaded area were thinner than those produced in full light. This is reflected
in their great specific leaf area or area per unit leaf weight (Table 2). The distribution of plant biomass
(such as leaf weight) indicated by leaf weight ratio (LWR) tend to increase with shade. These
concomitant increases in SLA and LWR resulted in substantial increases in leaf area ratio (LAR) or
amount of leaf area per unit of plant weight as shading was increased and it also supported by Corre
(1983) which stated that shading have directly proportional to leaf area (due to increase of SLA).
Increasing in LWR and LAR may be of adaptive significance of Ischaemum magnum as they
represented a greater input of plant biomass in photosynthethic tissues (Blackman, 1960; Blackman,
1968; Patterson, 1980). Shading significantly increases NAR and LAD in both harvest intervals (Table
3). Ipor (1992) also observed that the NAR and LAD values of Mikania micrantha were significantly
increased with increase in shading.
The amount of N, P and K in soil was decreased with time (Table 4). The amount of N, P and K was not
significantly differed between shade levels except for P at 4 month interval. Different percentage of
nitrogen distribution can be seen as in the Table 5, which 75% has the highest % of nitrogen within the
root and leaf, 0.217% and 0.154% respectively. Under 50% shades, nitrogen were found to be
abundant in I. magnum’s stem with 1.059%. Thus, it showed that more N was accumulated in plants
being exposed to higher shade levels compared to 0% area and this was supported by Wong and
Wilson (1980) on which N yield were rose of 8-weeks cut Panicum maximum under 50% shade cloth on
heavy clay soil at Narayan Research Station (Wilson et al., 1986).
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th
Generally, N content within I. magnum’s partition at the 28 days of planting were lower than P content
due to the immobility of P ion in soil (Cui & Caldwell, 1996), hence it does not leached easily through
soil medium. More P was found in both 50% shade with 1.884% in stem as compare with those at 0%
and 75% shade levels, 0.732% and 0.484%, respectively. Potassium was found lesser at 0% shade for
both at root and leaf parts than those from 50% and 75% shade (Table7). According to Table 3, DMP,
-2
-1
-2
NAR and LAD were highest at 75% shade with 8.48g, 511.0 mg cm day and 8544.9cm day,
respectively. However, Cruz (1997) reported dry matter production (DMP) of Dichantium aristatum were
inversely proportional to percentage of irradiance.
Table 1. Effect of shading on plant height, leaf number, leaf length and number of tillers of I. magnum,
7 weeks after transplanting
Shades (%)
Vegetative parts
0%
50%
75%
Plant height (cm)
64.3c
95.1b
112.1a
Numbers of leaf
334.9a
250.4b
84.1c
Leaf length (cm)
34.2c
4.0b
63.2a
Number of tillers
87.9a
59.1b
34.6c
*Within each row, values sharing the same letter are not significantly different at 5% (P= 0.05) level,
according to Duncan’s multiple range test.
Table 2. Effect of shading on vegetative growth, leaf area production and biomass allocation in I.
magnum
Shades (%)
0%
50%
75%
LWR
SWR
RWR
SLA
2
( cm /g)
0.48a
0.32ab
0.15b
0.09b
0.15ab
0.34a
116.03b
129.06b
404.14a
( g/g )
0.43a
0.53a
0.53a
LAR
50.09b
68.34a
203.50a
*Within each column, values sharing the same letter are not significantly different at 5% level, according
to Duncan’s multiple range test.
Table 3. Effect of shading on dry – matter production (DMP), net assimilation rate (NAR), leaf area
duration (LAD) of I . magnum
DMP
NAR
LAD
-2
-1
-2
(g)
(mg cm day )
(cm day)
0%
7.70a
210.2b
2748.3c
50%
7.62a
214.0b
5971.2b
75%
8.48a
511.0a
8544.9a
*Within each column, values sharing the same letters are not significantly different at 5% level,
according to Duncan’s multiple range test.
Shade (%)
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Table 4 . Distribution of nutrients in soil, 4 weeks after planting with I . magnum under different shade
levels.
N (%)
Shade (%)
0%
50%
75%
P(%)
K(%)
0 wk
4wk
0wk
4wk
0wk
4wk
0.079a
0.089a
0.073a
0.011a
0.017a
0.038a
0.065a
0.059a
0.069a
0.015ab
0.009b
0.028a
0.494a
0.518a
0.561a
0.124a
0.270a
0.364a
*Within each column, values sharing the same letter are not significantly difference at 5% level,
according to Duncan’s multiple range test.
Table 5. Distribution of nitrogen (% of tissue) in various vegetative parts of I . magnum grown under
different shade levels.
Nitrogen ( % tissue )
Shade (%)
Stem
Root
Leaf
0%
0 . 117a
0 . 079b
0 . 084b
50%
1 . 059b
0 . 061b
0 . 121ab
75%
0 . 126a
0 . 217a
0 . 154a
*Within each column, values sharing the same letter are not significantly different at 5% level, according
to Duncan’s multiple range test.
Table 6. Distribution of phosphorus (% tissue) in various vegetative parts of I . magnum grown under
different shade levels
Phosphorus ( % tissue )
Shade (%)
Stem
Root
Leaf
0%
50%
75%
0 . 732a
1 . 884a
0 . 484b
0 . 905a
0 . 675b
0 . 907a
0 . 611a
0 . 616a
0 . 733a
*Within each column, values sharing the same letter are not significantly different at 5% level, according
to Duncan’s multiple range test.
Table 7. Distribution of potassium ( % tissue ) in various vegetative parts of I . magnum grown under
different shade levels.
Shade (%)
0%
50%
75%
Potassium (% tissue)
Stem
Root
Leaf
2.082a
1.833a
2.376a
0.362b
2.161a
1.134a
0.478b
2.814a
2.412a
*Within each column, values sharing the same letters are not significantly different at 5% level,
according to Duncan’s multiple range test.
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4th Regional Conference on Natural Resources in the Tropics, 2012 (NTrop4):
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Figure 1. Seedling of I . magnum planted in the experimental plot at week-2.
Figure 2. I . magnum’s growth at different shading of 0%, 50% and 75% shade.
4. CONCLUSION
Shading is a primarily considered to affect a plant’s carbohydrate economy and adaptive morphologyν
an increase in tissue concentration of minerals in shaded plants is probably due to the allocation of
lower proportion of biomass to the roots. Under a lower light intensity, light will generally limit
photosynthesis. It seem logical that plants growing under a low light intensity will require small
adaptations to nutrient supply, because slow-growing plants have a lower absorption rate on the basis
of plant weight and thus require a smaller root weight ratio to maintain normal levels of nutrient,
provided that the absorption capacity on the basis of root weight is not affected. The actual absorption
rate on the basis of root weight may be low under low light intensity.
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Zul Helmey Mohamad Sabdin