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Volume 4, Number 2 

June 2010 

CANADIAN JOURNAL OF 

PURE AND APPLIED SCIENCES 

Richard Callaghan 

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University of Calgary, AB, Canada 

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David T Cramb 

Pratim K Chattaraj 


Indian Institute of Technology, Kharagpur, India 

Matthew Cooper 

Andrew Alek Tuen 

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Anatoly S Borisov 

Dale Wrubleski 

Kazan State University, Tatarstan, Russia 

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Ron Coley 

Dietrich Schmidt-Vogt 

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SS Alam 

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Indian Institute of Technology Kharagpur, India 

Geoffrey J Hay 

Biagio Ricceri 


University of Catania, Italy 

Chen Haoan 

Zhang Heming 

Guangdong Institute for drug control, Guangzhou, China 

Chemistry & Environment College, Normal University, China 

Hiroyoshi Ariga 

C Visvanathan 

Hokkaido University, Japan 

Asian Institute of Technology, Thailand 

Gongzhu Hu 

Indraneil Das 

Central Michigan University, Mount Pleasant, MI, USA 

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Gopal Das 

University of Haifa at Qranim, Tivon, Israel 

Indian Institute of Technology , Guwahati, India 

SA Isiorho 

Melanie LJ Stiassny 

Indiana University - Purdue University, (IPFW), IN, USA 

American Museum of Natural History, New York, NY, USA 

Bor-Luh Lin 

Kumlesh K Dev 

University of Iowa, IA, USA 

Bio-Sciences Research Institute, University College Cork, Ireland. 

Jinfei Li 

Shakeel A Khan 

Guangdong Coastal Institute for Drug Control, Guangzhou, China 

University of Karachi, Karachi, Pakistan 

Collen Kelly 

Victoria University of Wellington, New Zealand 

Xiaobin Shen 

Hamid M.K.AL-Naimiy 

University of Melbourne, Australia 

University of Sharjah, UAE 

Maria V Kalevitch 

Eric L Peters 

Robert Morris University, PA, USA 

Chicago State University, Chicago, IL, USA 

Xing Jin 

Roustam Latypov 

Hong Kong University of Science & Tech. 

Kazan State University, Kazan, Russia 

Leszek Czuchajowski 

Frances CP Law 

University of Idaho, ID, USA 

Simon Fraser University, Burnaby, BC, Canada 

Basem S Attili 

Guangchun Lei 

UAE University, UAE 

Ramsar Convention Secretariat, Switzerland 

David K Chiu 

Atif M Memon 

University of Guelph, Ontario, Canada 

University of Maryland, MD, USA 

Gustavo Davico 

SR Nasyrov 

University of Idaho, ID, USA 

Kazan State University,Kazan, Russia 

Andrew V Sills 

Russell A Nicholson 

Georgia Southern University Statesboro, GA, USA 

Simon Fraser University, Burnaby, BC, Canada 

Charles S. Wong 

Borislava Gutarts 

University of Alberta, Canada 

California State University, CA, USA 

Greg Gaston 

Sally Power 

University of North Alabama, USA 

Imperial College London, UK

LIFE SCIENCES 

CONTENTS 

Volume 4, Number 2 

June 2010 

Zhongping Chen, Zhisheng Wang and Anguo Zhou 

Fungal Protein Estimation of Aspergillus oryzae Growing in Solid State Cultivation Conditions .................................................... 1127 

Ulrich Vaconcelos, Maria Alice Gomes de Andrade Lima and Glicia Maria Torres Calazans 

Pseudomonas aeruginosa Associated with Negative Interactions on Coliform Bacteria Growth ...................................................... 1133 

Sarkar MA Kawsar, Sarkar MA Mamun, Md S Rahman, Hidetaro Yasumitsu and Yasuhiro Ozeki 

Inhibitory Effects of Rcg1, A Β-Galactoside-Binding Lectin from Rana catesbeiana (American bullfrog) Oocytes 

against Human and Phytopathogens ................................................................................................................................................... 1141 

M Zaheer Khan, Afsheen Zehra, Syed Ali Ghalib, Saima Siddiqui and Babar Hussain 

Vertebrate Biodiversity and Key Mammalian Species Status of Hingol National Park ..................................................................... 1151 

EA. Al Omireeni, NJ Siddiqi and AS Alhomida 

Effect of Magnesium Chloride and Sodium Fluoride on various Hydroxyproline Fractions in Rat Kidneys .................................... 1163 

AA Osowole, R Kempe, R Schobert and S A Balogun 

Synthesis, Characterisation and in-vitro Biological Activities of Some Metal(II) Complexes of 3-(-1-(4-Methyl-6-Chloro)-2- 

Pyrimidinylimino)Methyl-2-Napthol ................................................................................................................................................. 1169 

Ajanaku, KO, Dawodu, FA, James, OO, Ogunniran, KO, Ajani, OO and Nwinyi, OC 

Histological Studies of Brewery Spent Grains in Dietary Protein Formulation in Donryu Rats ......................................................... 1179 

PHYSICAL SCIENCES 

Falayi EO and Beloff, N. 

Estimating Geomagnetically Induced Currents at Subauroral and Low Latitudes to Assess 

their Effects on Power Systems ......................................................................................................................................................... 1187 

Bhanu Kumar, O S R U, S Ramalingeswara Rao and S S Raju 

Study of Interannual and Intra-Seasonal Variability of Summer Monsoon Circulation over India .................................................... 1199 

Magda A El-Bendary, Samiha M Abo El-OlA and Maysa E Moharam 

Enzymatic Surface Hydrolysis of Poly(Ethyleneterephthalate) by Lipase Enzyme and its Production .............................................. 1207 

Augustus Nzomo Wali 

On Submanifolds of Indefinite Complex Space Form ....................................................................................................................... 1217 

Hayder A Abdul Bari, Mohd Azimie Ahmad and Rosli Bin Mohd Yunus 

Experimental Study on the Reduction of Pressure Drop of Flowing Water in Horizontal Pipes using 

Paddy Husk Fibers .............................................................................................................................................................................. 1221 

Muhammad Asif Khan and Khalid Abdulmohsen Buragga 

Effectiveness of Accessibility and Usability of Government Websites in Saudi Arabia ................................................................... 1227

SENRA Academic Publishers, Burnaby, British Columbia 

Vol. 4, No. 2, pp. 1127-1131, June 2010 

ISSN: 1715-9997 

FUNGAL PROTEIN ESTIMATION OF ASPERGILLUS ORYZAE 

GROWING IN SOLID STATE CULTIVATION CONDITIONS 

Zhongping Chen, Zhisheng Wang and *Anguo Zhou 

Institute of Animal Nutrition, Sichuan Agricultural University, 46 Xinkang Road, Yaan 625 014, Sichuan, China 

ABSTRACT 

The estimation of fungal protein is an essential parameter for nitrogen metabolism studies and evaluating the biological 

efficiency of fungal protein synthesis during fermented soybean meal processing. The aim of this study was to validate 

the fungal protein of Aspergillus oryzae SICC 3.302 measurement in solid state cultivation conditions using glucosamine 

content. Factors influencing the correlation between the glucosamine amount and fungal protein content, including 

incubation time, culture conditions (liquid or solid medium), carbon and nitrogen source, have been measured. Results 

suggest that the correlation of the glucosamine level and fungal protein content, regardless of incubation time and culture 

conditions, was a nearly constant. The influence of the medium composition, in particular the nitrogen source on the 

correlation has occurred. Collectively, these results indicated that glucosamine could be considered as reliable fungal 

protein indicator under the same nitrogen source. 

Keywords: Glucosamine, fungal protein, Aspergillus oryzae, solid state cultivation. 

INTRODUCTION 

Soybean meal (SBM) is an important source of dietary 

protein in animal feed industry. However, variety of 

antinutritional factors, such as trypsin inhibitor, lectins 

and soya globulins, etc. contained in SBM limited the 

wide application of SBM in animal especially young 

animal feed (Dunsford et al., 1989; Li et al., 1990; Jiang 

et al., 2000). Fermentation with Aspergillus oryzae, a 

fungus widely used for hundreds of years in production of 

sake, miso, and soy sauce, could decrease antinutritional 

factors and improve the nutritional quality of SBM (Hong 

et al., 2004; Feng et al., 2007). Many biochemical 

reactions occurred during fermented SBM process 

involving substrate protein degradation and fungal protein 

synthesis, however, little is known about the method for 

distinguishing substrate protein and fungal protein when 

A. oryzae grow in solid state. The quantity of fungal 

protein is an essential parameter in nitrogen metabolism 

studies and for evaluating the biological efficiency of 

fungal protein synthesis during fermented SBM 

processing. 

In the case of solid state fermentation, direct measurement 

of fungal protein is very difficult because fungi penetrate 

into and bind themselves tightly to the solid substrate 

particles (Desgranges et al., 1991). Many authors have 

described indirect methods to estimate biomass in solid 

state fermentation. These indirect methods are based on 

measuring the content of certain cell components such as 

glucosamine (Nahara et al., 1982; Sparringa and Owens, 

1999; Wei et al., 2006), ergosterol (Nout et al., 1987; Wu 

*Corresponding author email: zhouanguo02@yahoo.com.cn 

1147 

et al., 2002), protein (Matcham et al., 1984; Córdova- 

López et al., 1996) and nucleic acids (Koliander et al., 

1984; Wei et al., 2006), and measuring the biological 

activity like enzymatic activity (Barak and Chet, 1986) 

and respiration (Oriol et al., 1988), or measuring the 

consumption of some media components (Matcham et al., 

1984). Meanwhile, previous studies have observed that 

the glucosamine content have a good correlations with 

sucrose consumption (Desgranges et al., 1991), 

amyloglucosidase and α-amylase level (Aidoo et al., 

1981). These provide a possible indirect method to 

measure fungal protein, by determining the quantity of 

glucosamine in solid state cultivation conditions. 

However, less information is available on the correlation 

relationship between fungal protein and glucosamine level 

in solid state cultivation conditions. 

Thus, the aim of the present work was to validate the 

fungal protein of Aspergillus oryzae measurement in solid 

state cultivation conditions using glucosamine content. As 

a fungal protein indicator, the relationship must ideally be 

constant throughout the fungal development. In the same 

way, culture conditions (liquid or solid medium) must not 

influence the relationship. If one of these two conditions 

is not fulfilled, this cell constituent cannot be considered 

as fungal protein indicator. In this study, agar plates 

covered with a membrane were used as a model system 

for solid state fermentation. This system prevents 

penetration of mycelium into the agar and allows the 

complete recovery of fungal biomass, so the content of 

glucosamine and fungal protein can be related to per gram 

dried biomass.

1128 

MATERIALS AND METHODS 

Microorganism 

Aspergillus oryzae SICC 3.302, was maintained on potato 

dextrose agar (PDA) slants. After two days incubation at 

30 o C, the slants were stored at 4 o C, for one month at most 

until use. 

Preparation of spore suspension 

Spore suspensions were prepared as previously (Córdova- 

López et al., 1996), the slants were suspended with 10ml 

0.1% tween 80 sterile water and counted in blood 

counting chamber. 

Submerged conditions 

The liquid medium were formed by five carbon sources 

(glucose, sucrose, maltose, glycerol and sodium acetate) 

at 16g C/l, four nitrogen sources ( bactopeptone, urea, 

(NH4)2SO4 and NaNO3) at 1.58g N/l and mineral element 

( 0.1 g/l K2HPO4, 0.05 g/l KCl, 0.05 g/l MgSO4·7H2O and 

0.001 g/l FeSO4) in this experiment. Carbon, nitrogen and 

mineral element solutions were sterilized separately at 

121 o C for 30min. 

The cultures were carried out in 250 ml Erlenmeyer flasks 

containing 95ml of different culture media. Each flask 

was inoculated with 5 ml of a spore suspension 

(Containing 10 6 spores ml -1 ), and incubated at 30±0.5 o C 

for 24, 48, 72, 96 and 120h. All cultures were incubated 

on an orbital shaker at 120rpm. The fungal were harvested 

by filtering through a weighed filter (Millipore HVLP 

04700, porosity 0.45µm) and the cake was washed with 

distilled water. Then, the dry weight is measured after 

drying in an oven at 65 o C for 48h. The mycelium was 

grounded in a mortar, redried at 65 o C for 24h and stored 

in a desiccator until analysed. 

Solid state conditions 

Because of the difficulty, or impossibility, of separating 

mycelium from solid state fermentation, the fungi was 

grown on agar medium to imitate the solid state 

conditions. The composition of agar medium was the 

same as the liquid medium except that each agar medium 

contained purified agar (15g/l). The sterilized and 

weighed filter (Millipore HVLP 04700, porosity 0.45µm) 

was placed on each agar plate, and it was inoculated 0.2 

ml spore suspension (Containing 10 6 spores ml -1 ). The 

agar plates were incubated at 30±0.5 o C in static 

conditions for 48h. The filter with the fungus was washed 

with distilled water and dried in an oven at 65 o C for 48h. 

The pulverization and storage was the same as Submerged 

conditions. 

Glucosamine determination 

For fungal chitin hydrolysis into N-acetyl glucosamine, 

20mg dried biomass was incubated with 2ml of H2SO4 

(72%) in a test tube. After standing on a rotary shaker 

Canadian Journal of Pure and Applied Sciences 

(130rpm) for 60min at 25 o C, it was diluted with 3ml of 

distilled water and autoclaved at 121 o C for 2h. The 

hydrolyzate was neutralized to pH 7.0 with 10M and then 

0.5M NaOH using a pH meter, and diluted to 100ml. 

Finally, glucosamine was assayed by the colorimetric 

method described by Tsuji et al. (1969) and modified by 

Ride and Drysdale (1972). 1ml diluted hydrolysate was 

mixed with 1ml of NaNO2 (5%) and 1ml of KHSO4 (5%) 

in a centrifuge tube. After shaking occasionally for 

15min, it was centrifuged at 3500rpm for 5min; 2ml of 

supernatant was mixed with 0.67ml of NH4SO3NH2 

(12.5%) and shaken for 3min. To the mixture was added 

0.67ml of 3-methyl-2-benzothiazolinone hydrazone 

hydrochloride (MBTH; 0.5%, prepared daily), and then 

the mixture was boiled for 3min and immediately cooled 

to room temperature; 0.67ml of FeCl3 (0.5%, prepared 

within 3 days) was added. After standing for 30min, the 

absorbance at 650nm was measured spectrophotometrically. 

The glucosamine content was calculated as 

milligrams per gram of fungal biomass according to the 

standard curve. 

Protein assay 

To determine the total protein of fungal cake, micro 

Kjeldahl method was used (AOAC, 1990). All reagents 

were of analytical grade, and determinations were 

conducted in triplicate unless stated otherwise. The total 

protein of fungal cake was expressed as milligrams per 

gram of fungal biomass. 

RESULTS AND DISCUSSION 

Effects of the age of the culture on fungal glucosamine 

and fungal protein content 

Biomass, glucosamine and fungal protein were 

determined from Aspergillus oryzae grown in liquid 

medium (sucrose and NaNO3) for 24, 48, 72, 96, 120h 

(Table 1). The fungal growth almost stopped after 48h 

cultivation and the biomass maintained at a relatively 

stable value. The glucosamine and fungal protein content 

of mycelium grown in liquid medium was 115 and 396 

millgram per gram dried biomass respectively for 24h, 

and maintained at a nearly constant in the monitoring 

time. Scotti et al. (2001) and Desgranges et al. (1991) 

have obtained exactly the same results of the glucosamine 

respectively for Cunninghamella elegans and Beauveria 

bassiana. Moreover, the ratio of glucosamine and fungal 

protein was almost a constant too. It appears that the 

glucosamine, fungal protein content and the correlation of 

glucosamine and fungal protein of Aspergillus oryzae are 

constant during all cultivation time. 

Effects of the culture conditions on fungal glucosamine 

and fungal protein content 

To compare the culture condition on fungal glucosamine 

and fungal protein content, the nutrition was the same in 

submerged and solid state conditions, except containing

Table1. Evolution of glucosamine and fungal protein content during cultivation time. 

15g/l agar in solid state condition. As shown in table 2 

and table 3, the glucosamine and fungal protein content of 

mycelium grown in submerged medium were higher than 

those obtained in solid state conditions on the 

corresponding medium. But, the correlation of 

glucosamine and fungal protein were very close between 

submerged (3.57mg fungal protein per millgram 

glucosamine) and solid state conditions (3.59mg fungal 

Chen et al. 1129 

Cultivation time(hours) 

Items 24 48 72 96 120 

Biomass(g) 0.17±0.02 0.60±0.06 0.65±0.02 0.64±0.06 0.65±0.06 

Glucosamine (mg/g) a 115.07±2.27 119.98±3.27 116.92±8.37 117.69±3.35 119.29±4.09 

Fungal protein (mg/g) b 396.18±17.85 391.43±8.34 398.22±6.49 400.92±3.97 398.33±9.61 

a c 3.44±0.21 3.26±0.13 3.42±0.29 3.41±0.06 3.20±0.13 

a b 

Expresses in mg glucosamine per gram of biomass dry weight. Expresses in mg fungal protein per gram of biomass dry weight. 

c 

Represents the correlation of fungal protein and glucosamine (expressed in mg fungal protein per millgram glucosamine). Data 

represents the means ± SEM, n=3. 

Table 2. Effects of carbon source on glucosamine and protein content in submerged and in solid state conditions, with 

similar nutrients. 

Culture conditions 

Bactopeptone 

+ carbon source 

Glucosamine (mg/g) a Fungal protein (mg/g) b a c 

Glucose 109.84±1.57 394.60±8.22 3.59±0.28 

Submerged 

conditions 

Sucrose 

Maltose 

Glycerol 

109.85±12.42 

111.80±2.81 

111.77±5.90 

395.39±9.42 

401.09±2.73 

400.62±6.26 

3.60±0.07 

3.59±0.07 

3.59±0.17 

Sodium Acetate 115.53±11.74 393.52±15.90 3.40±0.11 

Glucose 84.10±3.43 305.03±11.09 3.63±0.27 

Solid state 

conditions 

Sucrose 

Maltose 

Glycerol 

85.07±1.31 

85.30±0.18 

84.32±0.59 

306.64±9.74 

307.61±15.34 

296.62±48.25 

3.60±0.07 

3.61±0.17 

3.52±0.57 

Sodium Acetate 86.70±2.71 309.25±5.92 3.57±0.10 

a b 


c 



Table 3. Effects of nitrogen source on glucosamine and protein content in submerged and in solid state conditions, with 

similar nutrients. 

Culture conditions 

Sucrose 

+ nitrogen source 

Glucosamine (mg/g) a Fungal protein (mg/g) b a c 

Bactopeptone 109.85±2.42 395.39±1.42 3.60±0.07 

Submerged Sodium nitrate 111.11±18.82 392.80±29.64 3.54±0.32 

conditions Ammonium Sulfate 136.44±2.88 336.96±51.00 2.47±0.33 

Urea 135.10±5.74 321.39±36.31 2.39±0.36 

Bactopeptone 85.07±1.31 306.64±9.74 3.60±0.07 

Solid state Sodium nitrate 84.99±1.37 303.75±20.43 3.57±0.24 

conditions Ammonium Sulfate 93.11±5.56 226.30±13.31 2.43±0.13 

Urea 93.21±7.08 233.68±22.77 2.53±0.41 

a b 


c 



protein per millgram glucosamine) on the corresponding 

medium. Contrary to the results obtained by Ride and 

Drysdale (1971) on Fusarium oxysporum, our results 

permit to deduce that for Aspergillus oryzae, the 

glucosamine content is not influenced by the culture 

conditions. Scotti et al. (2001) reported the same result on 

Cunninghamella elegans.

1130 

Effects of the medium composition on fungal 

glucosamine and fungal protein content 

To show the influence of the medium composition on 

glucosamine and fungal protein content, two experiments 

were carried out in submerged and solid state conditions. 

The results (Table 2) show that the glucosamine, fungal 

protein content and the ratio of glucosamine and fungal 

protein were not influenced by carbon sources, whether in 

submerged or solid state condition. Thus, Aspergillus 

oryzae cultivated on bactopeptone and different carbon 

sources had the same glucosamine (117.76mg 

glucosamine per gram of biomass dry weight) and fungal 

protein content (397.05mg fungal protein per gram of 

biomass dry weight) in submerged condition, whereas it 

contained only a mean value of 85.10mg glucosamine per 

gram of biomass dry weight and 305.03mg fungal protein 

per gram of biomass dry weight in solid state condition. 

Moreover, the correlation of glucosamine and fungal 

protein were almost the same value. 

Consequently, within different carbon source, the 

glucosamine, fungal protein content can be considered as 

a good indicator of the biomass growth. Furthermore the 

glucosamine amount can be considered as a good 

indicator of fungal protein. 

The glucosamine, fungal protein content and the ratio of 

glucosamine and fungal protein, grown on media 

containing different nitrogen sources and sucrose as 

carbon sources, were determined. As shown in table 3, the 

glucosamine, fungal protein content and the ratio of 

glucosamine and fungal protein varied according to the 

carbon source. 

The results can be divided in two categories in submerged 

and solid state condition, respectively. Lowest 

glucosamine content and highest fungal protein content 

and the ratio of glucosamine and fungal protein values 

were obtained with bactopeptone and sodium nitrate, with 

a mean value of 110.48 and 85.03mg glucosamine per 

gram of biomass dry weight, 394.10 and 305.20mg fungal 

protein per gram of biomass dry weight and 3.57 and 3.59 

mg fungal protein per millgram glucosamine in 

submerged and solid state condition respectively. 

Ammonium sulfate and urea media give around 135.77 

and 93.16mg glucosamine per gram of biomass dry 

weight, 329.18 and 230.00mg fungal protein per gram of 

biomass dry weight and 2.43 and 2.48mg fungal protein 

per millgram glucosamine in submerged and solid state 

condition respectively. 

Obviously, the glucosamine amount can be considered as 

a good indicator of the fungal protein within each 

category of carbon source, whatever the culture 

conditions may be. Our results can be compared with 

those results obtained by Ride and Drysdale (1971) 


concerning Fusarium oxysporum and Desgranges et al. 

(1991) with Beauveria bassiana. Indeed, glucosamine 

content of Beauveria bassiana varied according to the 

medium composition, between 43 and 71mg of 

glucosamine per gram of fungal dry weight. But in this 

paper, comparison of glucosamine content was done for 

media having different compositions, as well for carbon 

and nitrogen sources as concentrations. Moreover, these 

results also show that glucosamine amount can be very 

different according to the strain. 

CONCLUSION 

The results of the experiments show that the glucosamine 

measurement of Aspergillus oryzae can be considered as a 

reliable indirect method for estimating fungal protein. In 

fact, the correlation of glucosamine and fungal protein 

content is constant whatever the age of the cultivation and 

culture conditions may be. On the other hand, the 

correlation appears to depend on the medium 

composition. Indeed, it was affected by the nitrogen 

source, but not by the carbon source. However, when the 

medium is well defined, this study demonstrates that the 

established results permit a good estimation of the fungal 

protein. 

ACKNOWLEDGEMENT 

We are grateful to the Program for Changjiang Scholars 

and Innovative Research Team in the University, No. 

IRT0555, and the Double-funded for 211 Project of 

Sichuan Agricultural University. 

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2001. Glucosamine measurement as indirect method for 

biomass estimation of Cunninghamella elegans grown in 

solid state cultivation conditions. Biochemical 

Engineering Journal. 7 (1):1-5. 

Sparringa, RA. and Owens, JD. 1999. Glucosamine 

content of tempe mould, Rhizopus oligosporus. 

International Journal of Food Microbiology. 47(1-2):153- 

157. 

Tsuji, A., Kinoshita, T. and Hoshino, M. 1969. Analytical 

chemical studies on amino sugar. II. Determination of 

hexosamines using 3-methyl-2-benzothiazolone 

hydrazone hydrochloride. Chemical and Pharmaceutical 

Bulletin. 17:1505-1510. 

Wei, PL., Cen, PL. and Sheng, CQ. 2006. Comparison of 

three biomass estimation methods in solid state 

fermentation. Journal of Food Science and Biotechnology. 

25(1):60-64. 

Wu, K., Yang, BH., Zhang, J., Liu, B., Cai, JM., Pan, RR. 

and Marinus, M. 2002. Trichoderma viride biomass assay 

and its cellulase synthesis Characteristic. Food and 

Fermentation Industries. 28(8):9-12. 

Received: Jan 19, 2010; Revised: April 26, 2010; Accepted: 

May 4, 2010


Vol. 4, No. 2, pp. 1133-1139, June 2010 

ISSN: 1715-9997 

PSEUDOMONAS AERUGINOSA ASSOCIATED WITH NEGATIVE 

INTERACTIONS ON COLIFORM BACTERIA GROWTH 

*Ulrich Vaconcelos 1 , Maria Alice Gomes de Andrade Lima 2 and Glicia Maria Torres Calazans 1 

1 Universidade Federal de Pernambuco, Centro de Ciências Biológicas, Departamento de Antibióticos, Av. Prof. Aurélio 

de Castro Cavalcanti, 79/104, CEP 51210-020, Recife-PE, Brazil 

2 Universidade Federal de Pernambuco, Centro de Tecnologia e Geociências 

Departamento de Engenharia Química, Recife-PE, Brazil 

ABSTRACT 

Screening aquatic Pseudomonads which inhibit growth of Escherichia coli and Enterobacter aerogenes produced sixteen 

strains of pyocyanin-producing Pseudomonas aeruginosa. Cell enumeration was carried out by the Most-Probable- 

Number technique for 96h by using diluted Müeller-Hinton broth. Conditions favouring pyocyanin production resulted 

in reduced growth of coliform strains. Representative strains were resistant to ciprofloxacin, norfloxacin and imipenem. 

It was also verified that a high population of coliform strains was reached when grown individually. Results show that 

there was an antagonistic phenomenon provided by Pseudomonas aeruginosa against coliform bacteria when pyocyanin 

was formed. This paper highlights the risks for human and environmental health that this phenomenon represents. 

Keywords: Pyocyanin-producing Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, antagonism. 

INTRODUCTION 

Pseudomonas aeruginosa (Migula, 1894) is a formidable 

widespread organism occurring naturally in water and 

other environments (Trivedi et al., 2008; Shrivastava et 

al., 2004). However it is most often described as an 

opportunistic pathogen involved in nosocomial infections 

and episodic outbreaks (Bou et al., 2009; Corvec et al., 

2008). 

Pyocyanin (phenazine 5-methyl-1-hydroxy phenazinium 

betaine) is a pigment produced by over 90% of strains 

(Mavrodi et al., 2001; Reyes et al., 1981) and some 

authors suggest that pyocyanin is particularly responsible 

for the antagonistic phenomenon against other 

microorganisms through the generation of reactive 

oxygen species which would be expected to exert 

antimicrobial activity toward coliform bacteria (D’áquila 

et al., 2000; Guilherme and Silva, 2000; Duncan et al., 

1999). 

The coliform bacteria comprise more than 30 

taxonomically different microorganisms. Genera 

Escherichia and Enterobacter are among the most 

important of the group. Since the 19 th century, coliform 

bacteria have been considered effective indicators of 

recent fecal pollution in water and E. coli (Escherich, 

1895) is the most important microorganism from this 

point of view (Foppen and Schijven, 2006). 

There are several common laboratory methods used for 

estimating concentration of viable cells. Most-Probable- 

*Corresponding author email: ulvasco@gmail.com 

Number (MPN) is a rapid and simple method and 

important reasons for choosing this method can be listed: 

1- when replacing a solid medium method due to the 

kinetic of growth of microorganisms that are highly 

variable and would obscure colonies of the organism of 

interest; 2- It is useful when microorganisms of interest 

produce some detectable product and cells can be 

estimated; 3- solid media may have factors such as heavy 

metals that alter the reliability of the count or interfere 

with the aim of the experimental plan and, 4- even if 

contaminating organisms overgrow the culture, the cells 

of interest can still be estimated (Gerhardt et al., 1994). 

Coliform enumeration may be affected by several 

physical and chemical sources such as temperature, pH, 

chloride content, salinity and metals which could mask 

the analysis leading to false-negative results. In addition, 

biological agents also interfere by inhibiting coliform cell 

growth through various mechanisms which includes 

amensalism, pigment concentration and competition for 

nutrients between non-coliforms and coliforms 

(Spangenberg, 2007). 

Several microorganisms may be antagonistic to the 

coliform group. Due to its opportunistic nature and 

metabolic versatility, P. aeruginosa strains are one of 

most important injury-promoting species of coliform 

bacteria inhibition in aquatic environments (Tamagnini 

and Gonzáles, 1997). Earlier data on negative interactions 

toward E. coli in water analysis identified some genera 

other than Pseudomonas may also be involved in this 

antagonistic phenomenon, such as Gram-positive

1134 

Actinomyces, Micrococcus and Sarcina, Gram-negative 

Flavobacterium and yeasts (Hutchinson et al., 1943). 

This paper reports a negative interaction of P. aeruginosa 

strains isolated from different aquatic sources against 

coliforms E. coli and E. aerogenes (Kruse, 1896). Tests 

were conducted to detect this phenomenon by measuring 

population of P. aeruginosa and coliform bacteria 

incubated simultaneously by simulating a recent coliform 

contamination in an environment where pyocyaninproducing 

P. aeruginosa strains were already established. 


Bacterial strains, media and identification methods 

A total of sixteen aquatic strains of P. aeruginosa were 

investigated in this study. All strains were isolated from 

environmental and clinical water samples surrounding 

Recife, capital of the state of Pernambuco, Brazil and data 

are shown in Table 1. Representative E. coli EC-5 and E. 

aerogenes EA-1, respectively, were isolated from distinct 

well water samples where P. aeruginosa was absent. The 

reference type strains of P. aeruginosa ATCC 27853, E. 

coli ATCC 25922 and E. aerogenes ATCC 15012 were 

also included in the study. 

All P. aeruginosa strains were identified by using 

asparagine and acetamide broth at 37±2°C for 24-48h of 

incubation, fluorescence at 360±20nm wavelength, 

growth on cetrimide Agar (Acumedia, Maryland, US) and 

enzymatic oxidase test (Probac do Brasil, São Paulo, 

Brazil). In order to guarantee conditions favouring the 


Table 1. The aquatic sources of Pseudomonas aeruginosa strains. 

enhancement of pyocyanin, P. aeruginosa strains were 

grown on Pseudomonas P medium. During the tests, the 

presence of pyocyanin was verified by the discoloration 

of acidic KMnO4 solution and by observing fluorescence 

at 360±20nm (Health Protection Agency, 2003; APHA et 

al., 1998). 

E. coli and E. aerogenes strains were identified by using 

lactose broth and green brilliant bile broth at 37±2°C for 

24-48h of incubation, and confirmed in EC broth and 

eosin-methylene-blue agar, respectively. Biochemical 

tests: indole production, methyl-red test, Vokes-Proskauer 

test and citrate utilization, were also performed (APHA et 

al., 1998). 

Antibiotic sensitivity was assessed using Müeller-Hinton 

agar (Merk, Darmstadt, Germany) and antibiotic disks 

(Sensidisc-DME, São Paulo, Brazil). 

Characterization of strains inhibitory to coliform 

The assay was conducted as described by Ichikawa et al. 

(1971). P. aeruginosa strains were subcultured onto the 

surface of Müeller-Hinton agar disks and incubated in 

sterilized Petri dishes at 37±2°C for 48h. Then, coliforms 

were spread on the surface of Müeller-Hinton agar and 

disks containing grown P. aeruginosa were transferred 

onto the agar surface and incubated at 37±1°C overnight. 

Tests were carried out in triplicate. The inhibition zones 

formed were measured. A greater inhibition zone 

diameter indicated more coliform susceptibility to P. 

aeruginosa. 

Strains Source Counts (MPN.100ml -1 ) 

Multi-drug resistance (from 8 

antibiotics tested) 

PA1 Várzea Cemitery (well #1) > 1600 0 

PA2 Várzea Cemitery (well #2) 17x10 3 

2 

PA3 Várzea Cemitery (well #3) > 1600 0 

PA4 Tap (hospital) 7x10 3 

0 

PA5 Bottled water 26x10 3 

0 

PA6 Storage tank (hospital) 17x10 4 

0 

PA7 Well (domestic) 11x10 2 

PA8 Water cooler (school) ND 0 

PA9 Tap (school) ND 0 

PA10 Tap (school’s kitchen) ND 0 

PA11 Tap (kinder care’s kitchen) ND 0 

PA12 Tap (hospital) 23x10 4 0 

PA13 Tap (UFPE campus bathroom) ND 0 

PA14 Landfill (influent tank) 6x10 5 

PA15 Landfill (facultative lagoon) 2x10 4 0 

PA16 Well (industry plant) ND 3 

ND – not determined. Strain was isolated without quantification 

0 

3

Inhibitory activity test in liquid-state 

The three most harmful strains in the selective inhibitory 

activity assay on solid medium were studied using diluted 

Müeller-Hinton Broth (Vetec, Rio de Janeiro, Brazil). The 

assay comprised the P. aeruginosa and coliform 

enumeration of the Most-Probable-Number for a period of 

96h at intervals of 24h by using the multiple tube 

technique with 5 tubes per dilution: 10.0, 1.0 and 0.1mL. 

A sample from P. aeruginosa suspension (around 10 2 

CFU/mL) was inoculated and incubated at 37±1°C for 

72h, after which either EA-1 or EC-5 inoculation 

occurred by transferring their suspensions (around 10 2 

CFU/mL) into the flask and re-incubating overnight. Tests 

were also conducted between reference type strains. 

Control tests with each strain grown individually were 

also carried out using the same conditions and time 

intervals. 


In this study we investigated the antagonistic interactions 

among pyocyanin-producing P. aeruginosa strains 

characterized by in vitro assays on solid and in liquid 

medium where the coliform bacteria represented a recent 

contamination in an environment where P. aeruginosa has 

already been established. 

All P. aeruginosa strains previously confirmed their 

ability to produce pyocyanin and further showed 

antimicrobial activity against coliform bacteria, detected 

by measuring the inhibition zone diameters formed. 

Fluorescein production was also observed in five strains. 

Diameters from 9 to 17±0.1mm for EC-5 and from 7 to 

Vaconcelos et al. 1135 

29±0.1mm for EA-1 (p

1136 

shown in figure 1. Among wild-type strains, reduction in 

number of cells ranged 18.3-29.8±0.1% for EA-1 and 

from 9.8 to 40.3±0.1% for EC-1 (p

log MPN/100mL 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

Our findings were consistent to a classical study on the 

incidence and significance of microorganisms 

antagonistic to the coliform group which demonstrated a 

reduction of 28-97% when antagonist and E. coli were 

inoculated simultaneously (Hutchison et al., 1943). The 

lower reduction in percentage of coliforms found in this 

study was possibly caused by the different culture 

medium and initial cell concentrations used. 

The use of diluted medium stimulated competition for 

carbon sources where coliform bacteria and P. aeruginosa 

were forced to overlap. Technically, coliform bacteria 

would have advantages due to shorter doubling-time 

when compared to P. aeruginosa (Tamagnani and 

Gonzáles, 1997; Camper et al., 1991). Nevertheless, 

pyocyanin-producing P. aeruginosa strains have proven 

to be more nutritionally versatile and capable of 

adaptation when competing with another microorganism. 

Moreover, literature reported that under competition for 

nutrients, coliform bacteria usually delay in lag time 


24 48 72 96 

Time (h) 

against PA2 against PA14 against PA16 Control 

Fig. 2. Patterns of EA-1 growth inhibition in the presence of pyocyanin-producing Pseudomonas aeruginosa strains. 

Control refers to EA-1 grown individually. Single values from experiments repeated on at least two separate 

occasions. 

log MPN/100mL 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

24 48 72 96 

Time (h) 

against PA2 against PA14 against PA16 Control 

Fig. 3. Patterns of EC-5 growth inhibition in the presence of pyocyanin-producing Pseudomonas aeruginosa strains. 

Control refers to EC-5 grown individually. Single values from experiments repeated on at least two separate 

occasions. 

which would also explain the susceptibility of that group 

in the present study (Evans et al., 1981). 

Although coliform bacteria are the universal indicators of 

fecal pollution in waters for human consumption, 

detection of P. aeruginosa as a complementary test is 

matter of concern because presence of P. aeruginosa and 

low coliform bacteria concentration would hide a major 

risk and would compromise the results from current 

methods used for water analysis. 

P. aeruginosa creates an element of concern, as this 

species can cause nosocomial infections or multi-drug 

resistant strains which may be untreatable. The three most 

harmful strains in this study were also resistant to at least 

three important antibiotics used for the treatment of P. 

aeruginosa infections, as follows (concentration tested in 

µg.ml -1 ): ciprofloxacine (5), norfloxacine (10) and 

imipenem (10), and were sensitive to amikacin (30), 

ceftazidime (30), gentamicin (10), ticarciline/clavulanate

1138 

(75/10) and tobramycin (10). The other thirteen remaining 

strains were sensitive to all antibiotics tested. Multi-drug 

resistance may also be related to selective pressures 

exerted by the environment influencing physiological 

evolution as discussed early. 

Some authors have also discussed that P. aeruginosa 

tends to persist in water for a longer period of time and it 

is metabolically able to resist caustic agents in nature, as 

opposed to coliform survival which decreases with 

sunlight and allows opportunistic pathogens to grow 

(Schultz-Fademrecht et al., 2008; Ahlén et al., 1998). 

Guilherme and Silva (2000) affirmed that pyocyanin is 

accepted as the main factor in the antagonistic role of P. 

aeruginosa. Other mechanisms of action of pyocyanin 

have been discussed in literature, which include 

involvement with the redox reaction and formation of 

superoxide and hydrogen peroxide with further ATP 

depletion (O’Malley et al., 2003; Denning et al., 1998). 

The P. aeruginosa bacteriocins suggest a potential role in 

environmental ecology, and further studies must be 

carried out to evaluate the effect of pyocyanin 

concentration in the process of coliform inhibition. The 

presence of pyocyanin-producing P. aeruginosa strains 

themselves would lead to inhibitory activity against 

coliform bacteria. If such microorganisms belong to the 

feacal coliform group, great attention must be given due 

to their epidemiological importance. 

Reduction in coliform growth induced by P. aeruginosa 

may lead to situations where the drinking water would be 

considered potable, however, under favorable conditions, 

coliform re-growth may develop and interspecies 

equilibrium would re-establish within the environment. 

Hence, coliform bacteria activation even in the presence 

of Psedomonads may possibly portray a hazardous 

scenario and may lead to real human and environmental 

health risks. 

This study also provided some contribution to the 

knowledge of the action of pyocyanin-producing P. 

aeruginosa related to environmental control of coliform 

bacteria. Although our findings do not represent a general 

rule and site specific studies are needed, the 

methodological approach used here can be a relevant 

support tool when designing water analysis strategies. 

CONCLUSIONS 

The results of the experimental work undertaken in the 

present study described one inhibitory activity of P. 

aeruginosa against coliform bacteria in solid and liquid 

media. This effect could lead to human and environmental 

health risks if, for example, total coliform concentrations 

in water samples were low or not detectable as a result of 

the presence of P. aeruginosa, but then subsequent 


favorable conditions allow for coliform re-growth, 

including potentially pathogenic strains. 

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APHA, AWWA, WEF. 1998. Standard methods for the 

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Bou, R., Lorente, L., Aguilar, A., Perpiñán, J., Ramos, P., 

Peris, M. and Gonzáles, D. 2009. Hospital economic 

impact of an outbreak of Pseudomonas aeruginosa 

infections. Journal of Hospital Infection. 71:138-142. 

Camper, AK., McFeters, GA., Characklis, WG. and 

Jones, WL. 1991. Growth kinetics of coliform bacteria 

under conditions relevant to drinking water distribution 

systems. Applied and Environmental Microbiology. 

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Corvec, S., Poirel, L., Espaze, E., Giraudeau, C., 

Drugeon, H. and Nordmann, P. 2008. Long-term 

evolution of a nosocomial outbreak of Pseudomonas 

aeruginosa producing VIM-2 metallo-enzyme. Journal of 

Hospital Infection. 68:73-82. 

D’áquila, PS., Roque, OCD., Miranda, CAS. and Ferreira, 

AP. 2000. Quality assessment of the public water supply 

in Nova Iguaçu, Rio de Janeiro. Public Health Report. 

16:791-798. 

Denning, GM., Wollenweber, LA., Railssback, MA., Cox, 

CD., Stoll, L. and Britigan, BE. 1998. Pseudomonas 

pyocyanin increases interleukin-8 expression by human 

airway epithelial cells. Infect Immunology. 66:5777-5784. 

Duncan, SH., Doherty, CJ., Govan, JRW, Neogrady, S., 

Galfi, P. and Stewart, CS. 1999. Characteristics of sheeprumen 

isolates of Pseudomonas aeruginosa inhibitory to 

the growth of Escherichia coli O157. FEMS 

Microbiology Letters. 180:305-310. 

Evans, TM., Waarvick, CE., Seidler, RJ. and 

LeChevallier, MW. 1981. Failure of the Most-Probable- 

Number technique to detect coliforms in drinking water 

and raw water supplies. Applied and Environmental 

Microbiology. 41:130-138. 

Foppen, JWA. and Shijven, JF. 2006. Evaluation of data 

from the literature on the transport and survival of 

Escherichia coli and thermotolerant coliforms in aquifers 

under saturated conditions. Water Research. 40:401-426. 

Gerhardt, P., Murray, RGE., Wood, WA. and Krieg, NR. 

1994. Methods for general and molecular bacteriology. 

American Society for Microbiology. Washington, DC, 

USA.

Guilherme, EFM. and Silva, JAM. 2000. Pseudomonas 

aeruginosa as an indicator of water pollution. Revista 

Higiene Alimentar. 14:43-47. 

Heath Protection Agency. 2003. Enumeration of 

Pseudomonas aeruginosa by membrane filtration. 

National Standard Methods issue 2.3, W6i2.3. London, 

UK. 

Hutchison, D., Weaver, RH. and Scherago, M. 1943. The 

incidence and significance of microorganisms 

antagonistic to Escherichia coli. Journal of Bacteriology. 

45:29. 

Ichikawa, T., Date, M., Ishikura, T. and Ozaki, A. 1971. 

Improvement of kasugamycin-producing strain by the 

agar piece method and the prototroph method. Folia 

Microbiologica. 16:218-224. 

Legnani, P., Leoni, E., Rapuano, S., Turin, D. and 

Valenti, C. 1999. Survival and growth of Pseudomonas 

aeruginosa in natural mineral water: a 5-year study. 

International Journal of Food Microbiology. 53:153-158. 

Mavrodi, DV., Bonsall, R., Delaney, SM., Soule, MJ., 

Phillips, G. and Thomashow, LS. 2001. Functional 

analysis of genes for biosynthesis of pyocyanin and 

phenazine-1-carboxamide from Pseudomonas aeruginosa 

PA01. Journal of Bacteriology. 183:6454-6465. 

O’Malley, YQ., Abdalla, MY., McCormick, ML., Reszka, 

KJ., Denning, GM. and Britigan, BE. 2003. Subcelluar 

localization of Pseudomonas aeruginosa pyocyanin 

cytotoxicity in human lung epithelial cells. American 

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1981. Identification of Pseudomonas aeruginosa by 

pyocynin production on Tech agar. Journal of Clinical 


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microorganisms both in river water and benthic biofilms. 

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P. and Chaturvedi, UC. 2004. Suboptimal Chlorine 

treatment of drinking water leads to selection of 

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corrugata. Microbiology Reviews. 163:329-336. 

Received: April 10, 2010; Accepted: May 14, 2010


Vol. 4, No. 2, pp. 1141-1149, June 2010 

ISSN: 1715-9997 

INHIBITORY EFFECTS OF RCG1, A β-GALACTOSIDE-BINDING LECTIN 

FROM RANA CATESBEIANA (AMERICAN BULLFROG) OOCYTES 

AGAINST HUMAN AND PHYTOPATHOGENS 

*Sarkar MA Kawsar 1 , Sarkar MA Mamun 2 , Md S Rahman 3 , Hidetaro Yasumitsu 1 and *Yasuhiro Ozeki 1 

1 Laboratory of Glycobiology and Marine Biochemistry, Department of Genome System Sciences, Graduate School of 

Nano Biosciences, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan 

2 Department of Botany, Faculty of Science, University of Chittagong, Chittagong-4331 

3 Department of Microbiology, Faculty of Science, University of Chittagong, Chittagong, Bangladesh 

ABSTRACT 

A β-galactoside-binding galectin-1 (RCG1) was purified from the oocytes of the American bullfrog, Rana catesbeiana 

by affinity column chromatography and was evaluated for its growth of inhibition effects on bacteria and fungi. Through 

SDS-PAGE and gel permeation chromatography, RCG1 was found to be a non-covalently-bonded dimeric protein that 

consisted of two 15 kDa polypeptide subunits. This lectin showed significant hemagglutinting activity against 

trypsinized human and rabbit erythrocytes and it was inhibited by asialofetuin, thiodigalactoside and lectose. RCG1 was 

screened for in vitro antibacterial activity against eleven human pathogenic bacteria and significantly inhibited the 

growth of gram-positive bacteria than the gram-negative bacteria. Bacillus subtilis (10±1 mm) and Bacillus cereus (8±1 

mm) were exhibited the highest growth of inhibition by the lectin (250 µg/disc). At the same time, RCG1 showed good 

growth inhibition against the gram-negative bacterium, Salmonella typhi but not others such as, Pseudomans sp., 

Escherichia coli and Vibrio cholerae. Antifungal activity also was investigated against six phytopathogenic fungi based 

on a food poisoning technique. Among the test fungi, the maximum inhibition of mycelial growth were observed in 

Fusarium equiseti (18.4±1%) followed by Colletotrichum corchori (13.3±1%) and Curvularia lunata (8.4±1%) at a 

concentration of the RCG1 100 µg/ml. These results suggest that future findings of lectin applications obtained from 

bullfrog oocytes may be of importance to clinical microbiology and have potential drug-resistant agents. 

Keywords: Rana catesbeiana, antibacterial, antifungal, inhibition zone, mycelial growth, lectin. 

INTRODUCTION 

Galectins, comprise S-type β-galactosyl binding proteins 

that are present in vertebrates and invertebrates and do not 

require divalent cations for their binding activity (Vasta, 

2009). The galectin family has been subdivided in several 

subgroups; ‘proto’, ‘tandem’ and ‘chimera’ (Rabinovich et 

al., 2007) and they are bind to lactose/Nacetyllactosamine 

(Ahmed and Vasta, 1994). Galectin has 

been found in many organs of many organisms including 

liver, placenta, lung, heart, spleen, testis, and even also 

electric organ of electric eel - however the physiological 

function of galectin is still unknown. Galectins have 

activity about apoptosis (Koh et al., 2008), cell adhesion 

(Stowell et al., 2008), immune response and cancer 

metastasis (Hernandez and Baum, 2002) in vitro, all 

suggesting that the protein has highly potency for 

creatures. In amphibians, galectins have been purified 

from the genus Rana (Ozeki et al., 1991 a ), Bufo (Ahmed 

et al., 1996) and Xenopus (Shoji et al., 2003) and proto 

type galectin (galectin-1) was isolated in abundance from 

oocytes of the American bullfrog Rana catesbeiana living 

*Corresponding author email: akawsarabe@yahoo.com 

in aqua (Ozeki et al., 1991 a ) and found to exist free from 

the endogenous ligand and abundant in oocytes. American 

bullfrog oocytes galectin-1 is located in the yolk platelets 

and is distributed in the extracellular matrices of these 

organs after development into adults (Uchiyama et al., 

1997). It was also shown to have potential cell adhesive 

activity to human rhabdomyosarcoma cells (Ozeki et al., 

1991 b ) and tissue fibronectin was determined to be a 

putative endogenous ligand (Ozeki et al., 1995). 

Gram-positive bacteria are those that are stained blue or 

violet by gram staining. This is in contrast to gramnegative 

bacteria, which cannot retain the crystal violet 

stain, instead taking up the counterstain (safranin) and 

appearing red or pink. Gram-positive organisms are able 

to retain the crystal violet stain because of the high 

amount of peptidoglycan in the cell wall. Gram-positive 

cell walls typically lack the outer membrane found in 

gram-negative bacteria. The cell wall of virtually all 

bacteria consists of a rigid peptidoglycan layer that is 

either overlaid by an outer lipopolysaccharide (LPS) layer 

in gram-negative bacteria or remains exposed on the

1142 

surface of gram-positive bacteria. Peptidoglycan is a 

polymer of alternating N-acetylglucosamine (GlcNAc or 

NAG) and N-acetylmuramic acid (NAM) units connected 

by short pentapeptides. The β-1,4-glycosidic bond of the 

N-acetylglucosamine-N-acetylmuramic acid peptideglycan 

backbone can be hydrolyzed by lysozyme 

(muramidase; mucopeptide N-acetylmuramoylhydrolase), 

a ubiquitous enzyme involved in innate immune reaction 

of numerous animal species (Ito et al., 1999). Galectins 

can interact directly with bacterial surface glycans. 

Furthermore, bacterial infection can modulate galectin 

expression, which in turn regulates leukocyte function and 

inflammatory responses. Both gram-positive bacteria, 

such as Streptococcus pneumoniae and gram-negative 

bacteria, such as Klebsiella pneumoniae display surface 

carbohydrate galectin ligands (Mey et al., 1996; Mandrell 

et al., 1994). Galectins can binds to bacterial 

lipopolysaccharides (LPSs) in a dual manner: the C 

terminus CRD binds to lactosyl moieties of K. 

pneumoniae LPS, whereas the non-carbohydrate-binding 

N-terminal domain of galectin-3 binds to the lipid A 

moiety of Salmonella enterica subsp enterica serovar 

Minnesota LPS (Mey et al., 1996). Galectins also binds 

mycobacterial phosphatidylinositol mannosides, 

glycolipids that accumulate on the membrane of 

Mycobacterium-containing phagosomes during infections 

(Barboni et al., 2005). The galectin BbtGal-L from the 

cephalochordate amphioxus (Branchiostoma belcheri) 

specifically recognizes Vibrio vulnificus but not Vibrio 

parahaemolyticus or Staphylococcus aureus and its 

expression is upregulated by bacterial challenge (Yu et al., 

2007). Galectins also can recognize surface glycans on 

the surface of saprophytic or pathogenic fungi and could 

function as a macrophage receptor for fungal pathogens 

(Fradin et al., 2000). Interestingly, the selective binding of 

galectin-3 to the Candida albicans cell wall glycans is 

fungicidal (Kohatsu et al., 2006) as function in innate 

immunity. It has been reported that many lectins from 

marine invertebrates show antibacterial activity. The 

lectin from the horse mussel M. modiolus exhibited strong 

antibacterial activity against tested vibrio strains 

(Tunkijjanukij and Olafsen, 1998). T-antigen binding 

lectin purified from sea cucumber showed strong broad 

spectrum antibacterial activity against both gram-positive 

and gram-negative bacteria (Gowda et al., 2008). 

We previously evaluated the cell adhesion activity by 

human rhabdomyosarcoma cells and determined the 

glycan-binding properties of the lectin by using frontal 

affinity chromatography technology (FACT) (Kawsar et 

al., 2009). In this paper, we evaluate the antibacterial and 

antifungal activities by the determination of growth 

inhibitory effects of the purified lectin, RCG1 from the 

American bullfrog Rana catesbeiana oocytes against 

human and phytopathogens. 



Drugs and reagents 

A lactosyl-agarose column was purchased from Seikagaku 

Kogyo Co. Ltd., Japan and standard protein marker 

mixture (Daiichi-III) for SDS-PAGE was purchased from 

Daiichi Pure Chem. Co. Ltd., Japan. A bicinchoninic acid 

(BCA) kit was purchased from Pierce Co. Ltd., USA. 

Superdex 75 and Sephadex G-75 were obtained from GE 

Healthcare, USA. Agar, dextrose, peptone, beef extract 

were purchased from Merck Ltd., India and BDH Ltd., 

Bangladesh and were of the highest purity grade. Female 

American bullfrogs (Rana catesbeiana) were purchased 

from an experimental animal company, Nippon Seibutsu 

Zairyo Co. Ltd., Tokyo, Japan. Eggs were stored at -80 ° C 

or used after collection according to the situation. 

Purification of the lectin (RCG1) 

RCG1, β-galactoside-binding lectin was purified from the 

oocytes of the American bullfrog (Rana catesbeiana) by 

affinity column chromatography (Ozeki et al., 1991 a ). 

Briefly, unfertilized oocytes (100 g w/v) were obtained 

from the abdominal cavity of a female by laparotomy, 

homogenized in a cooled pestle and frozen in 500 ml of 

acetone. During homogenization lipids located in the 

upper layer were discarded. After removal of lipids 

several times via frozen acetone, the precipitate was dried 

completely into powdered form by vacuum drying. 

Acetone-extracted oocyte powder (10 g) was 

homogenized with 20 times (w/v) TBS (10mM 

Tris(hydroxymethyl)aminomethane-HCl, pH 7.4, 

containing 150 mM NaCl) and centrifuged at 27,500 g for 

1 h at 4 ° C and the supernatant was applied to an affinity 

column of lactosyl-agarose (5 ml) that was fitted with a 

Sephadex G-75 pre-column (5 ml). After application of 

extracts, the lactosyl-agarose column was extensively 

washed with TBS. The lectin was eluted with 100 mM 

lactose in TBS and each 1 ml of elution was collected in 

tubes with a fraction collector. The eluted fractions were 

analyzed by spectrophotometer at 280 nm. The fractions 

were dialyzed against 1,000 times volumes of TBS to 

remove free lactose. 

Hemagglutinating activity 

The hemagglutinating activity was performed using 1% 

(w/v) trypsinized and 0.25% glutaraldehyde-fixed rabbit 

and human erythrocytes as described previously (Matsui, 

1984). Erythrocytes were suspended at a concentration of 

1% (w/v) in TBS. In the general assay, 20 µl each of TBS, 

TBS containing 1% Triton X-100, and erythrocytes were 

added to 20 µl of the two times-serially-diluted lectin with 

TBS in 96 well V-shape titer plates for 1 h. The 

hemagglutination activity of the lectin was expressed as 

the titer defined as the reciprocal of the highest dilution 

giving positive hemagglutination.

Sodium dodecyl sulfate-polyacrylamide gel 

electrophoresis 

The molecular mass of the polypeptide was determined by 

sodium dodecyl sulfate polyacrylamide gel electrophoresis 

(SDS-PAGE). Purified lectin was mixed with an 

equal amount of sample buffer (20 mM Tris-HCl, pH 6.8; 

0.2% SDS, and 20% glycerol) and then heated at 70 ° C for 

15 min. Aliquots of 30 µl were applied to the well of a 

mini-slab gel (gel size: 80mm × 100 mm with 1 mm 

thickness; 12% and 5% polyacrylamide were used in 

separation and upper gels, respectively, constant current at 

30 mA for 1h) according to a previous report (Laemmli, 

1970). The following polypeptides were used as 

molecular mass markers; phosphorylase b (Mr 94 kDa), 

bovine serum albumin (Mr 66 kDa), ovalbumin (Mr 42 

kDa), carbonic anhydrase (Mr 30 kDa), trypsin inhibitor 

(Mr 20 kDa), and lysozyme (Mr 14 kDa). After SDS- 

PAGE, the gel was stained with 0.1% (w/v) Coomassie 

Brilliant Blue (CBB) R-250 in 40% (v/v) and 10% acetic 

acid (v/v) followed by discoloration by excessive staining 

with 40% methanol and 10% acetic acid. 

Gel permeation chromatography 

The purified lectin was dissolved in 2.5% glycerol and 

subjected to gel permeation chromatography (GPC) 

utilizing a Superdex 75 column (1.0 × 65 cm) connected 

to an FPLC system (GE Healthcare, USA) in the presence 

of 50 mM lactose containing TBS. The elution time of the 

lectin from the column was detected by UV at an 

absorbance of 280 nm. Bovine serum albumin (66 kDa), 

ovalbumin (43 kDa), carbonic anhydrase (30 kDa), 

myoglobin (17 kDa) ribonuclease (14 kDa) and 

cytochrome c (6; 12 kDa) were used as standard 

molecular marker. 

Protein determination 

Protein concentrations were determined using BCA 

protein assay kit (Smith et al., 1985; Wiechelman et al., 

1988) with bovine serum albumin as the standard by 

measuring absorbance at 562 nm with spectrophotometer 

ND-1000 (Nano Drop Tech. Inc., USA). 

Tested pathogens 

The bacterial and fungal pathogens used in this study 

were obtained from the Microbiology Laboratory, 

Department of Microbiology, University of Chittagong, 

Bangladesh. Gram-positive bacterial strains were Bacillus 

subtilis BTCC 17, Bacillus cereus BTCC 19, Bacillus 

megaterium BTCC 18 and Staphylococcus aureus ATCC 

6538. Gram-negative bacterial strains were Salmonella 

typhi AE 14612, Salmonella paratyphi AE 146313, 

Shigella dysenteriae AE 14396, Shigella sonnei CRL 

(ICDDR,B), Escherichia coli ATCC 25922, Vibrio 

cholerae (CRL (ICDDR,B) and Pseudomonas sp. CRL 

(ICDDR,B). The fungal pathogens were Alternaria 

alternata (Fr.) Kedissler, Botryodiplodia theobromae Pat., 

Curvularia lunata (Wakker) Boedijin, Colletotrichum 

Kawsar et al. 1143 

corcori Ikata (Yoshida), Fusarium equiseti (Corda) Sacc 

and Macrophomina phaseolina (Tassi) Goid. 

Culture and media 

Standard NA (Nutrient Agar) medium was used for 

growing bacterial strains throughout the work whereby 20 

g of agar powder, 5 g of peptone, 3 g of beef extract and 

0.5 g of NaCl were added per liter of water. The medium 

was autoclaved for 15 minutes at 121 ° C with 15 psi. Older 

cultures were transferred to freshly prepared NA slants 

separately for each species via sterilized bacterial loop. In 

such a way, four tubes were freshly prepared for each 

bacterial pathogen. These tubes of inoculated slants were 

incubated at 35±2 ° C in incubator for 18-24 hours and each 

culture was used throughout for antibacterial screening 

studies. For preservation of the stock culture, one set of 

culture slants were kept in polythene bag, properly tied 

and preserved at 10 ° C. 

Antibacterial assay 

The in vitro growth inhibition assay against bacteria by 

RCG1 was carried out by the disc diffusion method 

(Bauer et al., 1966). In this method, sterilized paper discs 

of 4 mm in diameter and petridishes of 150 mm in 

diameter were used throughout the experiment. The 

autoclaved Mueller-Hinton agar medium, cooled to 45 ° C, 

was poured into sterilized petridishes to a depth of 3 to 4 

mm and after solidification of the agar medium; the plates 

were transferred to an incubator at 37 ° C for 15 to 20 

minutes to dry off the moisture that develops on the agar 

surface. The plates were inoculated with the standard 

bacterial suspensions (as of McFarland 0.5 standard) by 

help of sterilized glass and allowed to dry for three to five 

minutes. Dried and sterilized filter paper discs were 

treated separately with 20 µl (250 µg/disc) from 5% 

phosphate buffered saline (PBS, pH 7.4) solution of 

RCG1 using a micropipette, dried in air under aseptic 

condition and were placed at equidistance in a circle on 

the seeded plate. A control plate was also maintained in 

each case without any test material. These plates were 

kept for 4-6 hours at low temperature and the RCG1 

diffused from disc to the surrounding medium by this 

time. The plates were then incubated at 35±2 ° C for 24 

hours to allow maximum growth of the organisms. The 

antibacterial activity of the test agent was determined by 

measuring the mean diameter of zone of inhibitions in 

millimeter. Each experiment was repeated thrice. 

Galactose was used as negative control. All the results 

were compared with the standard antibacterial antibiotic 

ampicillin (20 µg/disc), (BEXIMCO Pharm., Bangladesh 

Ltd.). 

Antifungal activity 

The in vitro antifungal activity of the bullfrog oocytes 

lectin was determined by the poisoned food technique 

(Grover and Moore, 1962) with some modification (Miah 

et al., 1990). Potato dextrose agar (PDA) medium was

1144 

used for the culture of fungi. A required amount of PDA 

was taken in conical flasks separately and was sterilized 

by autoclave (121 ° C, 15 psi) for 15 minutes. Purified 

lectin (in PBS solution) was mixed with sterilized melted 

PDA medium to have 100 µg/ml in PDA and this was 

poured (about 20 ml/plate) in sterilized petridishes. At the 

center of each plate, 5 days old fungal mycelial block (4 

mm in diameter) was inoculated and incubated at 27 ° C. A 

control set was also maintained in each experiment. 

Linear mycelial growth of fungus was measured after 3-5 

days of incubation in triplicate. The average of two 

measurements was taken as mycelial colony diameter of 

the fungus in milimeter. All the antifungal results were 

compared with the standard antifungal antibiotic nystatin 

(100 µg/ml) in PDA. Galactose was used as negative 

control. The percentage inhibition of radial mycelial 

growth of the test fungus was calculated as follows: 

% Inhibition = (C-T/C) × 100 

Where, C = diameter of the fungal colony in the control 


petridish and T = diameter of the fungal colony in the 

treated petridish. 


Table 1. Purification of galactose-binding lectin from bullfrog Rana catesbeiana oocytes. 

Purification Steps 

Titer 

(HU) 

Volume 

(ml) 

Total 

activity 

The supernatant extract from acetone-powdered bullfrog 

oocytes showed strong hemagglutinating activity and the 

activity was cancelled by the co-presence of βgalactosides 

such as lactose but not by the α-galactose or 

melibiose and therefore it was applied to a lactosylagarose 

column. After column washing with TBS, the 

column bound lectin was specifically purified by the 

addition of 100 mM lactose in TBS (Fig. 1). Four 

milligrams of RCG1 was purified from 100g (wet weight) 

of unfertilized oocytes and it was concentrated 

approximately 410 fold by affinity chromatography 

(Table 1). The purified Rana catesbeiana oocyte galectin- 

1 (RCG1) showed strong hemagglutinating activity 

against trypsinized and glutaraldehyde-fixed human 

erythrocytes and was slightly more sensitive to human 

erythrocytes when compared to those from rabbit (Table 

Protein 

conc. 

(mg/ml) 

Specific 

activity 

Purificati 

on fold 

Recovery 

of activity 

(%) 

Crude extract obtained by 

acetone 

512 100 51200 2.1 2.4 1 100 

Purified lectin 4096 5 20480 0.83 987 411 40 

Total activity is shown by Titer × volume 

Specific activity was shown by titer/mg of protein. 

Fig. 1. Lactosyl-agarose affinity chromatography. 

Crude extract of R. catesbeiana oocytes were applied to 

an affinity column equilibrated with TBS. The column 

was extensively washed with TBS and the lectin was 

eluted with 100 mM lactose in TBS (arrow). The 

column bound fractions shown by the bar were 

collected and designated as purified lectin after dialysis 

against TBS. 

kDa 

97 

66 

42 

30 

20 

14 

NR R 

M L C L M 

kDa 

97 

66 

42 

30 

20 

14 

Fig. 2. SDS-polyacrylamide gel electrophoresis. C: 

crude extract of R. catesbeiana oocyte from acetone 

powdered; L: purified RCG1; NR: non-reducing and R: 

reducing conditions. 12% polyacrylamide was used as 

separating gel and the gels were stained with 

Coomassie brilliant blue. M: molecular weight markers 

(from top to bottom): phosphorylase b (97 kDa); bovine 

serum albumin (66 kDa); ovalbumin (42 kDa); carbonic 

anhydrase (30 kDa); trypsin inhibitor (20 kDa) and 

lysozyme (14 kDa).

2). RCG1 was purified as a single 15 kDa polypeptide 

under both reducing and non-reducing conditions by 

SDS-PAGE (Fig. 2, lane L). However, crude extract of 

the acetone powdered oocytes contained various proteins 

by SDS-PAGE (Fig. 2, lane C). On the other hand, RCG1 

appeared to have a molecular mass at 30 kDa in GPC 

(Fig. 3A & B), indicating that the lectin was a noncovalent 

bound dimeric protein consisting of two 15 kDa 

polypeptide subunits. A partial primary structure analysis 

of the lectin showed that the amino acid sequence of the 

protein belonged to a superfamily of galectins (Ozeki et 

al., 1991 c ) in addition to the carbohydrate binding 

specificity against β-galactoside. 

Table 2. Hemagglutinating activity of RCG1 by human 

and rabbit erythrocytes. 

Blood type* Titer (HU) 

Human (Type O) 2048 

Human (Type A) 2048 

Human (Type B) 1024 

Rabbit 1024 

*Trypsinzed and glutaraldehyde fixed erythrocytes were used. 

Absorbance at 280 nm 

0.3 

0.2 

0.1 

0 

A 

Marker 

1 2 3 4 5 6 

0 4 8 12 16 

Retention time (min) 


Molecular mass (da) 

Table 3. Antibacterial activity of RCG1 against grampositive 

bacteria. 

Name of bacteria 

Diameter of zone of inhibition in 

milimeter 

Lectin (250 Ampicillin* 

µg/disc) (20 µg/disc) 

Bacillus subtilis 10±1 20±1 

Bacillus cereus 8±1 19±1 

Bacillus 

megaterium 

4±1 18±1 

Staphylococcus 

aureus 

0 21±1 

Note: *Standard antibacterial antibiotic, Statistical analysis 

(RBD) at 1% level, organisms significant (F value 183.5), 

replica significant (F value 6.95). 

The inhibitory effects of eleven human pathogenic 

bacteria to RCG1 was tested and compared to that of the 

antibacterial antibiotic, ampicilin. The results of the 

sensitivity test are presented in Tables 3 and 4. It was 

found that the RCG1 (250 µg/disc) gave promising 

inhibitory effects against gram-positive bacteria such as 

Bacillus subtilis. The diameter zone of inhibition by the 

100000 

50000 

10000 

1 2 

3 

RCG1 

4 

5 

8 10 12 14 16 

Retention time (min) 

Fig. 3. Determination of the molecular weight by GPC. A: RCG1 (20 µg) was separated on a Superdex 75 column 

using FPLC system at a flow of 0.5 ml/min (chart speed is 0.5 cm/ml). B: Calibration line for the determination of 

molecular weight of RCG1 was determined using standard molecular mass marker proteins as; bovine serum albumin 

(1; 66 kDa), ovalbumin (2; 43 kDa), carbonic anhydrase (3; 30 kDa), myoglobin (4; 17 kDa), ribonuclease (5; 14 

kDa) and cytochrome c (6; 12 kDa). 

B 

6

1146 

addition of RCG1 was significantly effective for Bacillus 

subtilis and Bacillus cereus; 10 and 8 mm, respectively 

(Table 3) although the growth inhibition of Bacillus 

megaterium was less effective. On the other hand, RCG1 

showed good antibacterial activity against gram-negative 

bacterium Salmonella typhi but it did not inhibit the 

growth of other gram-negative bacteria such as 

Pseudomonas sp., Escherichia coli and Vibrio cholerae 

(Table 4), though the control antibiotic, ampicillin (20 

µg/disc) inhibited the growth against all gram-negative 

bacteria. Amongst all bacterial strains, the gram-positive 

bacteria were more susceptible to the lectin as compared 

to gram-negative bacteria. This result suggested that the 

structure of surface-exposed carbohydrates on grampositive 

bacteria were different even if they belong to the 

same genus within Bacillus. 

Table 4. Antibacterial activity of RCG1 against gramnegative 

bacteria. 

Name of bacteria 

Diameter of zone of 

inhibition in milimeter 

Lectin (250 Ampicillin* 

µg/disc) (20 µg/disc) 

Salmonella typhi 8±1 22±1 

Salmonella paratyphi 6±1 19±1 

Shigella sonnei 4±1 18±1 

Shigella dysenteriae 3±1 20±1 

Pseudomonas sp 0 19±1 

Escherichia coli 0 17±1 

Vibrio cholerae 0 18±1 

Note: *Standard antibacterial antibiotic, Statistical analysis 

(RBD) at 1% level, organisms significant (F value 183.5), 

replica significant (F value 6.95). 

A glycomics approach to determine the structure of 

surface glycans of bacteria may provide more useful 

clinical information to prevent disease using lectins. 

Indeed, galectins may be interesting candidates for 

interacting with bacteria owing to their specificity for βgalactosides 

in that they might be able to interact with 

some of the bacterial carbohydrates on the cell surface 

(Mandrell et al., 1994). The glycan binding profile of 

RCG1 was analyzed by frontal affinity chromatography 

(Kawsar et al., 2009) and it specifically recognizes 

branched complex type N-linked oligosaccharides having 

a lactosamine (Galβ1-4GlcNAc) structure. It seems 

reasonable that RCG1 may catch bacteria containing a 

lipopolysaccharide layer because the β-galactoside 

structure (Galβ1-4Glc) is contained in most of the major 

lipopolysaccharides at the non reducing terminal. In many 

galectins, a specific affinity moiety was reported against 

the N-acetyllactosamine and poly N-acetyllactosamine 

(Sharma et al., 1992), indicating that galectin family is 

available as a candidate molecule to trap gram-positive 

bacteria by binding to lipopolysaccharides that express 


the “asialo-lactoneo-series” structure. Recently, a 

lipopolysaccharide-binding lectin purified from the seeds 

of Eugenia uniflora showed antibacterial activity similar 

to RCG1, as it effectively inhibited growth against grampositive 

bacteria such as Bacillus subtilis (Oliveira et al., 

2008) and a β-galactoside binding pearl shell lectin 

purified from the marine bivalve, Pteria penguin showed 

antibacterial activity similar to RCG1 as well (Naganuma 

et al., 2006). Rhamnose-binding steelhead trout 

(Oncorhynchus mykiss) egg lectin inhibited the growth of 

gram-positive and gram-negative bacteria by recognizing 

lipopolysaccharide or lipoteichoic acid (Tateno et al., 

2002) and in addition, galectin interactions with 

lipopolysaccharides in the gram-negative bacteria as 

Salmonella minnesota inhibited the growth of same genus 

Salmonella in a similar fashion to RCG1 (Mey et al., 

1996). 

It has been reported (Yu et al., 2007) that host galectins 

can bind directly to glycoconjugates on the surface of 

bacteria, either facilitating or inhibiting pathogen entry, 

followed by positive and negative regulation of host 

innate and adaptive immunity. The presence of the lectin 

in the oocytes of R. catesbeiana led us to consider its 

possible biological involvement in the defense 

mechanisms of the species. RCG1 is the first lectin from 

frog reported to exhibit microbial growth inhibition. This 

activity was previously postulated for invertebrates 

(Inamori et al., 1999), which expressed soluble and bound 

membrane lectin forms that appeared to be one of the 

groups of molecules for recognition and defense. An 

instance of the above is provided by several types of 

hemocyte-derived lectins which may play a role of 

functional in the innate immunity. The same fact was 

suggested for mammalian lectins, such as mannosebinding 

lectins as collectin family, which play an 

important role in host-pathogen interactions by specific 

recognition of the cell surface substances of bacteria 

(Rabinovich and Gruppi, 2005). Some lectins seem to be 

useful for identification of pathogenic bacteria based on 

the specific binding moieties of lectins to the 

characteristic glycans on cell wall of such types of 

bacteria (Munoz-Crego et al., 1999). The characterization 

of glycans that presented the cell walls of gram-positive 

and gram-negative bacteria was characterized by lectins, 

finding to their peptidoglycan and lipopolysaccharides 

(Doyle, 1994). 

Contrastively, the interactions between lectins and 

glycans in fungi are not well known. In this study, we 

have found that RCG1 reduced the growth rate of some 

strains of fungi against six phytopathogenic strains by 

comparing with the antifungal antibiotic nystatin as 

positive control. The inhibition of fungal mycelial growth 

by RCG1 and nystatin are given in Table 5. From these 

results we observed that the mycelial growth of Fusarium 

equiseti was found to be inhibited (18.4±1%) by RCG1

(100 µg/ml), which showed highest inhibition towards the 

mycelial growth of all tested fungal strains, although the 

inhibition effect by the lectin was less than the case of 

bacteria. The growth of Colletotrichum corchori and 

Curvularia lunata (13.3-8.4±1%) was moderately 

inhibited but Botryodiplodia theobromae was least 

inhibited by RCG1. On the other hand, Alternaria 

alternata and Macrophomina phaseolina were never 

inhibited by the lectin, though the growth of all six fungi 

was totally inhibited by standard antibiotic, nystatin (100 

µg/ml). By now, antifungal activity has been reported in 

many plant lectins (Broekaert et al., 1989). Our results 

suggested that RCG1 has antifungal activity similarly to 

mannose-binding lectins from red cluster pepper 

(Capsicum frutescens) and Pisum sativum seeds that 

inhibited the growth of fungi Fusarium moniliforme and 

Fusarium oxysporum, Aspergillus flavus and Trichoderma 

viride (Nagi and Ng, 2007; Sitohy et al., 2007). 

Table 5. Antifungal activity of RCG1 from the bullfrog R. 

catesbeiana oocytes. 

Name of fungi 

Percentage inhibition of fungal 

mycelial growth 

Lectin (100 Nystatin* (100 

µg/ml PDA) µg/ml PDA) 

Fusarium equiseti 18.4±1 47.4±1 

Colletotrichum 

corchori 

13.3±1 50.3±1 

Curvularia lunata 8.4±1 46.5±1 

Botryodiplodia 

theobromae 

4.6±1 40.4±1 

Alternaria 

alternate 

0 39.4±1 

Macrophomina 

phaseolina 

0 52.2±1 

Note: *Standard antifungal antibiotic, Growth measured- radial 

growth in cm. 

RCG1 has belongs to the galectin family. Many galectins 

were reported to have multivalent functions such as in cell 

adhesion and apoptosis through the carbohydrate-binding 

activity. In this study, we have found that the lectin 

possess antimicrobial activity in addition to cell adhesive 

activity (Kawsar et al., 2009). Because the lectin is 

located on the outer membrane of frog eggs, it may play 

an integral role in defense against bacterial pathogens. 

Recently, a number of galactose-binding lectin were 

shown to act as host receptors for bacteria and fungi 

(Vasta, 2009; Ideo et al., 2009) by apparent direct or 

indirect galectin-dependent specific host-pathogen 

interactions. Since RCG1 can be purified in large amounts 

from bullfrog eggs, it may be a potential drug discovery 

target for both anti-cancer and anti-inflammatory agents. 


CONCLUSION 

The present study showed that RCG1 displayed 

significant growth inhibition effects against selected 

human and phytopathogens. American bullfrog Rana 

catesbeiana oocytes galectin-1 (RCG1) was screened for 

the first time for antibacterial and antifungal activities and 

these antimicrobial activities may provide an effective 

defense capability against invading microbes in the 

amphibian Rana catesbeiana. 

ACKNOWLEDGEMENTS 

We are grateful to the Chairman of the Department of 

Microbiology, University of Chittagong, Bangladesh to 

give us the opportunity to carry out the microbiology 

work. We are also thankful to Dr. Robert A. Kanaly (USA 

& YCU) for improving our manuscript. This work was 

supported by Grants-in Aid for Scientific Research from 

JSPS (Japan Society for the Promotion of Science). 

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Received: March 1, 2010; Revised: May 22, 2010; Accepted: 

May 24, 2010


Vol. 4, No. 2, pp. 1151-1162, June 2010 

ISSN: 1715-9997 

VERTEBRATE BIODIVERSITY AND KEY MAMMALIAN SPECIES STATUS OF 

HINGOL NATIONAL PARK 

M Zaheer Khan, Afsheen Zehra, Syed Ali Ghalib, Saima Siddiqui and Babar Hussain 

Department of Zoology (Wildlife & Fisheries) University of Karachi, Karachi-75270 

ABSTRACT 

Pakistan has recognized three categories of protected areas: National Parks, Wildlife Sanctuaries, and Game Reserves. 

Currently Pakistan has 22 National Parks of which Hingol National Park is the most unique and important because it 

consists of six ecosystems including Arabian Sea, Rugged Mountains, Desert, River, Estuary, and open plains. The park 

has rich biodiversity and diversified fauna of terrestrial, marine, and freshwater ecosystems. The park has many 

important mammals, birds, fish, reptiles and amphibian species, including Ibex (Capra aegagrus), Urial (Ovis vignei), 

Chinkara (Gazella bennettii), Dalmatian Pelican (Pelecanus crispus), Spotted-billed Pelican (Pelecanus philippinus), 

Houbara Bustard (Chlamydotis undulat), Imperial Eagle (Aquila heliaca), Sooty Falcon (Falco concolor), Flamingo 

(Phoenicopterus roseus), fish Mahsheer (Tor putitora), Marsh Crocodile (Crocodylus palustris), Green Sea Turtle 

(Chelonia mydas), Skittering frog (Rana cyanophlyctis) etc. During the study 2005 to 2009, 165 bird species (75 

resident and 90 migratory species), and 16 mammalian species were recorded. While Ibex, Urial and Chinkara are the 

key species of the park. Jungle Cat (Felis chaus), Desert Cat (Felis libyca), Desert Fox (Vulpes vulpes), Wolf (Canis 

lupus), and Asiatic Jackal (Canis aureus) have been observed. Due to aesthetic reason, and venues like mountains, 

wetlands, river, estuary, sea beach, mud volcano and sand dunes, the park has a potential to be developed as Eco-tourist 

Park. 

Keywords: Unique park, Hingol River, vertebrate biodiversity, green turtle, key mammalian species. 

INTRODUCTION 

Protected Areas are recognized as an important tool in 

conserving animal and plant species and ecosystems. 

These systems vary considerably from country to country, 

depending on priorities, and national needs, and on 

differences in institutional, legislative and financial 

support (Khan, 2004). Pakistan has 235 protected areas 

including 22 national parks (Table 1), 99 wildlife 

sanctuaries, 100 game reserves and 14 unclassified areas. 

Hingol National Park is located in the Province of 

Balochistan and covers an area of 619,043 hectares. This 

area falls at coordinates 26 o 00’ and 25 o 17’ North and 

65 o 10’ and 65 o 55’ East. Hingol National Park is named 

after the Hingol River, which flows through the center of 

the Park and empties into the Arabian Sea. 

In 2005, 600 km long Mekran Coastal Highway was built 

along most of the Southern Coastal Area of Balochistan, 

that runs for some 109 km through the HN Park area 

(Management Plan of HNP, 2006). Hingol National Park 

comprises the area from the Arabian Sea up to 5 fathom 

depth to the Dhrun Mountain with the Shak top at 1580 m 

above sea level. 

There are a number of plain valleys between hills in the 

HN park. The park area can be divided into areas West 

and East of Hingol River. The parallel mountain ranges 

West of the Hingol River follow an EW direction, 

*Corresponding author email: zaheerkhan67@yahoo.ca 

including the Shur Mountain Range, Hinglaj-Nani 

Mountains, Gurangatti and Rodaini - Kacho – Dhrun area. 

The mountain ranges East of Hingol River follow a more 

SSW-NNE direction including the Tranche Block and the 

Deo-Beharo Block. Further East, the mountain ranges 

show a NS direction including the Hala Range (Shir and 

Nawar Mountains) and the Haro Range, which flanks the 

Phore Valley respectively at the West and the East side. 

Between the large mountains blocks are small valleys 

including the Sham Valley located between Sangal-Kund 

Malir Range and the Shur Mountain Range, the Kundrach 

Valley between Shur and Hinglaj, the Harian-Maneji 

Valley between Hinglaj and Gurangatti (Zehra, 2009). 

The drainage of the park area is mainly to the Hingol 

River. The main rivers from the North are the Nal-Hingol 

River entering the park at the NW boundary, the Arra 

River entering the park between the Dhrun Mountains and 

the Washiaab, the Babro River entering the park at the NE 

boundary, North of Tranch. The drainage of Dhrun 

Mountain is to all directions. The major drainage, from 

the high plateau is in Eastern direction emptying in the 

Ara River South of Kukeri Bhent. The largest part of the 

Southern slopes drains into the Rodaini Kacho Valley and 

then to the Daraj-Kacho gorge into the Northern Plains 

and its Ara River (Management Plan of HNP, 2006; 

Zehra, 2009). 

In the HNP, 150 plant species have been reported (Zehra,

1152 

2009). The main vegetated areas are in the small zones of 

the valleys, the floodplains, riverbeds, and more extensive 

area of the coastal plains. Most ecological units are bare 

or almost bare including the mud flats, the salt plains, the 

clay Mountains and mud vent areas, the stone rippled 

terraces, and the smooth slopes of brown clay rock, 

mountains ridges, the steep mountain walls and the 

crusted valley floor and the active flood bank areas. The 

park is represented by six ecosystems including Arabian 

Sea, Rugged Mountains, River, Desert, Estuary, and open 

plains. The wildlife of the coastal area of HNP is diverse 

due to a combination of habitats found together at several 

areas. The coastal plains show rocky hill areas (Sappat 

Mountains, Agor Hills, Jabal Haro-Kund Malir), sand 

dunes, agricultural fields and river beds. 

The Arabian Sea, including the area bordering national 

park, is known to be highly productive. Nesting of Green 

turtle has been reported from the coastal area of HNP. A 

variety of water birds occurs at the coast line. Estuarine 

area of the River Hingol, supports a variety of resident 

and migratory water birds. Adjacent to coastline there is a 

large desert with prominent sand dunes. These sand dunes 

may be categorized as stable or fixed sand dunes. The 

desert area of the park has diversified biodiversity 

including birds, small mammals and reptiles. Occurrence 

of Houbara Bustard in winter is also reported in the dune 

areas (Azam, 2004). 

The objective of the present study was to record the 

vertebrate biodiversity and population status of three key 

mammalian species (Ibex, Urial and Chinkara) of Hingol 

National Park in the selected areas during 2005-2009. 


Based on preliminary studies in the Hingol National Park, 

Nani Mandir Complex, Rodaini Kacho, Harian Valley, 

Dhrun, Machii, Maneji, Ara Kaur, Babro Kaur , Agor, 

Kundrach, Qasim Goth, Wadh Bundar, Maneji, Nani 

Mandir/Nani Bent, Nala Jhakee, Kashee Goth, Kund 


Table 1. Current List of the National Parks of Pakistan (Khan and Siddiqui, 2005; Ahmad, 2009). 

Name of National 

Park 

Location 

Year of 

establishment 

Name of 

National Park 

Location 

Year of 

establishment 

Ayubia NWFP 1984 Kala Chitta Punjab 2009 

Central Karakoram Northern Areas 1995 Khirthar Sindh 1974 

Chinji Punjab 1987 Khunjerab Northern Areas 1975 

Chitral Gol NWFP 1984 Lal Suhanra Punjab 1972 

Deosai Northern Areas 1993 Lulusar NWFP 2003 

Deva Vatala AJK 2009 Machiara AJK 1996 

Ghamot AJK 2004 Margalla Hills Fed. Capital Territory 1980 

Gurez AJK 2009 Pir Lasora AJK 2005 

Handrap Shandoor Northern Areas 1993 Saif-ul-muluk NWFP 2003 

Hazarganji Chiltan Balochistan 1980 Sheikh Budin NWFP 1993 

Hingol Balochistan 1997 Toh Pir AJK 2005 

Malir, Kalair Goth, Nokoo Goth, Allah Buksh Goth, and 

Sanguri area were selected for the study of key 

mammalian and other vertebrate biodiversity (Table 2, 

Fig. 1, 2). The following methods and surveys techniques 

were employed for the observation, census and 

documentation of key mammalian species. 

1. Track Counts 

2. Point Surveys 

3. Roadside Counts 

4. Line Transects or Strip Census 

5. Pellet Counts 

Track Counts 

Tracks can be the first indication of the presence of 

animals in an area. Track counts especially after rain are 

useful in identifying different animals especially for 

nocturnal and secretive ones. A fresh rain eliminates the 

previous tracks, and the recent tracks of animals entering 

or leaving the study areas can be used as a measure of 

their abundance. During all studies the track count 

technique was applied at selected areas of HNP and this 

was more effective compare to other methods. 

Point Surveys 

In this method, observation points are established along 

roads, edges of ponds or marshes, at a higher place and 

other locations suitable for viewing the habitat. For a 

period of 1 to 5 hours at each observation point, the 

observer records all sightings of the mammals at that site 

and then an index of abundance of each species is 

expressed as the number of animals seen per hour of 

observations (Brower et al., 1990). Point surveys were 

conducted twice daily, first during early morning, i.e. one 

hour before sunrise until dawn and second, in the evening, 

i.e., begins one-half an hour before the sunset until dark. 

Roadside Counts 

Usually it is difficult to locate a mammal especially a 

large mammal by walking in its habitat, because it smells

Fig. 1. Study areas of key mammalian species and birds of Hingol National Park. 

Fig. 2. Study areas of small mammals and birds of Hingol National Park. 

human presence from a long distance. Hence, the method 

of roadside counts was applied to locate and estimate the 

population of different mammalian species. In this 

method, the observer travels by motor vehicle along roads 

Khan et al. 1153 

and trails while the sighted number of individuals of the 

species being estimated is tallied and related to the 

number of km travelled (Brower et al., 1990). This 

method has some advantages such as; travelling by

1154 

vehicle disturbs the animals and there is always a chance 

to observe the animals along the road / track from a 

distance of few meters. Secondly, by this method large 

areas can be covered quickly and easily by only two 

persons. The roadside counts technique was applied 

during survey along the coastal highway, mostly for 

nocturnal mammals like foxes, jackals, cats as well as for 

the diurnal mammals e.g., mongoose. For this purpose, 

4 x 4 vehicles were driven at a slow speed (7km/h) on 

inter-compartmental tracks, sandy plains river bank 

(4km/h) and on rough tracks along water channels at 

Hingol River (3km/h) in the park. These roadside counts 

were carried out during early morning, at dusk and during 

night by using search lights on top of the vehicle. 

Line Transects 

The line transect or strip census method of population 

estimation involves counting the animals seen by an 

observer traversing a predetermined transect line and 

recording the distances at which they were seen or 

flushed. The average of the flushing distance is 

determined and used to calculate the effective width of 

the strip covered by the observer. The population for the 

entire area is then considered to be the number of 

animals flushed, divided by the area of the strip and 

multiplied by the total area (Schemnitz, 1980). 

P = AZ / 2 XY 

Where P = population 

A = total area of study 

Z =number of animals flushed 

Y =average flushing distance 

X =length of strip 

Line transects or strip census method is particularly a 

useful technique when animals are difficult to see and 

must be flushed to be counted. This methodology was 

applied in HNP for estimating large mammals eg., Ibex, 

Urial, Chinkara etc. The transect method was also used 

for surveys of marine mammals in HNP. The transect was 

randomly selected from the chart prior to leaving shore. 

Large and medium sized boats were used during the 

survey. The boat speed was maintained below 12 knots 

and the width of transect was 25 m on either side of the 

boat. Two observers and one recorder worked at a time. 

Each observer watched 90 0 in an arc sweeping one quarter 

on front view from mid boat. The auto focus binoculars 

were used for observations. 

Pellet Counts 

Fecal material such as pellets counted in a specific area is 

a good technique for locating large mammals and 

assessing their population. This technique involves 

removing all pellet groups from plots and then estimating 

from subsequent observations on those plots the number 

of groups per hectare to compare animal use of areas 


between sampling periods. In some cases it is not 

possible to remove all the pellet groups from an area. 

Therefore, under such circumstances; an observer with a 

little practice can identify the fresh pellets depending on 

the color and dryness of the pellets. Ten to fifteen 100 sq. 

m plots (7.07 X 14.14) can be used for this purpose. 

These plots should be checked every three to seven days 

and the periods between sampling should not be so 

long that feces will decompose or be destroyed by 

weather or insects. A random selection of plots in the 

study area and the number of pellets groups in each plot 

were tallied and summed (Brower et al., 1990). 

The number of pellet group per unit area is determined as 

an index of density (ID) and determined as: 

ID = n / A 

where, n is the sum of pellet group counted over all plots 

and A is the total area sampled (i.e., the sum of the areas 

of all the plots). 

This method is effective in habitats with dry weather and 

little or no dung beetle activity where pellets groups 

remain preserved between sampling periods. After 

counting pellets, one must be assured that they will not be 

counted on successive sampling periods so they should be 

removed by the observer if they will not disappear by 

natural processes. Defecation rates for the species under 

the study are estimated if it is desired to convert pellet 

counts to number of animals. 

Techniques and tools for survey of small mammals 

Trapping 

Sherman Traps were used to collect the live specimens. 

Traps were set on a line approx 500 m long and approx 10 

m apart. Each trap was marked by a colored ribbon to 

locate the traps easily. The traps were set in the 

afternoon and checked early morning. The trapped 

animals were each carefully transferred into an already 

weighed transparent polythene bag. The species and sex 

of the trapped animals were noted. Other data for each 

trap, such as date of trap setting, date of data collection, 

habitat, location, elevation and weather conditions were 

recorded on a data sheet. After recording the data, the 

animals were released. Specimens with some doubt in 

identification were preserved in 10% formalin and 

brought to laboratory and identified. Specimens of 

each species were preserved as voucher specimens. 

A mixture of different food grains mixed with fragrant 

seeds was used as bait to attract small mammals. Wheat 

and rice were used as food grains while peanut 

butter coriander, oats and honey were used for 

fragrance. This bait was very successful in the study area 

probably due to overall food shortage and also because 

four ingredients were used for fragrance. Freshly prepared

ait was used on every trapping day. Only a small amount 

of bait was put on the platform near the traps. Because of 

limited time for surveys, trapping was done only at night 

at each of the sites and for best results trapping continued 

at least for 3 nights. To supplement low intensity trapping 

during field surveys, other data collection procedures such 

as active searching (day and night), spot light search, and 

interviews of local peoples, visible signs and literature 

review were also adopted. 

Spot Light Method 

This method is used at night for locating small and large 

mammals such as Hare, Porcupine, Hedgehog, Fox, Wild 

Cat, Jackal etc. because all these nocturnal animals move 

for food. In this way the populations of different species 

at different localities were estimated. 

Counting of Fresh Holes and Tracks 

According to Brower et al. (1990) the holes and tracks 

method can be used to determine the population range and 

status of small mammals. Fresh holes and tracks were 

counted in the study area of one sq. km, which provided 

population estimate. 

Several bird surveys were undertaken during the study. 

For bird identification, field guides such as Grimmett et 

al. (1988) and Khanum et al. (1980) were used. 


In this study, vertebrate biodiversity and key mammalian 

species status of Hingol National Park was determined for 

the selected sites, using several methods and survey 

techniques for the observation, census and documentation 

of the mammalian fauna, and other vertebrate biodiversity 

from 2005 to 2009. During the study, 16 mammalian 

species have been recorded including three large and 13 

small mammal species 

Population and Status of Key Mammalian Species 

Three key mammalian species have been recorded during 

2005 to 2007. Ibex (Capra aegagrus) was rated common, 

while Urial (Ovis vignei), and Chinkara (Gazella 

bennettii) less common. In 2005, Ibex was rated 79.64%, 

Urial 10.32%, and Chinkara 10.02% (Table 3). In 2006, 

Ibex was rated 78.53%, Urial 11.12%, and Chinkara 

10.34% (Table 4). In 2007, Ibex was rated 78.29%, Urial 

11.18%, and Chinkara 10.51% (Table 5). During 2008 

and 2009, Wild goat (Capra aegagrus) was rated as 

common, while Urial (Ovis vignei), and Chinkara 

(Gazella bennettii) less common. In 2008, Wild goat was 

rated 79.16%, Urial 11.45 %, and Chinkara 9.37% (Table 

6). In 2009, Wild goat was rated 80%, Urial as 11.6%, 

and Chinkara 8.4% (Table 7). The population of key 

species at Nani Mandir Complex, Rodaini Kacho, Harian 

Valley, Dhrun, Machii, Maneji, Ara Kaur, and Babro 

Kaur areas has also been summarized in tables 8-10. 


Ibex (Capra aegagrus) 

There are three key mammalian species in HNP, and 

Capra aegagrus (Ibex) is one of them. The estimated 

population of Ibex was observed was 540 in 2005, 600 in 

2006, 700 in 2007, 760 in 2008 and 800 in 2009 in 

different areas of the park (Table 8). Males, females, 

yearlings and young ones were also included in count. 

During the study period 2005 – 2009, the habitat 

conditions are better as both feed and shelter are in plenty 

in HNP. Even though the Ibex has to face the hunting 

pressure from different sources and disturbance at 

different times of the year from the pilgrims to Nani 

Mandir area and from the livestock herders, its population 

is still good. Livestock pressure in the area is the deciding 

factor in the distribution of the Ibex. In the face of 

competition with the livestock, Ibex either leave the area 

or their visitation is reduced. 

Urial (Ovis vignei) 

During the study period, Ovis vignei (Urial) was rated as 

less common. The estimated population of Urial was 

observed was 70 in 2005, 85 in 2006, 100 in 2007, 110 

in 2008 and 116 in 2009 in different areas of the park 

(Table 9). The Urial population has faced the hunting 

pressure due to easy access to its habitat and the 

competition with the livestock. However, during our 

study, no direct evidence of hunting pressure on Urial was 

found. The reason for the decline could be the 

deterioration in habitat conditions and competition with 

livestock. (Scanty population demands further studies and 

adoption of conservation measures). Urial habitat was 

observed in the foothill areas, close to the mountains 

where a reasonably good shelter may be available. 

Chinkara (Gazella bennettii) 

Gazella bennettii (Chinkara) was also rated as less 

common. Chinkara being distributed in the plain area is 

susceptible to hunting pressure as these areas are easily 

approachable even on motor cycles. This was evident by 

the presence of signs of motor cycles in many areas. The 

condition of the animals seen was good and did not 

suggest that they were not reproducing. The habitat 

conditions in the area were suitable. There was no 

indication of a competition with the livestock as no 

interaction was witnessed. Chinkara was observed in 

specific habitat viz., large plain areas in the valley 

bottoms and vast nullah beds. They move to the 

mountains or even to the foothill. Low population of 

Chinkara needs immediate attention and further studies 

especially into the population structure and nonrecruitment 

to the population are required. The estimated 

population of Chinkara was observed to be 68 in 2005, 79 

in 2006, 94 in 2007, 90 in 2008 and 84 in 2009 in 

different areas of the park (Table 10).

1156 

Population and Status of Small Mammals 

From 2005 – 2009, thirteen species of small mammals 

have been recorded. Based on the data, House Mouse 

(Mus musculus), Balochistan Gerbil (Gerbillus nanus), 

Palm Squirrel (Funambulus pennantii), Afghan Hedgehog 

(Hemiechinus auritus), House Rat (Rattus rattus), 

Porcupine (Hystrix cristatus), Indian Gerbil (Tatera 

indica), and Grey Spiny Mouse (Mus saxicola) were rated 

as common, while Cairo Spiny Mouse (Acomys 


Table 3. Population of key Mammalian Species of Hingol National Park in 2005. 

S. 

No 

Study Area 

Ibex 

(Capra aegagrus) 

Urial 

(Ovis vignei) 

Chinkara 

(Gazella bennettii) 

Total % 

1. NaniMandir Complex 325 - - 325 47.93 

2. Rodaini Kacho 110 11 10 131 19.32 

3. Harian Valley 40 09 13 62 9.14 

4. Dhrun 18 - - 18 2.65 

5. Machii 25 14 - 39 5.75 

6. Maneji 22 15 12 49 7.22 

7. Ara Kaur - 11 17 28 4.12 

8. Babro Kaur - 10 16 26 3.83 

Total 540 70 68 678 

% 79.64 10.32 10.02 


S. 

No 

Study Area 

Ibex 


Urial 

(Ovis vignei) 

Chinkara 


Total % 


2. Rodaini Kacho 120 13 12 145 18.97 

3. Harian Valley 44 10 16 70 9.16 

4. Dhrun 20 - - 20 2.61 

5. Machii 30 17 - 47 6.15 

6. Maneji 26 18 12 56 7.32 

7. Ara Kaur - 15 21 36 4.71 

8. Babro Kaur - 12 18 30 3.92 

Total 600 85 79 764 

% 78.53 11.12 10.34 


S. 

No 

Study Area 

Ibex 


Urial 

(Ovis vignei) 

Chinkara 


Total % 


2. Rodaini Kacho 140 14 15 169 18.90 

3. Harian Valley 50 12 20 82 9.17 

4. Dhrun 29 - - 29 3.24 

5. Machii 36 20 - 56 6.26 

6. Maneji 30 20 16 66 7.38 

7. Ara Kaur - 18 22 40 4.47 

8. Babro Kaur - 16 21 37 4.13 

Total 700 100 94 894 

% 78.29 11.18 10.51 

cahirinus), Cape Hare (Lepus capensis), Mouse like 

Hamster (Callomyscus bailwardi), and Indian Desert Jird 

(Meriones hurrianae) were rated as less common, and 

during 2006 – 2009, Persian Jird (Meriones persicus) was 

rated as rare. 

In 2005, House Mouse was rated as 12.17%, Balochistan 

Gerbil 8.86%, Cairo Spiny Mouse 5.79%, Palm Squirrel 

11.34%, Cape Hare 3.54%, Mouse like Hamster 4.49%,

Table 6. Population of key Mammalian species of Hingol National Park in 2008. 

Afghan Hedgehog 10.87%, House Rat 9.69%, Indian 

Desert Jird 6.38%, Porcupine 10.52%, Indian Gerbil 

9.10%, and Grey Spiny Mouse 7.21%. 



11.12%, Cape Hare 5.35%, Mouse like Hamster 4.55%, 



8.80%, Grey Spiny Mouse 7.88%, and Persian Jird 

1.01%. 





S. 

No 

Study Area 

Ibex 


Urial 

(Ovis vignei) 

Chinkara 


Total % 

1. Nani Mandir Complex 445 - - 445 46.35 

2. Rodaini Kacho 148 16 14 178 18.54 

3. Harian Valley 56 14 19 89 9.27 

4. Dhrun 34 - - 34 3.54 

5. Machii 42 22 - 64 6.66 

6. Maneji 35 22 16 73 7.60 

7. Ara Kaur - 19 21 40 4.16 

8. Babro Kaur - 17 20 37 3.85 

Total 760 110 90 960 

% 79.16 11.45 9.37 


S. 

No 

Study Area 

Ibex 


Urial 

(Ovis vignei) 

Chinkara 


Total % 


2. Rodaini Kacho 155 17 13 185 18.5 

3. Harian Valley 59 15 18 92 9.2 

4. Dhrun 38 - - 38 3.8 

5. Machii 44 23 - 67 6.7 

6. Maneji 37 23 15 75 7.5 

7. Ara Kaur - 20 20 40 4 

8. Babro Kaur - 18 18 36 3.6 

Total 800 116 84 1000 

% 80 11.6 8.4 

Table 8. Population of Ibex (Capra aegagrus) in HNP during 2005 to 2009. 

S. No. Study Area 2005 2006 2007 2008 2009 

1. Nani Mandir Complex 325 360 415 445 467 

2. Rodaini Kacho 110 120 140 148 155 

3. Harian Valley 40 44 50 56 59 

4. Dhrun 18 20 29 34 38 

5. Machii 25 30 36 42 44 

6. Maneji 22 26 30 35 37 

Total 540 600 700 760 800 



9.30%, Grey Spiny Mouse 8.58%, and Persian Jird 0.90% 

(Table 11). 







(Table 11). 


Gerbil 10.42%, Cairo Spiny Mouse 6.68%, Palm Squirrel

1158 





(Table 11). 

Other Vertebrate Biodiversity 

In the Hingol National Park the following vertebrates 

have been reported. However, none was seen during the 

study (Zehra, 2009): 


Table 9. Population of Urial (Ovis vignei) in HNP during 2005 to 2009. 

S. No. Study Area 2005 2006 2007 2008 2009 

1. Rodaini Kacho 11 13 14 16 17 

2. Harian Valley 09 10 12 14 15 

3. Machii 14 17 20 22 23 

4. Maneji 15 18 20 22 23 

5. Ara Kaur 11 15 18 19 20 

6. Babro Kaur 10 12 16 17 18 

Total 70 85 100 110 116 

Table. 10. Population of Chinkara (Gazella bennettii) in HNP during 2005 to 2009. 

S. No. Study Area 2005 2006 2007 2008 2009 

1. Rodaini Kacho 10 12 15 14 13 

2. Harian Valley 13 16 20 19 18 

3. Maneji 12 12 16 16 15 

4. Ara Kaur 17 21 22 21 20 

5. Babro Kaur 16 18 21 20 18 

Total 68 79 94 90 84 

Table. 10. Population of Chinkara (Gazella bennettii) in HNP during 2005 to 2009. 

S. No. Study Area 2005 2006 2007 2008 2009 

1. Rodaini Kacho 10 12 15 14 13 

2. Harian Valley 13 16 20 19 18 

3. Maneji 12 12 16 16 15 

4. Ara Kaur 17 21 22 21 20 

5. Babro Kaur 16 18 21 20 18 

Total 68 79 94 90 84 

Table 11. Population of Small Mammals in Hingol National Park 2005 to 2009. 

S. No 

Year 

House 

Mouse 

Baloch Gerbil 

Cairo Spiny 

Mouse 

Palm 

Squirrel 

Cape Hare 

Mouse like 

Hamster 

Afghan 

Hedgehog 

1 2005 103 75 49 96 30 38 92 82 54 89 77 61 0 846 

2 2006 110 99 60 110 53 45 110 88 48 98 80 78 10 989 

3 2007 151 110 68 72 45 36 125 115 67 110 103 95 10 1107 

4 2008 139 116 76 77 43 35 129 113 70 118 99 99 20 1134 

5 2009 142 120 77 80 42 33 133 121 73 122 104 88 16 1151 

Total 645 520 330 435 213 187 589 519 312 537 463 421 56 5227 

House Rat 

Indian Desert 

Jird 

Por-cupine 

Indian Gerbil 

Grey Spiny 

Mouse 

Persian Jird 

Total 

Desert Wolf (Canis lupus) 

The presence of Desert Wolf (Canis lupus) was reported 

in valleys viz Ara Kaur, Babro Kaur, adjoining Tranche 

valley, and Dhrun areas. 

Caracal or Bashoshah (Felis caracal) 

Caracal was reported only from Rodaini Kachho area.

Common Leopard (Panthera pardus saxicolor) 

Common Leopard signs were reported from at least two 

valleys (between Dhrun and Rodaini Kacho). Local 

people also talked about the occurrence of this animal. 

Asiatic Jackal (Canis aureus) 

Asiatic Jackal footprints were seen in Pachree valley. 

Pangolin or Scaly anteater (Manis crassicaudata) 

Local people reported Pangolin occurrence in other 

valleys also but this could not be confirmed. Its footprints 

were seen in Pacchri valley only. 

Wild Boar (Sus scrofa davidi) 

Wild Boar was recorded through the footprints in Harian 

and Pachree valley areas. 

Birds 

In Pakistan, 666 bird species have been recorded, out of 

which 380 species have been recorded in Balochistan 

(Ghalib et al., 2004). Azam (2004) reported 108 species 

belonging to 68 genera 37 families and 14 orders from 

HNP. The avifauna of the HNP consists of resident as 

well as migratory bird species. During the study, we have 

observed many bird habitats including; coastal area, 

estuarine area, river, mangrove area, mountains and 

desert. During the study 165 bird species were recorded in 

which 75 were resident and 90 were migratory species 

(Table 12). 

Table 12. Birds of Hingol National Park, observed during 

2005-2009. 

S. 

No. 

Scientific name Common name 

1. Phalacrocorax carbo Large Cormorant 

2. Phalacrocorax niger Little Cormorant 

3. Pelecanus crispus Dalmatian Pelican 

4. Pelecanus onocrotalus White or Rosy 

Pelican 

5. Pelecanus philippensis Spotted billed Pelican 

6. Dupetor flavicollis Yellow-throated 

black bittern 

7. Egretta alba Large Egret or Great 

Egret 

8. Egretta garzetta Little Egret 

9. Egretta gularis Indian Reef Heron 

10. Egretta intermedia Smaller or Median 

Egret 

11. Ardea cinerea Grey Heron 

12. Ardea goliath Giant Heron 

13. Ardea purpurea Purple Heron 

14. Ciconia nigra Black Stork 

15. Psuedibis papillosa Black Ibis 

16. Platalea leucoirodia Spoonbill 

17. Phoenicopterus roseus Flamingo 


S. 

No. 


18. Anas acuta Pintail 

19. Anas crecca Common Teal 

20. Anas clypeata Shoveller 

21. Anas penelope Wigeon 

22. Anas strepera Gadwall 

23. Milvus migrans Black Kite 

24. Haliastur indus Brahminy Kite 

25. Haliaeetus leucoryphus Pallas's Fishing Eagle 

26. Gypaetus barbatus Bearded Vulture 

27. Neophron percnopterus Egyptian Vulture 

28. Gyps fulvus Indian Griffon 

Vulture 

29. Circus aeroginosus Marsh Harrier 

30. Accipiter badius Central Asian Shikra 

31. Accipiter nisus Eurasian Sparrow 

Hawk 

32. Aquila heliaca Imperial Eagle 

33. Aquila rapax Tawny Eagle 

34. Pandion haliaeetus Osprey 

35. Falco columbarius Pallid Merlin 

36. Falco concolor Sooty Falcon 

37. Falco jugger Lagger Falcon 

38. Falco naumani Lesser Kestrel 

39. Falco peregrinus Peregrine Falcon 

40. Falco tinnunculus Kestrel 

41. Ammoperdix 

griseogularis 

See-see Partridge 

42. Francolinus 

pondicerianus 

Grey Patridge 

43. Conturnix conturnix Common Quail 

44. Gallinula chloropus Moorhen 

45. Fulica atra Coot 

46. Chlamydotis undulata Houbara Bustard 

47. Himantopus ostralegus Oyster Catch or Sea- 

Pie 

48. Himantopus 

himantopus 

Black-winged Stilt 

49 Dromas ardeola Crab Plover 

50. Burhinus oedicnemus Stone curlew/ 

Eurasian Thick-Knee 

51. Esacus recurvirostris Great Stone 

Plover/Thick-Knee 

52. Charadrius 

alexandrinus 

Kentish Plover 

53. Charadrius dubius Little Ringed Plover 

54. Charadrius hiaticula Ringed Plover 

55. Charadrius 

leschenaultii 

Large Sand Plover 

56.. Charadrius mongolus Lesser Sand Plover 

57. Vanellus gregarious Sociable Lapwing 

58. Vanellus indicus Red Wattled 

Lapwing 

59. Vanellus leucurus White-tailed Lapwing

1160 

S. 

No. 


60. Calidris alpinus Dunlin 

61. Calidris minutus Little Stint 

62. Calidris temminckii Temminck's Stint 

63. Calidris 

testaceus/ferruginea 

Curlew-Sandpiper 

64. Limicola falcinellus Broad billed 

Sandpiper 

65. Philomachus pugnax Ruff 

66. Capella gallinago Common or Fantail 

Snipe 

67. Limosa lapponica Ber tailed Godwit 

68. Limosa limosa Black tailed Godwit 

69. Numenius arquata Curlew 

70. Numenius phaeopus Whimbrel 

71. Tringa hypoleucos Common Sandpiper 

72. Tringa nebularia Greenshank 

73. Tringa ochropus Green Sandpiper 

74. Tringa stagnatilis Marsh Sandpiper 

75. Tringa terek Terek Sandpiper 

76. Tringa totanus Redshank 

77. Larus argentatus Herring Gull 

78. Larus brunnicephalus Brown-headed Gull 

79. Larus cachinans Yellow-legged Gull 

80. Larus fuscus Black-backed Gull 

81. Larus genei Slender billed Gull 

82. Larus ichthyaetus Great Black headed 

Gull or Pallas's Gull 

83. Larus ridibundus Black-headed Gull 

84. Gelochelidon nilotica Gull billed Tern 

85. Hydroprogne caspia Caspian Tern 

86. Sterna albifrons Little Tern 

87. Sterna bengalensis Lesser Crested Tern 

88. Sterna hirundo Common Tern 

89. Sterna repressa White-cheeked Tern 

90. Sterna sandvicensis Sandwich Tern 

91. Chlidonias hybrida Whiskered Tern 

92. Pterocles coronatus Caronetted Sandgrouse 

93. Pterocles indicus Painted Sandgrouse 

94. Pteroceles orientalis Imperial or Blackbellied 

Sandgrouse 

95. Columba livia Blue Rock Pigeon 

96. Streptopelia decaocto Ring Dove 

97. Streptopelia 

Little Brown or 

senegalensis 

Senegal Dove 

98. Psittacula krameri Rose Ringed 

Parakeet 

99. Caprimulgus 

Syke's or Sind 

mahrattensis 

Nightjar 

100. Athene brama Spotted Owlet 

101. Apus affinis House Swift 

102. Apus apus Common Swift 

103 Apus pallidus Pale Brown or Pallid 

Swift 


S. 

No. 


104. Alcedo atthis Common Small Blue 

Kingfisher 

105. Halcyon smyrnensis White breasted 

Kingfisher 

106. Merops orientalis Small Green Beeeater 

107. Merops superciliosus Blue-checked Beeeater 

108. Coracias bengalensis Ruller or Blue Jay 

109. Upupa epops Hoopoe 

110. Picoides assimilis Sind Pied 

Woodpecker 

111. Eremopterix grisea Ashycrowned Finchlark 

112. Eremopterix nigriceps Blackcrowned Finchlark 

113. Ammomanes deserti Indian Desert Finchlark 

114. Alaemon alaudipes Hoopoe Lark / 

Bifasciated Lark 

115. Calandrella 

Hume's Short-toed 

acutirostris 

Lark 

116. Galerida cristata Crested Lark 

117. Riparia paludicola Brown-throated Sand 

Martin 

118 Riparia riparia Collared Sand Martin 

119. Delichon urbica Common House 

Martin 

120. Hirundo daurica Redrumped Swallow 

121. Hirundo rustica Barn Swallow 

122. Hirundo smithi Wire-tailed Swallow 

123. Motacilla alba White or Pied 

Wagtail 

124. Motacilla cinerea Grey Wagtail 

125. Motacilla flava Yellow Wagtail 

125. Anthus trivialis Tree Pipit 

127. Pycnonotus cafer Red-vented Bulbul 

128. Pycnonotus leucogenys White-cheeked 

Bulbul 

129. Lucsinia svecica Bluethroat 

130. Oenanthe alboniger Hume's Chat or 

Wheatear 

131. Oenanthe deserti Desert Chat or Desert 

Wheatear 

132. Oenanthe isabellina Isabelline Wheatear 

133. Oenanthe monacha Hooded Chat or 

Wheatear 

134. Phoenicurus ochruros Black Redstart 

135. Saxicola ferea Gray Bushchat 

136. Saxicola torquata Collared Indian Bush 

Chat or Stone Chat 

137. Acrocephalus 

dumetorumn 

Blyth's Reed Warbler

S. 

No. 


138. Acrocephalus 

stantoreus 

Clamorous Warbler 

139. Phylloscopus collibita Chiffchaff 

140. Phylloscopus neglectus Plain Leaf Warbler 

141. Prinia buchanani Rufous Fronted Wren 

Warbler 

142. Sylvia curruca Lesser Whitethroat 

143. Sylvia nana Desert Warbler 

144. Ficedula parva Red throated 

Flycatcher 

145. Rhipidura aureola White browed 

Flycatcher 

146. Turdoides caudatus Common Babbler 

147. Nectarinia asiatica Purple Sunbird 

148. Lanius excubitor Grey Shrike 

149. Lanius isabellinus Rufous-tailed or 

Isabelline Shrike 

150. Lanius schach Rufous-backed 

Shrike 

151. Lanius vittatus Baybacked Shrike 

152. Dicrurus adsimilis Black Drongo or 

King Crow 

153. Corvus corax Common Raven 

154. Corvus ruficollis Dasert Raven 

155. Corvus splendens Indian House Crow 

156. Acridotheres tristis Indian Myna 

157. Passer domesticus House Sparrow 

158. Passer hispaniolensis Spanish Sparrow 

159. Passer pyrrhonotus Sind Jungle Sparrow 

160. Passer xanthocollis Sind Yellow throated 

Sparrow 

161. Lonchura malabarica Common Silverbill or 

White throated 

Munia 

162. Carpodacus erythrinus Common Rosefinch 

163. Emberiza cia Rock Bunting 

164. Emberiza 

Black headed 

melanocephala Bunting 

165. 

Emberiza striolata 

Striped or House 

Bunting 

Fish fauna 

Following fourteen fish species have been recorded: 

Cyprinion watsoni, Cyprinion microphthalmum, 

Cyprinion milesi, Schistura balochiorum, Tor pititora, 

Scaphiodom irregularis, Jalmius soldado, 

Pseudorhombus arsius, Mugil cascasia, Mugil cephalus, 

Scatophagus argus, Dastatis gugei, Aspidoparia morar 

and Crossocheilus diplocheilus. 

Amphibians and Reptiles 

During the study, two species of amphibian Rana 

cyanophlyctis (Skittering Frog) and Bufo stomaticus 


(Indus Valley Toad) have been recorded. Nine species of 

reptiles including two species of turtle Chelonia mydas 

(Green turtle), Eretmochelys imbricata (Hawksbill 

Turtle), five species of lizards Hemidactylus brooki, 

(House Gecko), Trapelus agilis (Brilliant Agama), 

Acanthodactylua cantrois (Indian Fringed-toed Lizard), 

Varanus griseus (Desert Monitor), Crossobamon 

orientalis (Sand Sind Gecko), one species of Crocodylus 

palustris (Marsh Crocodile) and one snake species Echis 

carinatus (Saw Scaled Viper) have been observed. 

Ecotourism and Hingol National Park 

Tourism is a principal export for 83% of developing 

countries. For the world’s poorest countries, tourism is 

the 2 nd most important source of foreign exchange, after 

oil (Hospodarsky and Lee, 2007). Due to aesthetic venues 

like mountains, wetlands, river, estuary, sea beach, mud 

volcano, sand dunes, and cultural attractions, the HN park 

has the potential to be developed as Eco-tourist Park, and 

eco-tourism can increase local jobs, income and other 

benefits for local peoples. The Hingol National Park area 

also has the Nani Mandar, a popular sacred place for the 

Hindus. 

ACKNOWLEDGMENTS 

This paper was presented in the International Congress 

Healthy Parks Healthy People held at Melbourne 

Convention Centre, Australia, in April 2010, and for 

paper presentation, traveling grant was provided by the 

Higher Education Commission (HEC), for which the 

authors deeply thank the HEC. We also thank Prof. Jamil 

Kazmi, Department of Geography, University of Karachi 

for preparation of study areas map. 

REFERENCES 

Ahmad, W. 2009. National Parks in Pakistan. Natura. 33 

(1):24-28. 

Azam, MM. 2004. Avifaunal Diversity of Hingol 

National Park. Rec. Zool. Surv. Pakistan. 15:7-15. 

Brower, J., Zar, J. and Ende, C. 1990. Field and 

laboratory methods for general ecology. Wm . C . Brown 

Publishers. 2460 Kerper Boulevard, Dubuque. A 52001. 

Hospodarsky, D. and Lee, M. 2007. Ecotourism and 

Natural Resources Management. International Seminar on 

Forest Administration and Management, USA. 

Ghalib, SA., Khan, MZ., Zehra, A. and Khan, AR. 2004. 

Current Population Status of the Birds of Balochistan, 

Pakistan. J. Nat. Hist. Wildlife. 3(2): 51-62. 

Ghalib, SA., Jabbar, A., Khan, AR. and Zehra, A. 2007. 

Current status of the mammals of Balochistan. Pakistan J. 

Zool. 39(2):117-122.

1162 

Grimmett, R., Inskipp, C. and Inskipp, T. 1988. Birds of 

the Indian sub-continent. Oxford University Press, 

Mumbai. 

Khanum, Z., Ahmed, MA. and Ahmed, M. 1980. A 

Check list of Birds of Pakistan with Illustrated Keys to 

their Identification. Rec. Zool. Surv. 9 (1&2):138pp. 

Khan, MZ. 2004. Protected areas with reference to 

Pakistan. J. Nat. Hist. Wildlife 3(1): 7-2. 

Khan, MZ. and Siddiqui, S. 2005. The Vertebrate 

biodiversity of Hazarganji Chiltan National Park, 

Balochistan. J. Nat. Hist. Wildlife. 4(1):93-99. 

Management Plan HNP (draft). 2006. Balochistan Forest 

and Wildlife Department. pp 198. 

Schemnitz, SD. 1980. Wildlife Management Techniques 

Manual. The Wildlife Society, Inc 5410 Grosvenor Lane, 

Bethesda, Maryland 20814. 

Zehra, A. 2009. Current Status of the Mammals of Hingol 

National Park. Ph.D thesis, Department of Zoology, 

University of Karachi, Karachi. 

Received: Jan 4, 2010; Revised: April 27, 2010; Accepted: May 

17, 2010 

Canadian Journal of Pure and Applied Sciences


Vol. 4, No. 2, pp. 1163-1168, June 2010 

ISSN: 1715-9997 

EFFECT OF MAGNESIUM CHLORIDE AND SODIUM FLUORIDE ON VARIOUS 

HYDROXYPROLINE FRACTIONS IN RAT KIDNEYS 

EA. Al Omireeni, *NJ Siddiqi and AS Alhomida 

Department of Biochemistry, College of Science, PO Box 22452 

King Saud University, Riyadh -11495, Saudi Arabia 

ABSTRACT 

Magnesium chloride (MgCl2) has been reported to protect against sodium fluoride (NaF) induced toxicity. This study 

was undertaken to study the effect of MgCl2 on NaF induced alteration in rat kidney hydroxyproline fractions and 

collagen. Four groups of rats were studied (each consisting of 4-6 rats) (i) normal rats: (ii) rats injected with MgCl2: (iii) 

rats injected with NaF: (iv) rats injected with MgCl2 followed by NaF. Results show that MgCl2 and NaF treatment alone 

and together caused a significant (p < 0.05) decrease in kidney protein, free, peptide–bound, protein-bound, total 

hydroxyproline and soluble collagen hydroxyproline. Administration of MgCl2 before NaF did not restore the altered 

parameters to normal levels. However administration of MgCl2 before NaF restored insoluble collagen hydroxyproline 

which was altered by NaF to near normal levels. Though MgCl2 has been reported to be protective against the toxic 

effect of NaF, it has no significant effect on NaF induced changes in kidney hydroxyproline/collagen except insoluble 

collagen Hyp. 

Keywords: Sodium fluoride; hydroxyproline; collagen; magnesium chloride. 

INTRODUCTION 

Fluorine occurs in environment in combination with other 

elements as a fluoride compound (Manna et al., 2007). 

Human beings are exposed to fluoride through food 

(Stannard et al., 1991; Dabek and McKenzie, 1995), 

drinking water (Zhao, 1996) and inhalation (Gritsan et al., 

1995). Frequent absorption of the fluoride causes tooth 

decay (Neurath, 2005), damage of kidneys (Lantz et al., 

1987), bones (Bezerra de Menezes et al., 2003), nerves 

(Shivarajashankara et al., 2002) and muscles (Cicek et al., 

2005). The adverse toxic effects of fluoride arise due to a) 

enzyme inhibition, b) collagen break down, c) gastric 

damage and d) disruption of the immune system (Ahmad 

et al., 2000). Magnesium is a mineral that is involved in 

over 300 reactions in the body. It is important for every 

organ in the body, particularly the heart, muscles, and 

kidneys. It also contributes to the composition of teeth 

and bones. Most importantly, it activates enzymes, 

contributes to energy production, and helps regulate the 

levels of other minerals in the body (Saris et al., 2000). 

The kidneys excrete waste products of metabolism and 

play an important role in maintaining the homeostasis by 

regulating the body water and solute balance. In addition 

to the excretory function, the kidneys also have an 

endocrine function producing hormones like renin, 

erythropoietin etc. The most commonly used medium for 

studying fluoride toxicity is urine. Acute exposure to high 

*Corresponding author email: nikhat@ksu.edu.sa 

doses of fluoride damages renal tissue and causes renal 

dysfunction (Zabulyte et al., 2007). In our previous 

studies (Al-Omireeni et al., 2009) we have shown that 

different doses of NaF have profound effect on various 

hydroxyproline (Hyp) fractions in rat kidneys. Hyp is a 

component amino acid of collagen and has been used as 

an index of collagen turnover (Reddy and Enwemeka., 

1996). Magnesium chloride (MgCl2) has been reported to 

exert a protective effect on sodium fluoride (NaF) induced 

mortality in rats (Luoma et al., 1984). The present study 

was carried out to study the effect of MgCl2 on NaF 

induced changes in Hyp fractions and collagen content in 

rat kidneys. 


Chemicals 

Chloramine-T, p-dimethylaminobenzaldeyde (Ehrlich’s 

reagent), L-hydroxyproline, sodium acetate, citric acid, 

perchloric acid, n-propanol, sodium hydroxide, and acetic 

acid were purchased from Sigma Chemical Company, St 

Louis, MO, USA. Double distilled water was used 

throughout the study. 

Animal Care 

Healthy adult male Wister rats each weighting 150-200g 

(four to six weeks old) were obtained from Breeding 

Laboratory, King Saud University, Riyadh, Saudi Arabia. 

The animals were labeled by identifying ear notches,

1164 

housed in clean cages, and placed in the animal care 

room. Ethical guidelines for animal care were followed. 

Effect of magnesium chloride and sodium fluoride on 

different Hyp fractions in rat kidneys 

The following groups of rats were studied (each 

consisting of 4-6 rats) (i) normal rats (Control group, n = 

4 - 6 rats); (ii) rats injected with MgCl2 through 

intraperitoneal route 30 mg/kg body weight dose (MgCl2 

treated group); (iii) rats injected with NaF through 

intraperitoneal route 10 mg/kg body weight dose (NaF 

treated group); (iv) rats injected with MgCl2 through 

intraperitoneal route 30 mg/kg body weight followed by 

NaF 10 mg/kg body weight through intraperitoneal route 

30 minutes after MgCl2 injection (MgCl2 + NaF treated 

group). 

Preparation of the sample 

Rats were killed by carbon dioxide asphyxiation 24 hours 

after the final injection. The kidneys were dissected out, 

cleared of adhering tissues and weighed. The kidneys 

were then homogenized in normal saline (10% W/V) and 

the homogenate was used for Hyp determination as 

described below. 

Extraction of Free, Peptide- and Protein-bound 

Hydroxyproline 

Free and protein-bound Hyp was extracted by the method 

of Varghese et al. (1981) with slight modification. 

Briefly, 0.5ml of the homogenate was treated with 3 X 2 

ml portion of re-rectified absolute alcohol and centrifuged 

at 3000rpm for 10min. The supernatants were pooled and 

evaporated to dryness. The residue was dissolved in 

suitable amount of distilled water and an aliquot of the 

extract was used for estimation of free Hyp. The peptidebound 

Hyp was determined after alkaline hydrolysis of 

the ethanol extractable fraction. The pellets were 

dissolved in distilled water and an aliquot of the extract 

was used for determination of protein-bound Hyp. The 

free Hyp fraction was measured on an aliquot of the 

ethanol extracted residue without alkali hydrolysis, 

whereas the peptide-bound Hyp was measured after 

alkaline hydrolysis. The precipitate obtained on ethanol 

treatment of the homogenate was subjected to alkali 

hydrolysis to determine protein-bound Hyp. Further 

details about the extraction of Hyp fractions have been 

described previously (Siddiqi et al., 2000). Hyp was 

determined in different fractions as described in the later 

section. 

Extraction of Soluble- and Insoluble-Collagen Hyp 

Soluble- and insoluble-collagen Hyp were extracted by 

the method of Kivirikko et al. (1965). Briefly, the tissue 


samples were homogenized (4ml/g tissue) in a cold 0.45 

M sodium chloride. The homogenate was extracted at 4 0 C 

for 24 hours with occasional stirring, followed by 

centrifugation at 13000rpm for 1hour. The supernatants 

obtained were precipitated with 4 volumes of a cold 

ethanol and were centrifuged twice with 80% ethanol, 

twice with absolute alcohol, twice with ether and twice 

with warm ethanol-ether (1:2). The residues were 

gelatinized with distilled water at 120 0 C for 3 hours and 

after filtration a sample of gelatine solution was used for 

soluble-collagen Hyp estimation as described below. 

The precipitates obtained after the above centrifugation 

were washed 3 times with 0.45 M NaCl and twice with 

distilled water, after which they were extracted with 

absolute ethanol, ether and ethanol-ether and gelatinized 

as above. A sample of gelatine solution was used for 

insoluble-collagen Hyp estimation as described in the 

following section. 

Determination of Hydroxyproline Concentration 

Hyp was measured by the modified alkaline hydrolysis 

method of Reddy and Enwemeka (1996). Briefly, an 

aliquot of the sample was added into NaOH (2 N final 

concentration) and the aliquot was hydrolyzed by heating 

in a boiling water bath for about 3-4 hours. An aliquot of 

56 mM chloramine-T reagent was added to the 

hydrolyzed sample and oxidation was allowed to proceed 

at room temperature for 25min. Then an aliquot of 1M 

Ehrlich’s reagent (p-dimethylaminobenzaldehyde) was 

added to the oxidized sample and the chromophore was 

developed by incubating the samples at 65 0 C for 20 

minutes. The absorbance was read at 550 nm using an 

Ultrospec 2000 UV/visible spectrophotometer (Pharmcia 

Biotech Ltd, Science Park, Cambridge, England). The 

Hyp concentration in the samples was calculated from the 

standard curve of Hyp. Further details about the 

optimization, linearity, specificity, precision and 

reproducibility of the method have been described 

previously (Siddiqi et al., 2000). 

Determination of Total Collagen 

Total collagen content was calculated from Hyp 

concentration assuming that Hyp constitutes 12.5% of 

total collagen (Edwards and O’Brien, 1980). 

Statistical Analysis 

Each sample was run in duplicate. The Hyp content was 

expressed as mean ± SD µg/gram wet tissue, for n = 4-6 

rats. Hyp levels between groups were compared using one 

way ANOVA analysis followed by Tukey’s for multiple 

comparison test. Values were considered significant if p < 

0.05. Statistical analysis was performed by means of 

InStat® package for personal computers (GraphPad TM 

Software, Inc., San Diego, USA).

RESULTS 

Table 1 shows the effect of MgCl2 and NaF on protein 

content of rat kidneys. MgCl2 and NaF alone caused a 

significant decrease of 70% (p

1166 

kidneys of all the experimental groups viz., MgCl2 alone, 

NaF alone and MgCl2 plus NaF treated groups when 

compared to control rats. 

S oluble colagen H yp 

g 

(µ 

/ 

gra m tissue) 

100 

75 

50 

25 

0 

*** 

*** 

Experimental Groups 


*** 

Control 

MgCl 2 

NaF 

MgCl 2+NaF 

Fig. 2. Effect of magnesium chloride and sodium fluoride 

on soluble collagen hydroxyproline in rat kidneys. 

Rats were injected sodium fluoride (10mg/kg body weight) and 

magnesium chloride (30mg/kg body weight) through 

intraperitoneal route. 

The rats were injected with sodium fluoride 30 minutes after 

magnesium chloride administration. 

The animals were sacrificed 24 hours after the sodium fluoride 

treatment. 

***P0.05) in insoluble collagen Hyp in rat kidneys. 

o 

s 

In 

lu 

b le colagen H yp 

g 

(µ 

/ 

gra m tissue) 

300 

200 

100 

0 

ns 

** 

Experimental Groups 

ns 

Control 

MgCl 2 

NaF 

MgCl 2+NaF 

Fig. 3. Effect of magnesium chloride and sodium fluoride 

on insoluble collagen hydroxyproline in rat kidneys. 

Rats were injected sodium fluoride and magnesium chloride 

through intraperitoneal route. 

The rats were injected with sodium fluoride 30 minutes after 

magnesium chloride administration. 

The animals were sacrificed 24 hours after the sodium fluoride 

treatment. 

ns 

Not significant as compared to control group (Tukey’s multiple 

comparison test); 

**P

animals may be either due to decreased synthesis or 

increased degradation by collagenase (Machoy – 

Mokrzynska, 2004). 

In the present study soluble collagen Hyp was found to be 

more susceptible to the effects of both MgCl2 as well as 

NaF. Both MgCl2 and NaF caused a decrease in soluble 

collagen Hyp in rat kidneys. Earlier studies of Prockop 

(1964) have shown that there exists at least 3 pools of 

body collagen with half lives of about 1day, 5days and 

50-100 days. The first two of these represent the soluble 

collagen fractions, i.e. fractions containing collagen not 

yet aggregated to insoluble-collagen fiber and the third 

pool, the insoluble collagen. In the present study the 

soluble collagen Hyp appeared to be susceptible to 

degradation by MgCl2 and NaF. This may be due to the 

fact that it has still not aggregated to form insoluble 

collagen fibers. Studies of Sharma (1982 b ) have shown 

that fluoride interferes with collagen crosslinking. Studies 

by the same author have also demonstrated that NaF 

interferes with maturation and normal metabolism of 

tissue collagen. The insoluble collagen Hyp was affected 

only by NaF treatment alone. Administration of MgCl2 

before NaF restored the altered insoluble collagen Hyp to 

near normal levels. This appears to be the only protective 

effect of MgCl2 on NaF induced change in kidney 

collagen. 

CONCLUSION 

Therefore the present study concludes that though MgCl2 

has been reported to be protective against the toxic effect 

of NaF it has no significant effect on NaF induced 

changes in kidney Hyp/collagen. However administration 

of MgCl2 before NaF restored insoluble collagen 

hydroxyproline which was altered by NaF to near normal 

levels. 


The authors would like to thank the Research Center, 

Center for Scientific and Medical Female Colleges, King 

Saud University, Riyadh and King Abdul Aziz City for 

Science and Technology (Grant Number 098-17 AT), 

Riyadh for financial support to EAA. 

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Al Omireeni et al. 1167 

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1168 

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Received: Jan 26, 2010: Revised: April 21, 2010; Accepted: 

April 29, 2010


Vol. 4, No. 2, pp. 1169-1178, June 2010 

ISSN: 1715-9997 

SYNTHESIS, CHARACTERISATION AND IN-VITRO BIOLOGICAL 

ACTIVITIES OF SOME METAL(II) COMPLEXES OF 

3-(-1-(4-METHYL-6-CHLORO)-2-PYRIMIDINYLIMINO)METHYL-2-NAPTHOL 

*A A Osowole 1 , R Kempe 2 , R Schobert 3 and S A Balogun 4 

1 Department of Chemistry, University of Ibadan, Ibadan, Nigeria 

2 Anorganische Chemie II, Universität Bayreuth, D-95440 Bayreuth, Germany 

3 Organische Chemie I, Universität Bayreuth, D-95440 Bayreuth, Germany 

4 Department of Microbiology, Adekunle Ajasin University Akungba–Akoko, PMB 001 

Akungba–Akoko, Ondo state, Nigeria 

ABSTRACT 

Synthesis, spectroscopic characterisation and in-vitro antibacterial and anticancer studies were done on the neutral 

bidentate (NO) Schiff base, 3-(-1-(4-methyl-6-chloro)-2-pyrimidinyl imino)methyl-2-napthol and its Mn(II), Co(II), 

Ni(II), Pd(II), Cu(II) and Zn(II) complexes. These complexes assumed a 4-coordinate tetrahedral /square-planar 

geometry as corroborated by room temperature magnetic data, IR and electronic spectral measurements. The 

conductance data confirmed their covalent nature while mass spectra and TGA data affirmed the proposed structure. The 

ligand was not sensitive to HL 60 (leukaemia) but resistant to 518A2 (melanoma) carcinomas. The Pd(II) complex had 

the best cytotoxic activities against HL 60 (Leukemia) and Melanoma (518A2) carcinomas with IC50 of 11.89 and 9.11 

µm at 48 and 72 h respectively, which was a third as, and four times more sensitive than CDDP (Cis-platin). The 

complexes generally exhibited good antibacterial activities against five Gram negative (E. cloacae, S. arizona, Serratia 

sp, E. Coli) and three Gram positive (C. violaceum, S. aureus, Bacillus sp) bacteria with inhibitory zones range of 7-19 

mm. Similar to gentamycin, the Cu(II) and Zn(II) complexes had broad-spectrum activity against the bacteria used, 

although much lower. 

Keywords: Antibacterial and anticancer activities, cis-platin –sensitive and –resistant cell lines, Schiff base. 

INTRODUCTION 

The interest in metal complexes as drugs has increased in 

the last decade due to the search for drugs with an 

enhanced therapeutic effect in combination with 

decreased toxicity (Zhaohua et al., 2001; Zhong et. al., 

2006). It has been documented that drugs containing 

pyrimidine moiety are more prominent in treating solid 

tumours and cancers due to their good anti-proliferation 

activity and low toxicity e.g. bevacizumab in combination 

with 5-fluoro uracil is used in treatment of metastatic 

colorectal cancer (Ramaling and Belani, 2007) while 

sorafenib and sunitinib, which are small-molecule 

multikinase inhibitors are used for the treatment of 

advanced renal-cell carcinoma (Atkins et al., 2006; 

Wilhelm et al; 2006). Currently, the drug pazopanib (5- 

(4-[(2,3-dimethyl-2H-indazoyl-6-yl)methylamino]-2pyrimidinyl]amino-2-methyl 

benzene sulfonamide) is 

undergoing clinical development for use in treating renalcell 

cancer and other solid tumours (Harries et al., 2008). 

In addition, pyrimidinyl Schiff bases are widely studied 

due to their ease of preparation, coordination to many 

metal ions, and the stability of such oxidation states. 

Furthermore, their good solubility in DMSO and DMF 

*Corresponding author email: aderoju30@yahoo.com 

makes them suitable for cell lines studies (Gorneva and 

Golovinsky, 2003; Shreelekha et al., 2006). In 

continuation of our studies on synthesis, characterisation 

and anticancer /antimicrobial properties of some 

metal(II) complexes of various pyrimidinyl Schiff bases 

(Osowole et al., 2009), we report our findings on 

anticancer and antibacterial properties of Mn(II), Ni(II), 

Co(II), Cu(II), Zn(II) and Pd(II) complexes of 3-(-1-(4methyl-6-chloro-2-pyrimidinyl 

imino)methyl-2-napthol 

(derived from condensation of 2-amino-4-methyl-6chloropyrimidine 

and 2-hydroxy-1-napthaldehyde) 

against cis-platin–sensitive (Leukemia) and –resistant 

(Melanoma) cell lines and, E. cloacae, S. arizona, 

Serratia sp, E. Coli) and three Gram positive (C. 

violaceum, S. aureus, Bacillus sp). Their spectroscopic, 

magnetic and thermal properties are also discussed. This 

ligand and its metal complexes are new, being reported 

here by our group for the first time. 


Chemicals and Instrumentation 

All the chemicals used were of reagent grade. 2-amino-4methyl-6-chloropyrimidine 

and 2-hydroxy-1napthaldehyde 

(Across), palladium(II) chloride, hydrated 

manganese(II) nitrate, cobalt(II) nitrate, nickel(II) nitrate,

1170 

copper(II) nitrate and zinc(II) nitrate (Aldrich) were used 

as received. The solvents were purified using 

conventional methods. C, H and N analyses were carried 

out using a GmbH VarioEl analyser. Palladium, 

Manganese, cobalt, nickel, copper and zinc were 

determined titrimetrically (Bassett et al., 1978). The 1 H 

nmr spectra were recorded on a 300 MHz Oxford Varian 

NMR instrument in CDCl3 at 295K. 1 H chemical shifts 

were referenced to the residual signals of the protons of 

CDCl3 and are quoted in ppm. Magnetic susceptibilities 

were measured on Johnson Matthey magnetic 

susceptibility balance at room temperature (27 o C) all 

susceptibilities were corrected for the diamagnetic 

contributions using Pascal’s constants (Earnshaw, 1980). 

The reflectance spectra were recorded on a Perkin-Elmer 

λ20 spectrophotometer equipped with an integrating 

sphere. The infrared spectra were measured as KBr discs 

on a Bruker-IFS 66V spectrometer in the range 4000-400 

cm −1 . Thermogravimetric analyses were done in static air, 

using a T6 Linseis thermal analyser with a heating rate of 

10°C/min in the range 30-700°C while electrolytic 

conductivities of the complexes (1 x 10 -3 Mdm -3 ) in 

nitromethane were determined using a Hanna HI 8828N 

conductometer and melting points (uncorrected) were 

done using a Stuart scientific melting point apparatus 

smp3. 

Biological Studies 

The human HL 60 leukaemia cells were obtained from the 

German National Resource Centre for Biological 

Materials (DSMZ), Braunschweig and the human 518A2 

melanoma cells were cultured in the Department of 

Oncology and Hematology, medical faculty of the Martin- 

Luther University, Halle, Germany. 

Cytotoxicity (MTT) Assay 

MTT[3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 

bromide] was used to identify viable cells which were 

capable of reducing it in their mitochondria by succinate 

dehydrogenase to a violet formazan product. 

Cell lines and Culture condition 

HL 60 (0.5 x 10 6 cells/mL) and 518A2 cells (1.7 x 10 5 

cells/mL) were seeded out and cultured for 24h on 96well 

microplates. Incubation (5% CO2, 95% humidity, 

and 37°C) of cells following treatment with test 

compounds was continued for 24, 48 and 72h. Blank and 

solvent controls were incubated under identical 

conditions. A 5 mg/mL stock solution of MTT in 

phosphate-buffered saline (PBS) was then added to a final 

concentration of 0.05%. After 2 h the precipitate of 

formazan crystals was redissolved in a 10% solution of 

sodium dodecylsulfate (SDS) in DMSO containing 0.6% 

acetic acid in the case of the HL 60 cells. For the adherent 

melanoma (518A2) cells, the microplates were swiftly 

Osowole et al. 

turned to discard the medium prior to adding the solvent 

mixture. The microplates were gently shaken in the dark 

for 30 min and left in the incubator overnight, to ensure a 

complete dissolution of the formazan. Finally the 

absorbance at wavelengths 570 and 630nm (background) 

was measured using an ELISA plate reader. All 

experiments were carried out in quadruplicate, and the 

percentage of viable cells quoted was calculated as the 

mean ± SD with respect to the controls set to 100%. 

Blank tests have shown that DMSO used in the 

preparation of the test compound does not have any effect 

on the cancer cell lines. 

Antibacterial assay 

The assay was carried out on the ligand and metal(II) 

complexes. The bacteria used were identified laboratory 

strains of E. cloacae, S. arizona, Bacillus sp, S. 

liquefaciens, S. aureus, Klebsiella sp, Salmonella sp, 

Serratia sp, Pseudomonas sp, E. coli, C. violaceum. The 

antibacterial susceptibility test was carried out using the 

Agar diffusion technique. The surface of the agar in a 

Petri dish was uniformly inoculated with 0.3 mL of 18 h 

old test bacteria culture. Using a sterile cork borer, 6 mm 

wells were bored into agar. Then 0.06 mL of 10 mg/mL 

concentration of each metal complex in DMSO was 

introduced into the wells and the plates were allowed to 

stand on bench for 30 min before incubation at 37 0 C for 

24 h after which inhibitory zones (in mm) were taken as a 

measure of antibacterial activity. The experiments were 

conducted in duplicates and gentamycin was used as a 

reference drug. 

Synthesis 

Preparation of Ligand 

The ligand, {[C10H6(OH)CH:N(C5H4N2Cl)]} HL, was 

prepared by refluxing a mixture of 0.017 mol (2.50 g) of 

2-amino-4-methyl-6-chloropyrimidine and 0.017 mol 

(3.00 g) of 2-hydoxy-1-napthaldehyde with 6 drops of 

acetic acid in 60 mL of absolute ethanol for 6 h. The 

yellow product, formed on cooling to room temperature, 

was filtered and recrystallized from ethanol and dried in 

vacuo over anhydrous calcium chloride. The yield of the 

resulting Schiff base was 3.62 g (70%). 1 H nmr (ppm) 

δ10.8 (s, OH), 9.38(s, 1H, HCN); δ 7.38-8.60 (m, 6H, 

C10H6); 6.70 (s, 1H, CHpyrimidine); 2.50 (s, 3H, CH3). 

Preparation of the Metal(II) Complexes 

A solution of the metal(II) nitrates (0.54 mmol, 0.10-0.16 

g M = Mn, Co, Ni, Cu, Zn ) in 20 mL ethanol was added 

to a stirring solution of the ligand (1.08 mmol, 0.32 g) in 

30mL ethanol at room temperature (26°C), followed by 

gradual addition of triethylamine (1.08 mmol, 0.15 mL). 

The resulting homogeneous solution was refluxed for 6h, 

and the products formed were later filtered, washed with 

ethanol and dried in vacuo over anhydrous CaCl2.

RESULTS AND DISCUSSIONS 

The reactions of the ligand with the metal (II) nitrates 

(Mn, Co, Ni, Cu, and Zn) gave mononuclear complexes in 

moderate-good yields (50-70%). The physical and 

analytical data, colours, % yields, melting 

points/decomposition temperature and room temperature 

magnetic moments of the compounds are presented in 

Table 1 and the proposed structures are shown in figure 1. 

The complexes were all air stable but decomposed on 

programmed heating and were soluble in methylene 

chloride, nitromethane, dimethylfornamide and 

dimethylsulphoxide. The elemental analysis, showed the 

stoichiometry of the complexes as 2L:M and confirmed 

the suggested molecular formula of the complexes. The 

decomposition pattern obtained from TGA curve 

corroborates the proposed formulation of the complexes. 

All complexes isolated adopted [ML2]xH2O stoichiometry 

{Mn, Pd: x = 2; Ni, Zn: x = 1} except, the Cu(II) complex 

which was anhydrous. The molar conductances of the 

complexes ranged between 12-20 Ω -1 cm 2 mol -1 in 

nitromethane, showing they were non-electrolytes. A 

value of 60-90 Ω -1 cm 2 mol -1 is expected for a 1:1 

electrolyte (Osowole et al., 2009). Attempts to isolate 

suitable crystals for single X-ray structural determination 

have not yet been successful. 

OH 

N 

N 

N 

Cl 


1171 

1 Hnmr spectra 

The ligand showed the napthyl ring protons as a multiplet 

at δ 6.94-9.45 ppm (m, 6H, C10H6). The phenolic proton 

was observed as a singlet at 10.8 ppm(s, 1H, OH) and the 

proton of the imine resonated as a singlet at δ 6.70 ppm 

(s,1H, H=CN) while that of the pyrimidine was seen as a 

singlet at δ 6.67 ppm(s, 1H, CH pyrimidine). The methyl 

protons resonated as a singlet at 2.50ppm(s, 3H, CH3). On 

coordination to Zn(II) ion, the napthyl ring protons were 

deshielded and resonated as a multiplet at δ 7.01-8.24 

ppm (m, 6H, C10H6). The phenolic proton was 

conspicuously absent, indicating the involvement of OH 

in chelating. The HC=N and CH(pyrimidine) protons 

resonated as singlets at δ 6.79 and δ 6.56 ppm 

respectively, while the methyl protons singlet shifted to δ 

2.36 ppm. These shifts indicate deshielding due to 

coordination of the imine nitrogen atom. Similarly, Pd(II) 

complex napthyl ring protons were deshielded and 

resonated as a multiplet at δ 7.24-9.33 ppm (m, 6H, 

C10H6). The phenolic proton was conspicuously absent, 

indicating the involvement of OH in chelating. The 

HC=N proton signet, appeared at δ 6.72 ppm, while the 

CH of the pyrimidine appeared at δ 6.69 ppm. The methyl 

protons singlet shifted to δ 2.51 ppm. These downfield 

shifts indicate coordination of the imine nitrogen atom. 

Cl 

N N 

(A) (B) 

A, Schiff base; B, Metal(II) Schiff base complexes 

Fig 1. Proposed structures for the ligand and its complexes. 

Table 1. Analytical data for the compounds. 

Compound 

(Empirical formula) 

HL 

(C16H12N3OCl) 

[MnL2]2H2O 

(MnC32H26N6O4Cl2) 

[CoL2]H2O 

(CoC32H24N6O3Cl2) 

[NiL2]H2O 

(NiC32H24N6O3Cl2) 

[CuL2] 

(CuC32H22N6O2Cl2) 

[ZnL2]H2O 


[PdL2]2H2O 

(PdC32H26N6O4Cl2) 

D = diamagnetic; * Ω -1 cm 2 mol -1 

O 

N 

M 

Cl 

N N 

N 

O 

xH 2O 

Formul 

a mass 

Color µeff 

% 

Yield 

Λm * 

M.p 

( o C) %C 

Analysis (Calculated) 

%H %N % M 

297.56 Yellow - 70 - 188-189 64.53 4.13 13.89 

- 

(64.58) (4.07) (14.12) 

684.08 Brownish 5.70 60 10.00 259-260 56.18 3.54 11.84 7.95 

green 

(56.19) (3.83) (12.29) (8.03) 

647.59 Brown 4.36 70 20.00 280-281 57.46 3.56 12.41 9.06 

(57.36) (3.61) (12.54) (9.10) 

629.30 Dark 3.41 70 25.00 283-284 57.16 3.55 12.51 9.20 

Orange 

(57.33) (3.61) (12.55) (9.07) 

616.70 Brown 1.91 70 18.00 238-239 58.67 3.40 12.50 10.25 

(58.53) (3.38) (12.80) (10.30) 

635.64 Orange D 60 12.00 268-269 57.44 3.56 12.20 10.17 

(56.90) (3.58) (12.40) (10.23) 

733.14 Brown D 50 15.00 183-184 52.67 3.40 11.57 14.24 

(52.50) (3.60) (11.50) (14.19)

1172 

Infrared spectra 

The relevant infrared data are presented in table 2. The 

assignments of the infrared bands were made by 

comparing the spectra of the compounds with reported 

literature on similar systems (Abd El Wahab, 2008; 

Osowole et al., 2005; Osowole et al., 2008; Sonmez and 

Haciyusufoglu, 2006; Sonmez et al., 2004). The strong 

band in the ligand at 3425 cm -1 , which disappeared in the 

spectra of all the complexes, is assigned as νOH. This 

indicates the involvement of the enolic O in bonding to 

the metal ions. Furthermore, the ν(C-O) enolic band in the 

ligand at 1447 and 1360 cm -1 were bathochromic shifted 

to 1281-1192 cm -1 also corroborating enolic oxygen´s 

coordination. A new broad band, ν(OH) at 3500 cm –1 , in 

the hydrated complexes is assigned to coordinated water. 

The uncoordinated C=N and C=C stretching vibrations in 

the ligand were expectedly coupled and observed at 1631- 

1539 cm –1 (Osowole et al., 2005). These bands suffered 

bathochromic shifts to 1630-1522 cm –1 in the metal 

complexes, thus confirming the involvement of the imine 

N atom in coordination to metal(II) ion (Singh et al., 

2001). The δC―H vibration of the ligand was observed at 

967 cm –1 and it was bathochromic shifted to 897-776 cm –1 

in the complexes due to the pseudo-aromatic nature of the 

chelates (Osowole et al., 2008). The bands due to 

ν(M―O) and ν(M―N) were observed at 499-417 and 

585-501 cm –1 respectively in the complexes (Sonmez et 

al., 2004). 

Electronic spectra and room temperature magnetic 

moments 

In the UV region, the reflectance electronic spectra (Table 

2) of all the complexes were similar, displaying strong 

absorption maxima between 26.50-50.00 kK, which are 

assigned to n- π*, π-π* and charge transfer transitions 

(Kwiatkowski and Kwiatkowski, 1980; Singh et al., 

2001). 

The Mn(II) complex showed two weak bands at 15.40 


Table 2. Relevant infrared and electronic spectral data of the complexes 

and 24.80 kK respectively, consistent with a fourcoordinate, 

tetrahedral geometry and are assigned to 6 A1 

→ 4 E1 (ν1) and 6 A1 → 4 A1 (ν2) transitions. The effective 

magnetic moment of Mn(II) complexes are expected to be 

close to the spin-only value of 5.90 B.M. since the 

ground term is 6 A1g and as such there is no orbital 

contribution. Consequently an observed moment of 5.70 

B.M. for this complex indicates that it is high spin and 

complementary of tetrahedral geometry (Chohan, 2001). 

The Co(II) complex exhibited two bands at 13.00 and 

19.50 kK, typical of a 4-coordinate tetrahedral geometry 

and are assigned to 4 A2 → 4 T1(F), (ν2), and 4 A2 → 4 T1(P), 

(ν3), transitions. The transition, 4 A2→ 4 T2, (ν1) in the range 

5-7 KK was not observed, being outside the range 

covered by the instrument. An observed moment of 4.36 

B.M is corroborative of tetrahedral geometry, since 

moments in the range 4.3-4.6 B.M are usually observed 

for tetrahedral Co(II) compounds (Singh et al., 2001; 

Sonmez and Haciyusufoglu, 2006; Sonmez et al., 2004). 

The reflectance spectra of the Ni(II) complex showed 

absorption bands at 12.00 and 17.00 kK which are 

assigned to 3 T1(F) → 3 T2,(ν2) and 3 T1(F) → 3 A2, (ν3) 

transitions, in a tetrahedral environment. Generally, 

square planar Ni(II) complexes are usually diamagnetic, 

while tetrahedral and octahedral complexes are 

paramagnetic with moments in the range 3.20-4.10 and 

2.90-3.30 B.M respectively. This complex gave a moment 

of 3.41 B.M. and hence is tetrahedral (Abd El Wahab, 

2008; Sonmez and Haciyusufoglu, 2006). 

The observance of two bands at 15.70 and 24.40 kK in the 

Cu(II) complex indicates square planar geometry with the 

assignment of the bands as 2 B1g → 2 A1g and 2 B1g → 2 E1g 

transitions. A moment of 1.9-2.2 B.M. is usually observed 

for mononuclear copper(II) complexes, regardless of 

stereochemistry, expectedly higher than the spin only 

moment due to orbital contribution and spin-orbit 

Compound νOH ν(C=N) + ν(C=C) νPh/C―O δC―H ν(M―N) ν(M―O) Electronic spectral (kK) 

[HL] 3425s 1631s 1572s 1539s 1447s 1360s 967s - - 28.00 32.00 39.00 

42.00 

[Mn(L)2]2H2O 3500b 1620s 1567s 1538s 1289s 1188s 897s 747s 537m 501m 486m 15.40 24.80 27.20 

450s 31.70 44.10 47.40 

[Co(L)2] H2O 3400b 1618s 1605s 1562s 1286m 1195m 832s 754s 568s 550m 481m 11.00 17.50 29.20 

1529s 

446m 34.51 42.30 

[Ni(L)2 ]H2O 3500b 1623s 1577s 1551s 1290m 1190m 875s 786m 544m 518m 458m 12.00 17.00 26.53 

423m 32.10 42.92 48.50 

[Cu(L)2] - 1619s 1604s 1290m 1195m 833s 747s 525m 504m 461m 15.70 24.40 26.50 

1581s 1539s 

417m 30.40 42.00 50.00 

[Zn(L)2]H2O 3500b 1620s 1605s 1579s 1281m 1196m 831s 755s 550m 544m 499m 20.00 30.00 33.30 

1522s 

456m 39.50 45.00 

[Pd(L)2]2H2O 3500b 1615s 1600s 

1575s 1522s 

b = broad, s = strong, m = medium; 1kK = 1000 cm -1 . 

1291m 1192m 827s 749s 585m 559m 486m 

452m 

16.00 23.30 29.90 

33.00 39.40 44.00

coupling. [CuL2] had a moment of 1.91 B.M. and is 

consequently mononuclear (Singh et al., 2001). 

The Zn(II) complex showed only the CT transitions from 

M→L at 20.00 kK as no d-d transition is expected. The 

bands at 30.00, 33.30, 39.50 and 45.00 kK are assigned as 

π-π* and intraligand charge transfer transitions 

respectively. This complex is expectedly diamagnetic, 

confirming its tetrahedral geometry (Singh et al., 2001). 

The Pd(II) complex showed absorption bands typical of a 

square-planar geometry at 16.00 and 23.25 kK, which are 

assigned to 1 A1g → 1 B1g and 1 A1g → 1 E2g transitions. Its 

diamagnetism is corroborative of square-planar geometry 

(Kwiatkowski and Kwiatkowski, 1980). 

Mass spectroscopy and Thermal studies 

The mass spectra of ligand and the complexes showed 

Table 3. Thermal data for the ligand and complexes. 

Compound Temperature 

range(°C) 

HL 

110-170 

(C16H12N3OCl) 

170-380 

380-700 

[MnL2]2H2O 

30-100 

(MnC32H26N6O4Cl2) 100-160 

160-230 

230-300 

300-410 

410-700 

[CoL2]H2O 

(CoC32H24N6O3Cl2) 

[NiL2]H2O 

(NiC32H24N6O3Cl2) 

[CuL2] 


[ZnL2]H2O 

(ZnC32H24N6O3Cl2) 

[PdL2]2H2O 

(PdC32H26N6O4Cl2) 

30-310 

310-410 

410-660 

30-100 

100-200 

200-390 

390-700 

30-170 

170-330 

330-700 

50-100 

100-230 

230-430 

430-700 

30-80 

130-360 

360-700 


1173 

peaks attributed to the molecular ions m/z at 297 [L] + ; 

647 [MnL2-2H] + ; 651 [CoL2-3H] + ; 652 [NiL2-2H] + ; 655 

[CuL2-3H] + ; 658 [ZnL2-H] + and 696 [PdL2-2H] + . The 

decomposition stages, temperature ranges, percentage 

losses in mass and assignment of decomposition moieties 

are given in table 3. 

The ligand, HL, decomposed in three stages. Stage one 

was between 110-170°C and corresponds to the loss of 

the fragment C2H2N, with mass losses of (obs. = 14.00%, 

calc. = 13.46%). The second stage involved the loss of the 

fragment C4H4Cl, with mass losses of (obs. = 29.89%, 

calc. = 29.43%) within a temperature range of 170-380°C 

and the final stage was the loss of the organic moiety, 

C10H6N2 at 380-700°C, with mass losses of (obs. = 

58.94%, calc. = 57.19%). 

The Mn(II) complex decomposed in six steps. The first 

TG weight loss(%) 

Calc. Found 

13.46 14.00 

29.43 29.89 

57.19 58.94 

9.95 9.83 

1.02 1.05 

2.64 2.93 

6.45 6.41 

7.69 7.72 

64.81 66.50 

5.99 6.03 

8.37 8.41 

69.75 70.02 

5.40 5.92 

10.60 10.57 

16.75 16.74 

58.57 65.68 

21.05 21.24 

10.80 11.21 

58.53 66.26 

14.62 14.72 

22.54 23.23 

10.80 10.54 

45.04 44.59 

10.66 10.72 

16.53 16.71 

58.57 65.68 

Assignments 

C2H2N 

C4H4Cl 

C10H6N2O 

2H2O + O2 

¼N2 

CH4+ H2 

C3H8 

C3H6N¾ 

C25H2N5Cl2 

Mn(residue) 

CH4 + 3H2 + H2O 

C2H4N2 

C25H8N4Cl2O2 

Co (residue) 

CH4 + 3H2 + H2O 

Cl2 

C6H12N2 

C25H4N4O2 

Ni(residue) 

C8H14N2 

Cl2 

C24H8O2N4 

Cu(residue) 

C6H6 + H2O 

Cl2 

C17H4O2N4 

Zn(residue) 

2H2O + C3H6 

C7H9+ H2 

C22H7N4O2Cl2 

Pd(residue)

1174 

step was the loss of two molecules of water and oxygen 

with mass losses of (obs. = 9.95%, calc. = 9.83%) in the 

temperature range 30-100°C. The second step involved 

the loss of 0.25 mole N2, with mass losses of (obs. = 

1.05%, calc. = 1.02%) between 100-160°C. The third step 

corresponds to the loss of methane and hydrogen at 160- 

230°C with, mass losses of (obs. = 2.93%, calc. = 2.64%). 

The fourth step involved the loss of propane, with mass 

losses of (obs. = 6.41%, calc. = 6.45%) at 230-300°C. The 

fifth was the loss of the fragment C3H6N¾ in the 

temperature range 300-410°C, with mass losses of (obs. = 

7.72%, calc. = 7.69%). The final step involved loss of the 

organic moiety C25H2N5Cl2 at 410-700°C, with mass 

losses of (obs. = 66.50 %, calc. = 64.81%), leaving behind 

Mn as the residue. 

The thermal degradation of the Co(II) complex occurred 

in three steps. The first step was within the temperature 

range of 30-310°C and corresponds to the loss of a 

molecule of water and methane, and three molecules of 

hydrogen with mass losses of (obs. = 6.03%, calc. = 

5.99%). The second step occurred between 310-410°C 

and was characterized by loss of the fragment, C2H4N2, 

with mass losses of (obs. = 8.41%, calc. = 8.37%). The 

final step involved the loss of the organic moiety 

C25H8O2N4Cl2, within a temperature range of 410-700°C 

with corresponding mass losses of (obs. = 70.02 %, calc. 

= 69.75%). The final product was cobalt. 

The Ni(II) complex decomposed in four steps. The first 

step was the loss of a molecule of water, methane and 

hydrogen at 30-100°C, with mass losses of (obs. = 5.92%, 

calc. = 5.40%). The second step was in the temperature 

range of 100-200 °C, and is attributed to the loss of Cl2 

(obs. = 10.57%, calc. = 10.60%). The third step involved 

the loss of the fragment C6H12N2, at 200-390°C, with 

mass losses of (obs. = 16.74%, calc. = 16.75%). The final 

step was within the temperature range of 390-700°C and 


is assigned to the loss of the organic moiety C25H4N4O2, 

(obs. = 66.10%, calc. = 58.57%). The remaining residue 

was Ni. 

The Cu(II) complex decomposed in three ways. The first 

decomposition, was the loss of the fragment, C8H14N2 at 

30-170°C, with mass losses of (obs. = 21.24%, calc. = 

21.05 %). The second step occurred within a temperature 

range of 170-330°C and is attributed to the loss of Cl2 

(obs. = 11.21%, calc. = 10.80%). The final stage was 

within a temperature range of 460-700°C, assigned to the 

loss of the organic moiety C24H8O2N4 (obs. = 66.26%, 

calc. = 58.53%). The remaining fraction was Copper. 

The Zn(II) complex decomposed in four stages. The stage 

one was between 50-100°C, which indicated the loss of a 

mole of water and benzene respectively, with mass losses 

of (obs. = 14.72%, calc. = 14.62%). The second stage was 

from 100-230 °C and is assigned to the loss of the 

fragment, C9H12N2, with mass losses of (obs. = 23.32 %, 

calc. = 22.54%). The third stage involved the loss of a 

mole of chlorine at 230-430°C, with mass losses of (obs. 

= 10.54%, calc. = 10.80%). The final stage was within a 

temperature range of 430-700°C, attributed to the loss of 

the organic moiety C17H4N4O2, (obs. = 44.59%, calc. = 

45.04%). The remaining residue was Zn. 

The Pd(II) complex decomposed in three steps. The first 

step involved the loss of two molecules of water and a 

molecule of propene at 30-130°C, with mass losses of 

(obs. = 10.72%, calc. = 10.66 %). The second was within 

a temperature range of 130-360°C, which corresponds to 

the loss of the fragment, C7H9N2 with mass losses of (obs. 

= 16.71%, calc. = 16.53%). The final stage was within a 

temperature range of 360-700°C and corresponds to the 

loss of the organic moiety C22H7O2N4Cl2 with mass losses 

of (obs. = 65.68%, calc. = 58.70%). The remaining 

fraction was Pd. The high mass residue (~7%) observed in 

Table 4. IC50 values of the ligand and its complexes against Melanonoma (518A2) and Leukemia (HL60) cells. 

Compound IC50 [µM] 

Melanoma cells(518A2) 

IC50 [µM] 

Leukaemia cells (HL 60) 

24h 48h 72h 24h 48h 72h 

CDDP 35 - - 3.5 - - 

H2L 74.12±15.82 78.01±15.56 56.43 ±7.33 >100 >100 >100 

[Mn(L)2]2H2O >100 37.02±4.29 31.93±3.96 32.39 29.58 18.80 

[Co(L)2]H2O >100 >100 >100 >100 35.35 26.91 

[Ni(L)2]H2O >100 >100 >100 >100 16.43 28.48 

[Cu(L)2] >100 >100 >100 14.04 75.33 39.29 

[Zn(L)2]H2O >100 >100 >100 >100 >100 >100 

[Pd(L)2]2H2O 10.68±1.47 12.51±0.88 9.11±0.55 20.56 11.89 22.55 

30µm = resistant; 

>100 µm = not active; CDDP = cis-platin; - = Not determined

Melanoma (518A2) cells [µm] 

the Ni(II), Cu(II) and Pd(II) complexes were attributed to 

carboneous material (Sharma and Srivastava, 2006). 

Biological studies 

The growth inhibitory effects of the ligand and its 

complexes on the cis-platin (CDDP) resistant 518A2 

(Melanoma) and (CDDP) sensitive HL 60(Leukemia) cell 

80 

70 

60 

50 

40 

30 

20 

10 

0 

CDDP 


HL 

Compounds 

[MnL 2 .2H 2 O] 

Fig 2. The inhibitory effect of the ligand, Mn(II) and Pd(II) complexes against Melanoma cells. 

Leukemia(HL 60) cells [µm] 

80 

60 

40 

20 

0 

CDDP 

[MnL 2 .2H 2 O] 

[CoL 2 .2H 2 O] 

[NiL 2 ]H 2 O 

Compounds 

Fig 3. The inhibitory effect of some of the complexes against Leukemia cells. 

[CuL 2 ] 

24 h 

48 h 

72 h 

[PdL 2 ] 

24 h 

48 h 

72 h 

[PdL 2 ] 

1175 

lines are shown in table 4, figures 2 and 3. The ligand, HL 

(74.12-78.01µm) was about twice as resistant as CDDP 

(35.0µm) against Melanoma cells during the first 48h of 

experiment, while its resistance dropped by about 20% at 

72 h of exposure. In contrast, the Co(II), Ni(II), Cu(II) 

and Zn(II) complexes showed no anti-tumor activity 

throughout the duration of experiment. It is important to

1176 

note that though the Mn(II) complex does not show 

antitumor activity at 24 h, it exhibited comparable activity 

to CDDP (35.0µm) at 48 and 72 h, with IC50 values of 

37.02 and 31.93µm respectively. Notably, [PdL2] was 

about three times more sensitive than CDDP with IC50 of 

10.68 and 12.51 µm, at 24 and 48 h of experiment 

respectively. Further exposure of the cell line to the metal 

complex at 72 h, showed that the sensitivity increased to 

four times that of CDDP. Consequently, for further 

studies with the Pd(II) complex in drug development 

against melanoma cancers, the most effective time for 

exposure is 72 h. 

The ligand and the Zn(II) complex were not sensitive to 

CDDP (3.5 µm) sensitive HL 60 Leukemia cells 

throughout the duration of experiment (Fig. 3). The 

Mn(II) complex was resistant with IC50 of 32.39 µm at 24 

h. Further exposure at 48 and 72 h showed improved 

weak and moderate activity with IC50 of 29.58 and 


Table 5. Inhibitory zones(mm) of the ligand and complexes against various bacterial isolates. 

Complexes 

E. Cloacae 

S.arizona 

Bacillus sp 

S.Liquefaciens 

HL 7.0±0.1 R R 8.0±0.3 R R 7.0±1.2 R 11.0±0 11.0±0 

[Mn(L)2]2H2O 10.0±0.5 7.0±0.3 8.0±0.1 10.0±0.1 10.0±0.01 9.0±1.2 14.0±1.2 R 15.0±0 7.0±0.2 

[Co(L)2]H2O 7.0±0.01 10.0±0.5 R R 8.0±0.01 R 7.0±0.4 R 19.0±0.1 12.0±0.6 

[Ni(L)2]H2O 8.0 ±0.03 15.0±0.2 8.0±0.01 R 8.0±0.1 R 9.0±2.0 R 18.0±0.2 8.0±0 

[Cu(L)2] 11.0±0.1 10.0±0.1 9.0±0.2 11.0±0.1 13.0±0.1 11.0±2.0 14.0±2.1 10.0±0.6 15.0±1.1 12.0±1.1 

[Zn(L)2]H2O 12.0±0.3 12.0±0.1 7.0±1.0 13.0±0.1 7.0±0 9.0±0.6 15.0±1.1 7.0±0.3 19.0±0.1 7.0±1.2 

[Pd(L)2]2H2O 8.0±0.1 10.0±0.4 13.0±1.1 13.0±0.2 9.0±0.3 7.0±2.1 19.0±1.4 R 16.0±0.3 10.0±0 

DMSO* R R R R R R R R R R 

Gentamycin + 40.0±0.1 30.0±2.0 28.0±1.2 26.0±1.4 40.0±0.8 30.0±1.3 29.0±1.2 40.0±1.6 45.0±1.2 43.0±1.6 

R = Resistant, * = Negative standard, + = Positive standard, E. cloacae = Enterococcus cloacae; S. arizona = Salmonella arizona; 

S. liquefaciens = Serratia liquefaciens; S. aureus = Staphyloccus aureus; E. Coli = Escherichia coli; C. violaceum = Chromobacter violaceum 

Actitity(mm) 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

HL 

MnL 2 

CoL 2 

NiL 2 

CuL 2 

Complexes 

S. aureus 

ZnL 2 

Klebsiella sp 

PdL 2 

Serratia sp 

Gentamycin 

18.80µm respectively. Similarly, the Co(II) complex had 

no activity at 24 h, but became resistant at 48 h (35.35 

µm) and weakly active (26.91µm) at 72 h respectively. 

The Ni(II) complex was not effective at 24 h but showed 

moderate activity at 48 h (16.43 µm) and became weakly 

active at 72 h (28.48 µm). On the other hand, the Cu(II) 

complex exhibited moderate activity at 24 h (14.04 µm), 

which was a quarter of that of CDDP (3.5 µm). However 

it became resistant at 48 (75.33 µm) and 72 (39.29 µm) h 

respectively, with the latter having overcome the former’s 

resistance by about 50 %. The Pd(II) complex was 

sensitive throughout the duration of the experiment. It 

was weakly active (20.56 µm) at 24 h. It then became 

moderately active (11.89 µm) at 48 h and finally at 72 h, 

activity dropped back to weakly active (22.55 µm). The 

activity at 48 h was a third of that of CDDP (3.5 µm). 

It was evident from this cytotoxic studies, that the Pd(II) 

complex had the best anticancer activity against both 

Pseudomonas sp 

E.Coli 

E.cloacae 

S.arizona 

Bacillus sp 

S.liquefaciens 

S.aureus 

Klebsiella sp 

Serratia sp 

Pseudomonas sp 

E. coli 

C.violaceum 

Fig. 4. The comparative activities of the complexes against bacteria strains and standard antibiotic. 

C.violaceum

Melanoma (518A2) and HL60 (Leukemia) carcinomas invitro 

with IC50 values of 9.11 and 11.87 µm at 72 h 

and 48 h of exposure respectively, which was four times 

more sensitive than, and a third as sensitive as CDDP. 

The activities of the Cu(II) and Pd(II) complexes may be 

attributed to their planar structure. It has been 

documented that complexes with such geometry avoid 

possible steric hindrance during physiological actions, 

and are consequently more active than complexes of other 

geometries (Bolos et al., 1998). 

Antibacterial studies 

The results of the antibacterial activities of the 

compounds are presented in figure 4 and Table 5. Five 

organisms, E. cloacae, S. liquefaciens, Serratia sp, E. 

coli, S. aureus and C. violaceum were sensitive to the 

ligand with inhibitory zones range of 7-11 mm. The metal 

complexes are generally more active than the ligand 

against the organisms with the exception of [CoL2]H2O 

which had the same activity (7 mm) as the ligand against 

E. cloacae and Serratia sp. [MnL2]2H2O, [NiL2]H2O and 

[ZnL2]H2O had lower activities (7

1178 

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41 (9):1090-1092. 



Vol. 4, No. 2, pp. 1179-1185, June 2010 

ISSN: 1715-9997 

HISTOLOGICAL STUDIES OF BREWERY SPENT GRAINS IN DIETARY 

PROTEIN FORMULATION IN DONRYU RATS 

*Ajanaku, K O 1 , Dawodu, F A 2 , James, O O 1 , Ogunniran, K O 1 , Ajani, O O 1 and Nwinyi, O C 3 

1 Department of Chemistry, College of Science and Technology, Covenant University 

PMB. 1023, Ota, Ogun State 

2 Department of Chemistry, Faculty of Science, University of Ibadan, Oyo State 

3 Department of Biological Sciences, College of Science and Technology, Covenant University 

P.M.B. 1023, Ota, Ogun State, Nigeria. 

ABSTRACT 

The increasing production of large tonnage of products in brewing industries continually generates lots of solid waste 

which includes spent grains, surplus yeast, malt sprout and cullet. The disposal of spent grains is often a problem and 

poses major health and environmental challenges, thereby making it imminently necessary to explore alternatives for its 

management. This paper focuses on investigating the effects of Brewery Spent Grain formulated diet on haematological, 

biochemical, histological and growth performance of Donryu rats. The rats were allocated into six dietary treatment 

groups and fed on a short-term study with diet containing graded levels of spent grains from 0, 3, 6, 9, 12 and 100% 

weight/weight. The outcome demonstrated that formulated diet had a positive effect on the growth performance of the 

rats up to levels of 6% inclusions, while the haematological and biochemical evaluation revealed that threshold limit 

should not exceed 9% of the grain. However, the histological study on the liver indicated a limit of 3% inclusion in feed 

without serious adverse effect. Thus invariably showing that blend between ranges 1-3% is appropriate for the utilization 

of the waste in human food without adverse effect on the liver organ. The economic advantage accruing from this waste 

conversion process not only solves problem of waste disposal but also handle issues of malnutrition in feeding ration. 

Keywords: Brewery spent grains, waste utilization, donryu rats, dietary treatment. 

INTRODUCTION 

Brewery Spent Grains (BSG) is one of the voluminous 

solid residuals that remain after the mashing process. It 

has received little attention as a marketable commodity 

apart from being used primarily as ruminant feed and its 

disposal is often a problem. Its present disposal methods 

which include dumping, use as animal feed and biomass 

are no longer sustainable for the environment with 

devastating level of pollution. Nutritionally, the grain is 

far from spent since the residual protein level is in the 

range of 26-30% and crude fibre content up to 13% 

(Qzturk et al., 2002). BSG is a safe feed when it is used 

fresh or properly stored. The wet grain spoils quickly and 

should be used fresh or stored in an air tight compartment. 

However, BSG may vary with barley variety, time of 

harvest, characteristics of hops and other adjunct added as 

well as brewing technology. The waste management 

problems then require developing new ways to use the 

spent grains considering the adverse impacts on 

environment and health. 

There have been advances on the importance of fibre in 

diets as well as protein being used as supplements in food 

(Trowell et al. (1975). Other researches Bays (1977); 

*Corresponding author email: kolatooyin@yahoo.com 

Tacon and Ferms (1978); Ahn (1979); Enweremade et al. 

(2008); and Bi-Yu et al. (1998) also focused on 

alternative uses of brewery by – products and waste 

minimization from brewery processes. There was also a 

growing interest in the use of BSG in human foods such 

as flour mixes, bread and meat product (Morgan et al., 

1984; Chiou et al., 1995; Kellems and Church, 1998; 

Finley and Hanamoto, 1980). However not much has been 

carried out in the area of histopathological effect in 

human foods when used as protein supplement. In the 

light of the above findings, this study has been undertaken 

to determine the effect of using BSG as dietary feed and 

the histological effect it will have on human organs if 

utilized. 


Chemicals and Reagents 

All chemicals and reagents were of analytical grade. 

Sources of Materials 

Brewery Spent Grains was obtained from Nigerian 

Breweries Plc, Ibadan, Nigeria. The spent grain was a 

mixture of sorghum and barley. Maize, soyabean meal, 

wheat offal, fish meal, bone meal, salt, lysine, methionine 

and premix (Growers) were obtained from International 

Institute of Tropical Agriculture (IITA) Ibadan, Nigeria to

1180 

prepare the rats feed. The rats for the experiment were 

obtained from the animal farm of Cocoa Research 

Institute (CRIN) Ibadan having life average weight range 

of 49.92 ± 5.69g. The rats were in the family of Donryu 

species and were four weeks old before the 

commencement of the experiments. The ethnic use of the 

animals was also obtained from the Institute. 

Treatment of Brewery Spent Grains Sample 

The BSG samples were collected in wet form, sundried 

and later dried at 40 o C for six hours in an electric oven. It 

was then stored in an airtight container till the time of use. 

The dried BSG was milled to increase the surface area. 

The moisture content, ash content, crude fat, crude 

protein, crude fibre and the nitrogen-free extract of the 

BSG were determined. The BSG were mixed with rats 

feed at levels of 0, 3, 6, 9, 12 and 100% w/w. The 0% was 

the control, while the 100% serves as the extreme use of 

BSG alone. 

Treatment of Animals 

The thirty six (Donryu) rats were allocated into six dietary 

treatment groups of six rats each and confined into 

individual cages during the experimental period. The 

animals were free from externals and internal parasites. 

The study was conducted during the rainy season and the 

cages were built for easy collection of the faeces and 

urine. They were weighed at the beginning of the 

experiment as zero (0) day; fed according to their group 

levels with the BSG compounded feeds and subsequently 

weighed at daily intervals in a short time study of fifteen 

days. 

Preparation of Blood Samples 

On the sixteenth day of the experiment, the rats were 

humanely slaughtered using cervical dislocation method 

of Euthanasia Klaunberg et al. (2004). Their blood 

samples were collected into two heparinized tubes for the 

studies; one tube contains ethylene diaminetetracetic acid 

(EDTA) with calcium serving as anticoagulant in the 

blood samples for the haematology tests while the second 

tubes, which did not contain EDTA, were stored at -20 o C 

for the biochemical studies. The internal organs of the rats 

(liver, heart and kidney) were collected and weighed. The 

microscopic slide of the liver organs were then prepared 

and observed. 

Analysis of Heamatological and Biochemical 

Parameters 

Red blood Cell (RBC) and white blood cell (WBC) 

counts were determined using Neubauer haemocytometer. 

Packed cell volume (PVC) was determined using 

haematocrit centrifuge. Haemoglobin was determined by 

cyanmethemoglobin method (MCH), Mean Corpuscular 

Haemoglobin (MCH) and Mean Corpuscular 

Haemoglobin Concentration (MCHC) were determined 

according to the methods of Jain (1986). Glutamate 


Pyruvate Transaminose (GPT), Glutamate Oxaloacetate 

Transaminase (GOT), Globulin (GLB), Albumin (ALB) 

and Alkaline Phosphatase (ALP) were analysed 

spectrophotometrically by using commercially available 

diagnostic kits. 

STATISTICAL ANALYSIS 

The data collected were subjected to statistical analysis of 

variance and means compared by the Duncan’s Multiple 

Range Test (Steel and Torrie, 1980). 

RESULT AND DISCUSSION 

The proximate analysis of the Brewery Spent Grains 

(BSG) samples is presented in table 1. The carbohydrate 

level was high with value of 51.38% and coupled with the 

38% of nitrogen-free extract and crude protein of 23.19% 

gives a clue of a balanced formulation if the BSG is 

compounded as feed blends. The high protein values 

observed in the sample was due to the protein rest and 

washing operation of the grains during the brewing 

process. 

Table 1. Proximate Analysis of the Brewery Spent Grains 

(BSG) Samples. 

Analysis 

Crude Protein 

Crude Fibre 

Crude Fat 

Moisture Content 

Ash Content 

Carbohydrate 

Total Nitrogen 

Nitrogen – Free Extract 

Percentage Mean 

Amount (%) 

23.20 

12.85 

2.79 

6.14 

16.99 

51.39 

3.71 

38.03 

The ration formulation for the feed diet is shown in table 

2 while the effect of spent grains on the weight of the rats 

and the average weights of the rats are shown in tables 3 

and 4. The effect of BSG blend on the weight of the rats 

was commendable. The 3 and 6% BSG blends gave an 

average daily weight gain of 3.810 and 3.520g 

respectively, which was higher than the value of 3.706g 

obtained for the control (0% BSG). There was 

significance increase (p < 0.05) in the average weight 

gained by the rats fed in all the groupings except in the 

100% group. This implied a high feed-efficiency due to 

increased level of blending of the spent grains. The 

statistical analysis shows that the results fits a linear 

model of 0.532024 - 0.154247 x Weight Gain in order to 

describe the relationship between the feed-efficiency and 

weight gain. This also revealed 84.58% of the variability 

in feed-efficiency while the correlation coefficient 

indicated a strong relationship between the variables. 

Since the p-value is < 0.05, there is a statistically

significant relationship between feed-efficiency and 

weight gained at the 95.0% confidence level. It was 

observed that increasing levels of BSG in the diet resulted 

in decreased body weight with threshold limit in the range 

of 9% (Fig. 1). 

The rats in the 100% BSG group experienced daily 

weight loss of -2.080 g per day, and their metabolic 

wastes concentration was very high and toxic to inhale. 

The loss in the body weight might be due to low level of 

crude fat (2.79%) in the feed. These findings agreed with 

Ajanaku et al. 

Table 2. Ration formulation of the treatment feed diets (g/100g). 

Ingredients 

Diets 

1 2 3 4 5 6 

% BSG inclusion in diets 

0% 3% 6% 9% 12% 15% 

Maize 2.64 2.57 2.49 2.41 2.33 2.25 

BSG 0.00 0.07 0.15 0.23 0.31 0.39 

Soybean Meal 1.20 1.20 1.20 1.20 1.20 1.20 

Palm Kernel Cake / Wheat offal 1.60 1.60 1.60 1.60 1.60 1.60 

Blood meal 0.4 0.4 0.4 0.4 0.4 0.4 

Bone meal 0.04 0.04 0.04 0.04 0.04 0.04 

Salt 0.04 0.04 0.04 0.04 0.04 0.04 

Lysine 0.03 0.03 0.03 0.03 0.03 0.03 

Methionine 0.03 0.03 0.03 0.03 0.03 0.03 

Premix 1 0.02 0.02 0.02 0.02 0.02 0.02 

Total (g) 6.00 6.00 6.00 6.00 6.00 6.00 

1 

Contained vitamins A (10,000,000iu); D (2,000,000iu); E (35000iu); K (1900mg); B12 (19mg); Riboflavin (7,000mg); Pyridoxine 

(3800mg); Thiamine (2,200mg); D pantothenic acid (11,000mg); Nicotinic acid (45,000mg); Folic acid (1400mg); Biotin (113mg); 

and trace elements as Cu (8000mg); Mn (64,000mg); Zn (40,000mg); Fe (32,000mg); Se (160mg); I2 (800mg); and other items as 

Co (400mg); Choline (475,000mg); Methionine (50,000mg); BHT (5,000mg) and Spiramycin (5,000mg) per 2.5kg 

Table 3. Mean body weight of Rats for each day (Grammes). 

Days/Blends 0% 3% 6% 9% 12% 15% 100% 

0 day (g) 

1 st (g) 

2 nd (g) 

3 rd (g) 

4 th (g) 

5 th (g) 

6 th (g) 

7 th (g) 

8 th (g) 

9 th (g) 

10 th (g) 

11 th (g) 

12 th (g) 

13 th (g) 

14 th (g) 

15 th 52.55 ±2.250 55.50 ±2.700 49.55±3.350 49.15 ±4.351 44.10 ±3.900 42.29 ±2.100 48.65 ±4.110 

52.95 ±4.751 55.75 ±3.851 51.00 ±5.201 51.40 ±3.901 45.45 ±5.020 42.13 ±3.220 46.00 ±3.951 

52.35 ±3.150 56.45 ±3.551 55.85 ±4.251 54.10 ±6.001 46.35 ±5.651 43.26 ±2.510 41.80 ±3.751 

55.85 ±1.850 57.93 ±3.826 60.00 ±4.101 59.65 ±5.751 47.05 ±6.451 43.12 ±4.280 38.35 ±3.301 

58.55 ±0.6501 70.80 ±4.201 63.80 ±4.301 61.75 ±6.051 48.30 ±4.801 43.64 ±2.110 37.35 ±4.601 

63.30 ±2.20 83.15 ±5.51 68.50 ±4.301 64.25 ±5.051 50.55 ±4.551 44.32 ±3.320 34.90 ±4.351 

66.10 ±2.50 86.30 ±3.001 71.10 ±4.801 68.10 ±4.301 51.35 ±5.051 44.24 ±4.110 32.70 ±3.351 

69.95 ±3.050 90.15 ±3.751 74.25 ±4.351 70.90 ±4.701 51.40 ±5.801 45.13 ±4.350 29.60 ±3.751 

75.50 ±4.001 93.95 ±3.451 75.80 ±4.801 74.70 ±4.001 52.60 ±4.401 45.35 ±2.600 30.00 ±3.751 

80.30 ±4.101 95.70 ±3.501 83.05 ±3.451 78.45 ±3.351 52.55 ±4.751 45.68 ±3.140 26.10 ±3.651 

84.50 ±5.101 98.65 ±4.451 85.15 ±3.151 79.15 ±2.150 53.45 ±4.851 46.32 ±2.101 25.20 ±3.851 

88.50 ±5.351 99.15 ±4.451 90.30 ±2.10 81.85 ±0.850 54.50 ±4.101 47.01 ±5.210 22.95 ±3.001 

93.65 ±4.951 105.25 ±3.051 94.00 ±2.700 83.70 ±3.101 55.20 ±4.501 48.15 ±2.130 20.35 ±3.401 

99.70 ±6.201 108.65 ±3.051 95.90 ±0.90 84.25 ±3.451 55.45 ±3.651 48.98 ±3.323 17.85 ±2.701 

102.10 ±4.701 112.60 ±1.10 97.70 ±2.30 84.90 ±2.50 56.15 ±2.350 49.32 ±2.220 17.50 ±2.951 

(g) 108.15 ±4.551 112.65 ±1.750 102.40 ±2.70 85.15 ±2.550 57.70 ±2.40 49.71 ±3.170 17.45 ±2.901 

1181 

the reports of Ironkwo and Oruwari (2004) who reported 

that fat supplementation significantly improve feed 

conversion efficiently. The acceptable limit is in the range 

of 1% to 6% w/w of the blends. For obese patient, the 

range between 9-12% is a good recommendation. The 

statistical significance of the mean body weight of the rats 

(p

1182 

The result of the mean internal organs weight of the rats 

fed with the blends is presented in table 5. The mean liver 

weight for the control group was 5.41 ± 0.11 g, while 

those fed with only BSG dropped to 2.01 ± 0.685 g. The 

same was observed for the mean kidney weights, while 

others were within the range of the control 0.970 ± 0.05 g. 

Those fed in 100% BSG have their kidney weight reduced 

to 0.69 ± 0.09 g. Generally, the rats fed with 100% BSG 

have their liver, kidneys, lungs and heart weight reduced 


Table 4. Weight gain by rats for each feed formulation per day. 

Feeding Weight at 0 day (g) Weight at 15 th day (g) Weight difference (g) Weight gain/day 

0% 52.55 ±2.250 108.15 ±4.551 55.60 3.706 

3% 55.50 ±2.700 112.65 ±1.750 57.15 3.810 

6% 49.55 ±3.350 102.40 ±2.70 52.85 3.520 

9% 49.15 ±4.351 85.15 ±2.550 36.00 2.400 

12% 44.10 ±3.900 57.70 ±2.400 13.6 0.907 

15% 42.29 ±2.100 49.71 ±3.170 7.42 0.495 

100% BSG 48.65 ±4.100 17.45 ±2.901 -31.20 2.080* 

* Weight loss 

Fig. 1: Graph showing weight gain by rat against % BSG in the feed formulation. 

Table 5. Mean internal organ weight (grammes) of the rats. 

Blends 

Liver 

Internal organs 

Kidneys Lungs Heart 

weight (g) weight (g) weight (g) weight (g) 

0% 5.41 ±0.11 0.970 ±0.05 0.86 ±0.060 0.53 ±0.025 

3% 4.54 ±0.06 0.75 ±0.01 0.80 ±0.01 0.46 ±0.035 

6% 5.01 ±0.59 0.91 ±0.04 1.30 ±0.13 0.42 ±0.005 

9% 4.87 ±0.035 0.91 ±0.07 1.09 ±0.01 0.51 ±0.01 

12% 5.11 ±0.685 0.75 ±0.255 1.03 ±0.175 0.51 ±0.005 

15% 4.42 ±0.535 0.77 ±0.450 1.10 ±0.295 0.39 ±0.710 

100% BSG 2.01 ±0.685 0.69 ±0.090 0.52 ±0.030 0.25 ±0.035 

when compared with the control and other groups, 

because of the absence of enrichment that could sustain 

the blend. This implies that, if the blend is appropriately 

enriched, there could be a positive response as it was 

observed in the other groupings. 

In tables 6 and 7, the heamatological and the biochemical 

studies of the rats used for the experiment were 

respectively presented. The normal packed cell volume

(PCV) of the Donryu rat was in the range of 36 – 54 %. 

The lower end of the range is normal in juveniles, but not 

in adult rats. The rats fed with 3% and 6% BSG blends 

experienced a significant increase in haemoglobin 

concentration of 13.8 ± 0.6 and 13.8 ± 0.2 g% 

respectively. The observed value for packed cell volume 

(PCV), 39.00 ± 1.18% for 3% blend and 38.00 ± 2.17% 

for 6% blend; red blood cell counts (RBC) was 4.78 ± 

0.45 10 6 /mm 3 as against the control 3.68 ± 0.42 10 6 /mm 3 ; 

white blood cell counts (WBC) was in the range of 7.20 – 

7.10 10 3 /mm 3 in 3% to 9% respectively. 

Platelets had the highest value of 210 ± 12.32 10 6 /mm 3 in 

6% as against 155.00 ± 11.20 10 6 /mm 3 observed in the 


Table 6. Haematological studies of BSG blended feed in Rats. 

Parameter / 

Blends 

0% BSG 3% BSG 6% BSG 9% BSG 12% BSG 15% BSG 

Normal 

Value 

Hb (g%) 10.90 ± 0.2 13.8 ± 0.6 13.80 ± 0.2 7.60 ± 0.3 11.60 ± 0.4 12.8 ± 0.1 16.1 ± 0.4 

PCV (%) 31.70 ± 1.22 39.00 ± 1.18 38.00 ± 2.17 30.00 ± 1.11 24.00 ± 1.42 30.70 ± 2.23 40.6 ± 0.27 

RBC 

3.68 ± 0.42 5.52 ± 0.22 4.78 ± 0.45 3.80 ± 0.34 4.50 ± 0.23 3.85 ± 0.54 8.21 ± 0.14 

(x10 6 /mm 3 ) 

MCV (U 3 ) 95.00 ± 6.10 87.00 ± 4.20 83.00 ± 3.02 82.00 ± 5.40 85.00 ± 3.25 91.00 ± 2.50 56.2 ± 0.6 

MCH (Ug) 33.00 ± 1.22 32.00 ± 3.10 29.00 ± 3.12 27.00 ± 1.12 30.00 ± 2.72 32.00 ± 1.11 14.7 - 15.9 

MCHC (%) 33.00 ± 2.10 37.00 ± 2.11 36.00 ± 1.10 35.00 ± 3.11 34.00 ± 3.40 34.20 ± 3.25 32.4 ± 0.4 

Neutro (%) 6.00 ± 0.15 20.00 ± 0.55 2.00 ± 0.18 4.00 ± 0.10 26.00 ± 0.49 12.00 ± 1.20 10 – 55 

Lympho (%) 94.00 ± 5.35 80.00 ± 6.82 98.00 ± 6.82 96.00 ± 4.57 74.00 ± 3.45 88.00 ± 5.10 40 – 90 

Eosino (%) 0 0 0 0 0 0 0 

Mono (%) 0 0 0 0 0 0 0 

Baso (%) 0 0 0 0 0 0 0 

Platelets 

(x10 3 / mm 3 ) 

WBC 

(x10 3 /mm 3 ) 

155.00 ± 

11.20 

198.00 ± 

10.30 

210.00 ± 

12.32 

180.00 ± 

13.16 

184.00 ± 

10.10 

168.00 ± 

11.12 

54.5 ± 13.6 

5.00 ± 0.11 7.20 ± 0.14 6.60 ± 0.17 7.10 ± 0.26 5.20 ± 0.13 6.20 ± 0.10 5.3 ± 0.5 

Platelets(10 3 /mm 3 ), Neutrophil (%) Eosinophil (%); Lymphocytes (%); Monocytes (%); Basophil (%) Hb = Haemoglobin, concerntration (g%); PCV 

= Packed cell volume (%), RBC = Red Blood Cell Counts (x10 6 /mm 3 ), WBC = White Blood cell count (x10 3 /mm 3 ), MCV = Mean Corpuscular 

Volume (U 3 ) , MCH = Mean corpuscular Haemoglobin (UUg); MCHC = Mean Corpuscular Haemoglobin Concentration (%). IU/L = International 

unit per litre; 

Table 7. Biochemical studies of BSG blended feed in Rats. 

Parameter/ 0% 3% 6% 9% 12% 15% Normal 

Blends 

Value 

ALP (IU/L) 250.00 ± 302.00 ± 300.00 ± 298.00 ± 275.00 ± 213.00 ± 43.2 ± 0.38 

35.10 30.12 12.20 16.30 28.20 30.11 

GOT (IU/L) 66.00 ± 5.40 72.00 ± 4.90 71.00 ± 3.20 70.00 ± 2.50 68.00 ± 4.50 57.00 ± 3.11 7.3 ± 0.4 

GPT (IU/L) 46.00 ± 4.22 36.00 ± 3.61 44.00 ± 1.84 46.00 ± 2.61 48.00 ± 2.22 46.00 ± 2.62 NA 

AP (g/dL) 65.00 ± 4.80 68.00 ± 2.56 67.00 ± 1.68 66.00 ± 3.22 59.00 ± 3.81 62.00 ± 1.12 NA 

TP (g/dL) 7.30 ± 0.57 6.60 ± 0.77 6.90 ± 0.83 6.40 ± 0.10 6.50 ± 0.22 7.10 ± 0.45 0.65 ± 0.02 

ALB (g/dL) 3.90 ± 0.71 4.30 ± 0.90 4.10 ± 0.21 4.00 ± 1.00 3.80 ± 0.51 3.80 ± 0.60 0.43 ± 0.02 

GLB (g/dL) 3.40 ± 0.55 2.30 ± 0.11 2.80 ± 0.30 2.40 ± 0.41 2.70 ± 0.12 3.30 ± 0.45 NA 

ALB/GLB 

RATIO 

1.15 ± 0.01 1.87 ± 0.12 1.46 ± 0.11 1.67 ± 0.55 1.41 ± 0.31 1.15 ± 0.50 

ALP = Alkaline phosphatase (IU/L); g/dL = gramme per deciliter; GOT = Glutamate Oxalacetate Transaminase (IU/L); ALB = Albumin (g/dL); 

GLB = Globulin (g/dL); ALB/GLB = Albumin – Globulin ratio; GPT = Glutamate Pyrovate Transaminase (IU/L); TP = Total Protein (g/dL); AP = 

Acid Phosphatase (IU/L). 

1183 

control. Mean corpuscular haemoglobin concentration 

(MCHC) of the entire group was higher than the control 

group. Alkaline phosphatase (ALP), glutamate 

oxalacetate transminase (GOT), acid phosphatase (AP), 

and albumin (ALB) also showed significant increase as 

compared with the 0% BSG blend. The resistance of the 

body system to infection in 3% and 6% rats’ blood was 

high because there are direct actions of antibodies 

attacking the antigenic invaders, due to antibodies or anti 

infection properties that is present in the blood. Blood of 

the rats fed with 9% BSG blend had a reduced 

haemoglobin concentration, packed cell volume, but there 

was high value in WBC, lymphocytes, platelets, alkaline 

phosphates and albumin, compared with the 0% blend.

1184 

Table 8. Histopathology result of rats’ liver. 

The rats fed with 12% blend had a low value in PCV, 

MCV, MCH, lymphocytes, AP, but increased value in 

ALP, Hb, RBC and WBC in comparison with the control. 

The 15% blends had a reduced MCV, MCH, ALP and 

lymphocytes values but an increased PCV, Hb, RBC, 

MCHC, WBC and platelets values. This result revealed 

that BSG presence activates oxygen in the blood cell 

thereby making the haemoglobin in the red blood cell 

inactive, a state which is termed hypoxia, and this 

enhances increase in the production of red blood cell 

counts and haemoglobin stimulating synthesis. It was 

observed that the histopathology result varies with the 

weather, climate, and region under which the experiment 

was carried out. The temperate and tropical region may 

have slight difference from each other in their blood 

result. In spite of this, it was observed that the rats fed 

with high concentration was adversely affected, hence 

low concentration of the blend is appropriate without side 

effects. Eosinophils count, monocytes and basophils 

counts are not significantly different in the blood of the 

rats, hence no significant change. The statistical 


Formulation 

Sinusoids 

Histology 

Central Vein 

0% Normal No visible lesions 

3% Normal Mild periportal lymphocytic infiltrates 

6% Widening Epithelia lining affected 

9% Almost disappeared Epithelia lining affected 

12% Inflammatory Hepatitis 

15% Compacted Hepatitis 

Plate 1. Microscopic view of rats’ liver fed with BSG compounded feeds. 

significance of the histophatology on blood of the 

different blends at the 0.01 level (2-tailed) of 99% 

confidence interval showed significant difference, but 

they are not significantly different at 0.05 level (2-tailed) 

at 95% confidence interval, p

the sinusoids experienced a hepatitis alteration. The 

sinusoid was more compacted and there was serum 

hepatitis in the central vein in the categories of 15% 

blend. The histological study of the kidney also showed 

that the kidneys of the control, 3, 6, 9 and 12% are all 

normal, while the kidney of 100% blend had a 

nephrotoxic effect, which is fatty degradation in the 

cellular tubules and glomerular region. This could be 

attributed to high protein, though the basis was not 

understood and the phenomenon that it was due to high 

protein was not confirmed. It could be due to some other 

substances that are present in BSG. The summary of the 

histopathology result suggest that the use up to 3% 

concentration will not have adverse effect on human liver 

and that the concentration of the blend should be kept 

minimal. 

CONCLUSION 

In this study, it has been shown that blends from 1-3% 

BSG could be used as protein supplement with 3% BSG 

as the threshold limit by virtue of the histological effect 

on rats liver. Thus invariably showing that blend between 

ranges 1-3% is appropriate for the utilization of the waste 

in human food without adverse effect on the liver organ. 

These levels of incorporation will reduce the number of 

people suffering from micronutrient deficiency related 

disease in developing nations as well as propose an 

additional utilization alternative to the disposal of 

brewery spent grains worldwide. 

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by-products. MBAA Teach Quart. 14:47-49. 

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Brewer’s Grain or Bean Curd Pomance as Partial 

Replacement of Soya bean meals lactating cows. J. 

Animal Feed Science and Technology. 5:120-128. 

Chiou, PWS., Chen, KJ., Kuo, KS., Hsu, JC. and Yu, B. 

1995. Studies on the protein degradabilities of feedstuffs 

in Taiwan. Anim. Feed Sci. Technol. 55:215-226. 

Enweremade, CC., Waheed, MA., Adekunle, AA. and 

Adeola, A. 2008. The energy potential of Brewer’s Spent 

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and Applied Sciences. 3(2):175-177. 

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baking properties of dried brewer’s spent grains. Cereal 

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Ironkwo, MO. and Oruwari, BM. 2004. Influence of the 

dietary Palm oil, Groundnut oil and Corn meal on 


1185 

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2004. Euthanasia of Mouse Fetuses and Neonates. 

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the pig in the determination of apparent digestibility of 

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Fibre Content of Cookies. Journal of the Institute of 

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Received: Aug 5, 2009; Accepted: April 19, 2010


Vol. 4, No. 2, pp. 1187-1197, June 2010 

ISSN: 1715-9997 

ESTIMATING GEOMAGNETICALLY INDUCED CURRENTS 

AT SUBAURORAL AND LOW LATITUDES TO ASSESS 

THEIR EFFECTS ON POWER SYSTEMS 

Falayi EO and Beloff, N. 

Space Science Centre, University of Sussex, Falmer, East Sussex, BN1 9QJ, UK 

ABSTRACT 

During large magnetic storm the geomagnetically induced current has a negative impact on ground conducting 

technology systems. The time derivative of the horizontal component of the geomagnetic field (dH/dt) is greater than 

30nT/min for induced currents causing undesirable consequence in power grids. Multiple regression analyses were 

developed to predict the level of geomagnetic disturbance using time derivatives of the horizontal geomagnetic field, east 

and north components of the geoelectric field, auroral electrojet and disturbance storm times from 1994-2007 at low and 

subauroral latitudes. The statistical test RMSE (Root Mean Square Error) and MBE (Mean Bias Error) were employed to 

evaluate the accuracy of the geomagnetic disturbance. Different variables have been used to develop different types of 

models. Values of the correlation coefficient and the coefficient of determination were high, which indicates that the 

results are good. The equations produced the best correlations at subauroral and low latitudes, and the best correlation 

was obtained with low values of RMSE and MBE. 

Keywords: Time derivatives of the geomagnetic field, geomagnetically induced current, latitudes. 

INTRODUCTION 

Grounds based technologies especially electric power, are 

susceptible to geomagnetic storms and geomagnetically 

induced current. This arises from the changes in the 

Earth’s magnetic field, caused by high energy particle 

streams from the Sun. It creates voltage between 

grounding points in the grid, which in turn induces a 

small, irregular dc current to flow along electric power 

lines and into transformers. Both space-borne and 

ground-based technology can experience problems due to 

space weather (Pirjola et al., 2005). 

Geomagnetically induced current (GIC) are directly 

related to the horizontal time derivatives of the 

geomagnetic field strength produced by the changing of 

electrical current in the ionosphere and magnetosphere. 

These changes in the geomagnetic field in turn produce an 

electrical current that flows around the Earth’s surface. 

Disturbances in power grid systems are directly related to 

geomagnetic storms and are caused by voltages induced at 

ground level by variations in ionospheric and 

magnetosphere currents. The magnetosphere and 

ionospheric electric current induces changes in the 

magnetic field and varying voltage in the crust of the 

Earth and this in turn drives a direct current through 

transformers, called (GIC). These GIC can harm power 

equipment and even cause a collapse of the power system 

(Coles et al., 1992; Makinen, 1993; Viljanen, 1998, 2001; 

*Corresponding author email: olukayodefalayi@yahoo.com 

Kappenman, 2006). Observations also confirmed that 

geomagnetic field disturbances usually associated with 

equatorial region current intensification can be a source of 

large magnitude and long duration GIC in power grids in 

low and equatorial regions (Erinmez et al., 2002). 

Kappenman (2005) established the fact that GIC studied 

during an October 2003 storm that caused geomagnetic 

disturbance in low and equatorial areas were due to ring 

current intensification that served as a source for GIC in 

the mid latitude regions. 

The fundamental principle of the flow of GIC in ground 

based technology is well understood using the Faraday 

law of induction. Electric fields drive current in ground 

technology networks. The geomagnetic variation and the 

geoelectric field observed at the Earth’s surfaces which 

primarily depend on the magnetosphere and ionospheric 

current, determine the space weather conditions in the 

Earth’s environment. Also, surface fields are affected by 

the current and charges induced in the Earth (Viljanen, 

1997; Trichtchenko and Boteler, 2006). The geomagnetic 

field variations are associated with geoelectric field 

variation at the surface of the Earth which is influenced 

by the conductivities of different structures of the Earth’s 

interior. The induced electric field is directly related to the 

rate of change of the geomagnetic field, which implies 

that many researchers have used time derivatives of the 

geomagnetic field as a measure of GIC strength. Boteler 

et al. (2000) concluded that geomagnetic disturbances are

1188 

directly related to auroral electrojet and an assessment of 

the effects on the ground based technological systems 

requires that appropriate models of the electrojet are 

available. In the predicted levels of geomagnetically 

induced currents in power systems, it is significant that 

the electrojet model allows rapid calculations of the 

Earth-surface electric fields. 

The geomagnetic disturbance that triggered the Hydro- 

Quebec collapse during the March 13, 1989, storm 

reached an intensity of 479nT/min. Power pools serving 

the entire north-eastern of Canada also came perilously 

close to a comparable calamity, with similar cascading 

system failures, during the same geomagnetic storm; its 

intensity ranged between 300 and 600nT/min. Malmo in 

Sweden experienced GIC problems leaving 50,000 

customers with power blackouts on October 30, 2003, 

which lasted between 20 and 50 minutes. In South 

Africa, it was observed that the October and November 

2003 geomagnetic disturbance damaged transformers 

(Campbell, 2003; Wik et al., 2008; Kappenman, 2006; 

Poppe and Jordan, 2006). In Africa and South America 

the GIC effect was also observed at low and mid latitudes 

(Barkers and Skinner, 1980; Ogunade, 1986; Osella and 

Favetto 1999; Vodjannikov et al., 2007). Koen and Gaunt 

(2002) carried out dH/dt measurement simultaneously 

with GIC records in the South African power grid. 

Comparison of GIC and dH/dt has shown that when dH/dt 

>30 nT/min, the induced current has an effect on the 

power grid. 

Most of the ground technological systems located in 

higher latitude areas are prone to GIC effects. Pirjola 

(2004) reported that equatorial regions are affected by the 

equatorial electrojet current, and GIC investigation has 

been performed in Kenya, Nigeria and Argentina. 

This paper presents the significance of GIC by measuring 

variation of the horizontal time derivatives of the 

geomagnetic field, at the threshold of dH/dt > 30nT/min 

at mid and low latitude. Multi regression analysis was 

also performed between the variables used. 


METHODS 

Table 1. Lists of geographic and geomagnetic corrected coordinates. 

Abbreviation Name Geographic 

Latitude (N) 

Data analysis 

We used Dst and AE indices obtained from 

http://isgi.cetp.ipsl.fr and http://swdcwww.kugi.kyotou.ac.jp/index.html 

to define the substorms events from -90 

up to -1800nT. The geomagnetic field parameters and 

telluric electric field (Ex and Ey) are obtained from the 

Canadian magnetic observatory for subauroral zones, and 

geomagnetic fields for low latitudes were obtained from 

INTERMAGNET. Table 1 gives the geographic and 

geomagnetic corrected coordinates (IGRF model 

http://www.iugg.org/IAGA/iaga_pages/pubs/igrf.htm) for 

the 6 observatories selected for this study which are: 

Ottawa, Victoria, St John, Addis Ababa, Bangui and 

M’bour. Ottawa, Victoria and St John are in the 

subauroral zone in the American longitude sector, M’bour 

is in tropical zone and Addis Ababa and Bangui are in the 

equatorial zone of the African longitude sector. The data 

used in this study covers the disturbed periods from 1994 

-2007. We have taken a value as a threshold for the 

definition of total time of existence in the power grids of 

the selected geomagnetic observatories of the appreciable 

induced current, when dH/dt values are greater than 

30nT/min, which appears to be significant in South 

Africa. Table 1 lists geographic and geomagnetic 

corrected coordinates. 

A regression and correlation analyses were carried out 

between the time derivatives of the horizontal 

geomagnetic field (dH/dt), AE magnetic index related to 

auroral electrojets, Dst magnetic index related to the 

storm development, and North and East components of 

the geoelectric field (Ex and Ey). The regression values 

and correlation coefficients are reported in table 2 (a, b, c) 

and 2 (d, e, and f). The accuracy of the estimated values 

was tested by calculating the RMSE (Root Mean Square 

Error) and MBE (Mean Bias Error) for the variables. 

[ ( dH / dt pred dH / dt ) ] 

− 

∑ 

[ ∑( 

dH / dt − dH / dt ) ] / n 

1 

2 2 

{ / n} 

RMSE obs 

= (1) 

MBE pred 

obs 

Geographic 

Longitude (E) 

= (2) 

Geomagnetic 

Latitude (N) 

Geomagnetic 

Longitude (E) 

AAE Addis Ababa 9.0 38.8 5.28 111.79 

BNG Bangui 4.3 18.6 4.16 91.14 

MBO Mbour 14.4 343.0 20.13 57.44 

OTT Ottawa 45.4 284.5 55.63 355.38 

STJ St John 47.6 307.3 57.15 23.98 

VIC Victoria 48.5 236.6 54.12 297.6

RMSE and MBE are statistical instruments used to 

compare the models of geomagnetic distribution 

prediction. Low values of RMSE are desirable, but a few 

errors in the sum can produce a significant increase in the 

indicator. Low values of MBE are also desirable. It is also 

possible to have large RMSE values at the same time as a 

small MBE or vice versa. 

Distribution of time derivatives of the horizontal 

magnetic field (dH/dt) 

The large scale auroral ionospheric electric currents flow 

mainly in an east- west direction thus mostly affecting the 

X- Z components. Horizontal currents of small scales and 

amplitudes and field aligned currents also contribute to Y. 

Falayi and Beloff 1189 

Mid latitude 

Table 2a. Shows equation, correlation coefficient, correlation of determination, MBE and RMSE. 

Equations 

(Number) 

Equations (Ottawa) r R 2 MBE RMSE 

4 dH/dt=-3.179- 0.197Ex+0.366Ey+0.00378AE- 

0.136Dst 

0.959 0.920 1.416E-15 13.171 

5 dH/dt=3.064- 0.0017Ex +0.256Ey+0.0012AE 0.953 0.908 -3.64E-15 12.963 

6 dH/dt=4.233 +0.095Ex+0.263Ey 0.947 0.897 2.1E-5 13.759 

7 AE=92.422+ 9.74Ex+0.567Ey 0.926 0.857 -5.507E-14 397.7 

8 Dst=-51.639 -1.876Ex+0.777Ey 0.906 0.821 2.344E-13 45.278 

Table 2b. Shows equation, correlation coefficient, correlation of determination, MBE and RMSE. 

Equations 

(Number) 

Equations (Victoria) r R 2 MBE RMSE 

9 dH/dt=6.479+0.335Ex+0.302Ey+0.0021AE- 

0.096Dst 

0.966 0.933 1.200E-15 6.175 

10 dH/dt=3.80+0.4027 Ex -0.1322Ey+0.00254AE 0.958 0.917 -2.387E-15 6.866 

11 dH/dt=3.803 +0.4007Ex+0.129Ey 0.960 0.92 -2.609E-15 6.867 

12 AE=-11.15+ 8.057Ex+13.15Ey 0.909 0.825 -7.240E-14 479.3 

13 Dst=-27.59 +0.502Ex-2.089Ey 0.950 0.903 2.575E-14 33.35 

Table2 c. Shows equation, correlation coefficient, correlation of determination, MBE and RMSE. 

Equations 

(Number) 

Equations (St John) 

r R 2 MBE RMSE 

14 dH/dt=-1.66+0.1286Ex+0.1109Ey+0.00092AE- 

0.0189Dst 

0.989 0.977 -6.106E-16 4.194 

15 dH/dt=-0.863+0.1386 Ex +0.1284Ey+0.0094AE 0.988 0.970 4.441E-16 4.26 

16 dH/dt=0.95 +0.22Ex+0.208Ey 0.980 0.960 7.216E-16 5.44 

17 AE=193.45+ 8.71Ex+8.54Ey 0.950 0.900 5.684E-14 360.94 

18 Dst=-43.084-0.566Ex-0.966Ey 0.925 0.856 1.0658E-14 40.65 

Low latitudes 

Table 2d. Shows equation, correlation coefficient, correlation of determination, MBE and RMSE. 

Equations 

(Number) 

Equations (Addis Ababa) r R 2 MBE RMSE 

19 dH/dt= -1.42054 +0.0070AE -0.0286Dst 0.859 0.739 -2.498E-15 6.493 

20 dH/dt= 0.489 +0.00934AE 0.853 0.727 11.703 6.633 

The distribution of dH/dt provides a strong indication that 

the occurrence of large value time derivatives is strongly 

coupled with the occurrence of great magnetic storms. 

Figure 1 illustrates the distributions of dH/dt for 4 

intervals, in the 3 observatories Ottawa, Victoria and St 

John. It was observed that the power lines disruption 

which occurred when the geomagnetic rate of change 

exceeded 30nT/min posed a serious threat to high voltage 

power line circuits. Between 1994 and 2007, mid latitudes 

percentages of the horizontal component of time 

derivatives of the geomagnetic field (dH/dt) which were 

greater than 30nT/min were: 78.48 %( Ottawa); 64.56 %( 

Victoria); and 38.75 %( St John).

1190 

Distribution of dH/dt for Ottawa Observatory from 1994-2007 

30 

25 

20 

Number of 

15 

Occurences 

10 

5 

0 

30 

25 

20 

Number of 

15 

Occurences 

10 

5 

0 

0-30nT/min 30-60nT/min 60-90nT/min 90nT/min> 

dH/dt (nT/min) 

Distribution of dHdt for Victoria Observatory from 1994-2007 

50 

40 

Number of 

30 

Occurences 

20 

10 

0 

0-30nT/min 30-60nT/min 60-90nT/min 90nT/min> 


Distribution of dH/dt for St John from 1994-2007 

0-30nT/min 30-60nT/min 60-90nT/min >90nT/min 

dH/dt (nT) 

Fig. 1. Distributions of dH/dt at mid latitude of Ottawa, 

Victoria, and St John geomagnetic field Observatories. 


Table2 e. Shows equation, correlation coefficient, correlation of determination, MBE and RMSE. 

Equations 

(Number) 

Equations (Bangui) r R 2 MBE RMSE 

21 dH/dt= -2.051 -1.1E-06AE -0.0495Dst 0.952 0.907 3.60 1.641 

22 dH/dt= 1.1306 +0.0031AE 0.791 0.626 -4.545 3.28 

Table 2f. Shows equation, correlation coefficient, correlation of determination, MBE and RMSE. 

Equations 

(Number) 

Equations (Mbour) r R 2 MBE RMSE 

23 dH/dt= 0.6232 +0.004AE +0.0012Dst 0.917 0.841 8.327E-16 2.488 

24 dH/dt= 1.072 +0.0050AE 0.912 0.832 5.274E-16 2.555 

25 

20 

Number of 

15 

Occurences 

10 

Distribution of dH/dt for Addis Abba from 1994-2007 

5 

0 

40 

35 

30 

25 

Number of 

20 

Occurences 

15 

10 

5 

0 



Distribution of dH/dt for Bangui Observatory from 1994-2007 

50 

40 

Number of 

30 

Occurences 

20 

10 

0 



Distribution of dH/dt for Mbour from 1994-2007 



Fig. 2. Distributions of dH/dt at mid latitude of Bangui, 

Mbour and Addis Ababa geomagnetic field observatories.

Figure 2 is similar to Figure 1, for the observatories of the 

low latitudes in the African longitude sector and 

illustrates the data recorded at Bangui, M’bour and Addis 

Ababa. From figure 2, the geomagnetic disturbances are 

not significant in value at Bangui and M’bour; they more 

significant at low latitude in Addis Ababa. Between 1994 

and 2007, low latitudes percentages of the horizontal 

component of time derivatives of the geomagnetic field 

(dH/dt) which were greater than 30nT/min were: 9.6 %( 

Bangui); 24.2 %( M’bour); and 45.2 %( Addis Ababa). 

Figures 3-8, further illustrate the comparison between 

observed and predicted values of the correlation 

coefficient. 

Figure 3 illustrates the comparison between observed and 

predicted values of the correlation coefficient at Ottawa. 

For equations (4/ Table 2a), a correlation coefficient of 

0.959 exists between time derivatives of the horizontal 

geomagnetic field, north and east components of the 

geoelectric field, auroral electrojet and disturbance storm 

time. The coefficient of determination of 0.920 which 

implies 92.0% of time derivatives of the horizontal 

geomagnetic field can be accounted for using the auroral 

electrojet index, disturbance storm time, and north and 

east components of the geoelectric field. 

Ottawa 

dH/dt nT/min (obs) 

dH/dt nT/min (Obs) 

200 

150 

100 

50 

Equation 4 


r = 0.959 

R 2 = 0.920 

0 

0 50 100 

dH/dt nT/min (pred) 

150 200 

200 

150 

100 

50 

0 

Equation 5 

r = 0.953 

R 2 = 0.9081 

0 50 100 150 200 

dH/dt nT/min (Pred) 

dH/dt nT/min 

(Obs) 

AE nT 

(Obs) 

Dst nT (Obs) 

200 

150 

100 

50 

0 

6000 

4000 

2000 

500 

400 

300 

200 

100 

0 

Equation 6 

r =0.947 

R 2 = 0.8965 

0 50 100 


150 200 

Equation 7 

r =0.926 

R 2 = 0.857 

0 1000 2000 3000 4000 5000 

AE nT (Pred) 

Equation 8 

r = 0.906 

R 2 = 0.821 

0 

0 100 200 300 400 500 

Dst nT/min (Pred) 

Fig. 3. Comparison between observed and predicted 

values of the correlation coefficient at Ottawa. 

Figure 4 is devoted to the comparison between observed 

and predicted values of the correlation coefficient at 

Victoria. Equation (9/ Table 2b) shows the correlation 

coefficient of 0.966 that exists between time derivatives 

of the horizontal geomagnetic field, north and east 

components of the geoelectric field, the auroral electrojet 

and disturbance storm time. The coefficient of 

determination of 0.933 which implies 93.3% of time 

derivatives of the horizontal geomagnetic field can be 

accounted for using the auroral electrojet index, 

disturbance storm time, and north and east components of 

the geoelectric field.

1192 

Victoria 




AE nT(Obs) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

4500 

4000 

3500 

3000 

2500 

2000 

1500 

1000 

500 

0 

Equation 9 


r = 0.966 

R 2 = 0.933 

0 10 20 30 40 50 60 70 80 90 100 


Equation 10 

r = 0.958 

R 2 = 0.917 

0 10 20 30 40 50 60 70 80 90 100 

dH/dt nT/min (pred) 

Equation 11 

r = 0.958 

R 2 = 0.917 

0 10 20 30 40 50 60 70 80 90 100 


Equation 12 

r =0.909 

R 2 = 0.826 

0 500 1000 1500 2000 2500 3000 3500 4000 4500 

AE nT (Pred) 

Dst nT(Obs) 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

Equation 13 

r= 0.950 

R 2 = 0.9028 

0 50 100 150 200 

Dst nT (Pred) 

250 300 350 400 


values of the correlation coefficient at Victoria. 

Figure 5 shows the comparison between observed and 

predicted values of the correlation coefficient at St John. 

Equation (14/ Table 2c) shows a correlation coefficient of 

0.988 that exists between time derivatives of the 

horizontal geomagnetic field, north and east components 

of the geoelectric field, auroral electrojet and disturbance 

storm time. The coefficient of determination of 0.977 

which implies 97.7% of time derivatives of the horizontal 

geomagnetic field can be accounted for by using the 

auroral electrojet index, disturbance storm time, and the 

north and east components of the geoelectric field. 



120 

100 

80 

60 

40 

20 

Equation 14 

r = 0.988 

R 2 = 0.977 

0 

0 20 40 60 


80 100 120 

120 

100 

80 

60 

40 

20 

Equation 15 

r = 0.988 

R 2 = 0.9761 

0 

0 20 40 60 80 100 120 

dH/dt nT/min (Pred)


AE nT (Obs) 

Dst nT(Obs) 

120 

100 

4500 

4000 

3500 

3000 

2500 

2000 

1500 

1000 

500 

0 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

80 

60 

40 

20 

Equation 16 


r = 0.980 

R 2 = 0.961 

0 

0 20 40 60 80 100 120 


Equation 17 

r = 0.949 

R 2 = 0.901 

0 500 1000 1500 2000 2500 3000 3500 4000 4500 

AE nT (Pred) 

Equation 18 

r = 0.925 

R 2 = 0.856 

0 50 100 150 200 250 300 350 400 450 

Dst nT (Pred) 


values of the correlation coefficient at St John. 

Figure 6 illustrates the comparison between observed and 

predicted values of the correlation coefficient at Addis 

Ababa. From equation (19/ Table 2d) a correlation 

coefficient of 0.859 exists between time derivatives of the 

horizontal geomagnetic field, the auroral electrojet and 

disturbance storm time. The coefficient of determination 

of 0.739 implies 73.9 % of time derivatives of the 

horizontal magnetic field can be accounted for by using 

the auroral electrojet and disturbance storm time. 

Addis Ababa 



40 

35 

30 

25 

20 

15 

10 

5 

Equation 19 

r = 0.859 

R 2 = 0.7387 

0 

0 5 10 15 20 25 30 35 40 


40 

35 

30 

25 

20 

15 

10 

5 

Equation 20 

r = 0.853 

R 2 = 0.727 

0 

0 5 10 15 20 25 30 35 40 



values of the correlation coefficient at Addis Ababa. 

Figure 7 shows the comparison between observed and 

predicted values of the correlation coefficient at Bangui. 

Equation (21/ Table 2e) shows a correlation coefficient of 

0.952 that exists between time derivatives of the 

horizontal geomagnetic field, the auroral electrojet and 


of 0.907 implies 90.7% of time derivatives of the 

horizontal magnetic field can be accounted for by the 

using auroral electrojet and disturbance storm time. 

Bangui 

dH/dt nT/min ( Obs) 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

-2 

Equation 21 

r = 0.952 

R 2 = 0.907 

-2 0 2 4 6 8 10 12 14 16 18 

dH/dt nT/min (Pred)

1194 


18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

Equation 22 


r = 0.791 

R 2 = 0.626 

0 2 4 6 8 10 12 14 16 18 



values of the correlation coefficient at Bangui. 

Figure 8 is devoted to the comparison between observed 

and predicted values of the correlation coefficient at 

Mbour. Equation (23/ Table 2f) shows a correlation 

coefficient of 0.917 that exists between time derivatives 

of the horizontal geomagnetic field, auroral electrojet and 


of 0.841 implies 84.1% of time derivatives of the 

horizontal magnetic field can be accounted for by using 

the auroral electrojet and disturbance storm time. 

M’bour 



30 

25 

20 

15 

10 

5 

Equation 23 

r = 0.917 

R 2 = 0.8407 

0 

0 5 10 15 20 25 


30 

25 

20 

15 

10 

5 

Equation 24 

r = 0.912 

R 2 = 0.832 

0 

0 5 10 15 20 25 



values of the correlation coefficient at Mbour. 

The statistical tests, RMSE and MBE were used to 

evaluate the accuracy of the predicted time derivatives of 

the geomagnetic fields. They provide the long and short 

term performances of an equation. The smaller the values, 

the better the equation. 


During geomagnetic activity periods, the amplitude of 

auroral electrojets flowing along the auroral oval 

increases and the auroral oval can extend toward and 

usually reach subauroral zones. Sometimes during very 

strong magnetic storms the auroral oval extends toward 

middle and equatorial latitudes, occasionally becoming 

visible from tropical latitudes. Many physical processes 

are involved in auroral zones: the precipitation of particles 

which increase the electric conductivities in the E region, 

the field aligned currents which close magnetospheric 

electric currents and the dawn dusk potential drop 

imposed by the solar wind flowing around the 

magnetosphere. All these processes contribute to an 

increase in the electric currents flowing in the ionosphere 

(Kikuchi et al., 1996; Abduet et al., 1998; Kobea et al., 

1998). 

As the geomagnetic storm is intense the edge of the 

auroral boundary moves to a lower geographical latitude. 

Geomagnetic activity, even when there is not a big storm, 

affects the whole Earth from auroral to low latitudes. 

Kobea et al. (2000), shows the latitudinal profile of a 

magnetic disturbance observed on May 27, 1993 (Fig. 4 

of the paper). The amplitude of this disturbance is ~ 150- 

200nT at auroral latitudes ~ 80-100nT (half) at subauroral 

latitudes and still ~30nT at equatorial latitudes. 

Kappenman (1996, 2005) reported that the power grids 

are seriously affected by geomagnetic storms at high 

latitude due to large impulsive geomagnetic disturbance 

driven by auroral intensification, but there are signs that 

space weather poses significant risks to ground 

technology at low latitude (Addis Ababa) and subauroral 

latitudes (Ottawa and Victoria); it is reported in our 

investigation that when the rate of change of geomagnetic 

disturbance exceeded 30nT/min it posed a threat to power 

grids. Vanhamaki et al. (2005) established that changes in 

ionospheric current will give rise to induction current in 

the conducting ground which can significantly contribute 

to magnetic and electric fields. It has been reported that 

auroral electrojet, substorm onsets, geomagnetic pulsation 

and sudden impulses are responsible for large GIC 

(Boteler, 2001; Lam et al., 2002; Kappenman, 2003; 

Pulkkinen et al., 2003, 2005; Viljanen et al., 2006). 

Recently, Watari et al. (2009) confirmed that GIC 

associated with substorms were detected in Japan over a 2 

year GIC measurement period, during the solar minimum 

although intense GIC do occur mostly during 

geomagnetic storms.

Tables 2 (a-f) contain summaries of various linear 

regression analyses. It is clear that the correlation 

coefficient r, correlation of determination R 2 , MBE 

(nT/min), and RMSE (nT/min) vary from one variable to 

another. Generally, correlation coefficients (0.990 - 

0.779) are high for all the variables. This implies 

relationships between the time derivatives of the 

horizontal geomagnetic field (dH/dt), auroral electrojet 

(AE) index, disturbance storm time (Dst), and north and 

east components of the geoelectric field (Ex and Ey). This 

is further demonstrated by high values of the coefficient 

of determination R 2 (0.980 - 0.607) across the variables. 

There is a significant relationship between ionospheric 

response and ground based parameters. 

Comparing the output, we can see that all regressions give 

good results. For better analysis we considered high 

values of the correlation coefficient and coefficient of 

determination and a low value of RMSE. The following 

equations produced the best subauroral correlations: Eq. 4 

(Table 2a, Ottawa), Eq. 9, (Table 2b, Victoria) and Eq. 14 

(Table 2c, St John). At low latitude: Eq. 19 (Table 2d, 

Addis Ababa), Eq. 21 (Table 2e, Bangui), and Eq. 23 

(Table 2f, M’bour) were considered the best correlations 

with low values of RMSE and MBE. 

Our result has demonstrated that temporal variation of 

time derivatives of the horizontal geomagnetic field 

shows high correlation with the geoelectric field and 

ionospheric response (AE and Dst indices) at different 

stations in mid and low altitudes. The variation in 

correlation coefficient may be a result of geographical 

orientation of the power grids and also Earth 

conductivities. 

Pulkkinen et al. (2006) suggested that GIC magnitudes 

also depend on grid topology, configuration and 

resistances and vary greatly from site to site in a network. 

Pulkkinen et al. (2001) reported that GIC flow in the 

pipeline responds differently for the east-west and northsouth 

geoelectric fields in association with temporal 

changes of the magnetic field. Also Pirjola (2000) 

recommended that power grids should be aligned as close 

to north-south as possible rather than east–west direction. 

This is because of the auroral electrojet which is 

significant in connection with magnetic disturbance 

inducing GIC in an east-west direction. Our analysis has 

shown that geomagnetic storm effects were not so strong 

at St John (subauroral), M’bour and Bangui (low latitude), 

and the effect on consumers was small during weak and 

mild geomagnetic disturbance. 

CONCLUSION 

Kataoka and Pulkkienen (2008) reported that the 

horizontal geomagnetic field (dH/dt) is an excellent 

indicator of GIC and the relationship between the GIC 


and dH/dt is always the same in a very large dynamic 

range of about three orders of magnitude. This study 

examined the possibility of geomagnetic induced current 

(GIC) using time derivatives of the horizontal 

geomagnetic field (dH/dt) at the threshold of 30nT/min at 

mid and low latitude. When the rate of change of 

geomagnetic disturbance exceeded 30nT/min it posed a 

serious threat for power grids. Strong relationships 

between time derivatives of the horizontal magnetic field 

(dH/dt), auroral electrojet (AE) index, disturbance storm 

time (Dst), and the geoelectric field (Ex and Ey) led to the 

conclusion that auroral activity influences GIC down to 

low latitudes. 

An interesting phenomenon was also detected in the 

Addis Ababa region, which showed much higher GIC 

activity when compared to other typical low latitude 

regions (Bangui and M’bour). This may be due to current 

moving at equator called equatorial electrojet. 

Ionospheric current systems exist which can occasionally 

affect equatorial electrojet. This current is generated at 

high latitudes in the vicinity of the auroral zones as a 

result of motions in the magnetosphere (Onwumechilli 

and Ogbuechi, 1967). During magnetically disturbed 

conditions this current system may undergo considerable 

enhancement and may extend to equatorial latitudes. 

Akasofu and Chapman (1963) showed that polar 

geomagnetic storms can greatly enhance the equatorial 

electrojet current and Rastogi (1977) suggested that when 

there is fluctuation in the magnetic field during SSC at 

low latitude station are due to the imposition of electric 

field over the equatorial ionosphere leading to equatorial 

electrojet. Kappenman (2003) showed that during the 

global burst (the sudden beginning of a magnetic storm), 

the intensity of the geomagnetic field can be a reason of 

significant GIC at all geomagnetic latitudes, including the 

equatorial region. 

The results obtained in this paper are applicable to the 

estimation of geomagnetically induced currents GIC using 

time derivatives of the horizontal geomagnetic field in 

connection with research of space weather effects. 


The authors acknowledge World Data Centre (WDC) for 

geomagnetism, Space Physics Interactive Data Resources 

(SPIDR) for Dst and AE indices data, Canadian magnetic 

observatory and INTERMAGNET for geomagnetic fields. 

We also thank Christine Amory Mazaudier of CEPT for 

her valuable comments. 

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Vol. 4, No. 2, pp. 1199-1206, June 2010 

ISSN: 1715-9997 

STUDY OF INTERANNUAL AND INTRA-SEASONAL VARIABILITY OF 

SUMMER MONSOON CIRCULATION OVER INDIA 

* Bhanu Kumar, O S R U, S Ramalingeswara Rao and S S Raju 

Department of Meteorology and Oceanography, Andhra University, Visakhapatnam, India 

ABSTRACT 

In this study satellite derived Outgoing Long wave Radiation (OLR) and National Centre for Environmental Prediction 

(NCEP) reanalysis zonal wind (U-850 and U-200 hPa levels) datasets for 27-years are used to examine important and 

unique characteristics of summer monsoon circulation variability across India. Among mean 73 pentads of annual cycles 

for OLR and zonal wind components, large scale monsoon convective activity, monsoon westerlies at 850 hPa and 

strengthening of easterlies at 200 hPa levels are conspicuously represented by P-30 to P-55 pentads over India. More 

details of monsoon circulation change are inspected in spatial distribution of OLR and wind fields for the climatological 

annual cycles. The OLR field is inversely related with U-850 hPa, while it is directly related with U-200 hPa levels; both 

are statistically significant at 0.1% level in this study. Annual cycles of the OLR and wind fields (anomaly) reveal 

striking interannual monsoon circulation variability in El Nino (2002) and La Nina (1998) years. The monsoon seasonal 

circulation changes in above contrasting years are highlighted and they reveal that U-850 hPa is almost a mirror image of 

U-200 hPa distribution of wind fields over the monsoon region. Finally the strength of ISO index in terms of Monsoon 

Hadley Index (MHI) and Madden and Julian Oscillation (MJO) index are evaluated to study year-to-year monsoon 

variability across India. Above ISO indices are related with Indian summer monsoon rainfall and the relationships are 

statistically significant. 

Keywords: Convective activity, circulation changes, variability. 

INTRODUCTION 

The interannual variability of combined atmosphereocean 

system is manifest in changes of the atmospheric, 

oceanic circulations and extreme events like monsoon 

rainfall in the regional climate. Strong seasonality in 

terms of rainfall over the tropical monsoon region is 

observed due to interannual variability in the form of the 

mean annual cycle (Mooley and Parthasarathy, 1984). 

Goswami (1997) opined that modulation of the Intra- 

Seasonal Oscillations (ISO) by the annual cycle could 

give rise to an internal quasi-biennial oscillation in the 

tropical atmosphere. It is well documented that the most 

dominant feature in the tropics is the ISO, which is a 

naturally occurring component of both coupled oceanatmospheric 

phenomenon. ISOs represent an entire cycle 

of 30-50 days and fluctuations in tropical rainfall, which 

is driven by internal feedback mechanism between 

convection and dynamics (Madden and Julian, 1971). It is 

also called as Madden-Julian Oscillation (MJO) or 40-day 

wave. They have originated from western Indian Ocean, 

which propagate quite vigorously in eastward or 

northward direction and exhibits remarkable interannual 

and intra-seasonal variability of rainfall (Ramamurthy, 

1969). The ISO moves northward across India resulting in 

wet and dry spells within the Indian monsoon season, 

which is known as active and break monsoon conditions. 

It is, however, varied its activity from year-to-year over 

Southeast Asia in general and India in particular. 

*Corresponding author email: osrubhanukumar@yahoo.com 

Northward propagation of ISO using convective fields 

during Indian summer monsoon is studied by (Yasunari, 

1979 and 1980; Sikka and Gadgil, 1980) with a limited 

data. Singh and Kripalani (1990) and Singh et al. (1992) 

used long records of daily rainfall data over the Indian 

continent and examined the 30-50 day oscillation. They, 

however, could not come to a clear conclusion regarding 

relationship between the ISOs and the interannual 

variability of the Indian summer monsoon rainfall. 

Fenessey and Shukla (1994) showed that the spatial 

structures of the interannual variability and intraseasonal 

variability are quite similar, while Goswami (1994) 

proposed a conceptual model of how the ISOs influence 

the seasonal mean and interannual variability of the 

Indian monsoon. Later Annamalai et al. (1999) examined 

the relationship between intraseasonal oscillations and 

interannual variability using NCEP and European Centre 

for Medium range Weather Forecast (ECMWF) reanalysis 

and they concluded that there was not a common mode 

that described the intraseasonal and interannual 

variability. Apart from the above, several studies have 

indicated that the 30-50 day oscillation is a dominant 

phenomenon in the interannual variability of Indian 

summer monsoon rainfall (Julian and Madden, 1981; 

Krishnamurthi et al., 1985; Wang and Rui, 1990; 

Goswami and Mohan, 2001; Annamalai and Slingo, 2001; 

Kemball-Cook and Wang, 2001; Goswami et al., 2003; 

Rao and Sikka, 2007; Susmitha et al., 2008; Bhanu 

Kumar et al., 2008). Several modeling studies showed

1200 

that a significant fraction of the interannual variability of 

the seasonal mean Indian summer monsoon is governed 

by internal chaotic dynamics (Goswami, 1998; Hazarallah 

and Sadourny, 1995; Rowell et al., 1995; Stern and 

Miyakoda, 1995). Most of these studies, however, do not 

provide any detailed insight regarding the origin of the 

internally generated interannual variability. Recently a 

few studies of course made use of MJO and Monsoon 

Indices to express Asian monsoon circulation variability 

with limited data (Webster and Yang, 1992; Degtyarev et 

al., 2007; Manoel et al., 2006; Goswami et al., 1999; Li 

and Zeng, 2002). So far there are no detailed studies to 

explain variability of monsoon circulation based on 

convection, circulation changes and above said indices. 

Hence the aim of this paper is to investigate interannual 

and intra-seasonal monsoon variability over India. 


We know that marked interannual and intra-seasonal 

variations of the atmosphere-ocean system are an 

essential characteristic of tropical climate. In this study 

satellite derived daily OLR and NCEP zonal wind fields 

(2.5°x2.5°) at 850 and 200 hPa levels are downloaded 

from the NOAA website (www.cdc.noaa.gov) to study 

interannual and intra-seasonal variability of Indian 

monsoon circulation changes for the period, 1979-2005 

and the study region (50°-100°E and 0°-38°N) is shown 

in figure 1. These datasets are used to prepare mean 73 

pentads in order to examine climatological annual cycles 

in the form of histograms and spatial distribution formats 

over the study region. Anomaly annual cycles of OLR and 

wind fields are also prepared for El Nino (2002) and La 

Nina (1998) years to find out contrasting convection and 

circulation changes if any. Mean convective and 

circulation changes over study region during monsoon 

season (June-September) are also prepared for above 

contrasting years. The MJO index is obtained from the 

Climate Prediction Centre (http://www.cpc.noaa.gov) at 

70°E, 80°E and 100°E longitudes for the monsoon period, 

which is evaluated from pentad velocity potential at 200 

hPa level using extended empirical orthogonal function 

analysis across Indian longitudes for intra-seasonal 

variations. And mean MJO index variations are calculated 

for the study period and related with monsoon rainfall. 

Similarly MHI is evaluated by taking the difference 

between U-850 and U-200 hPa wind flow over the study 

region during the monsoon season and above indices are 

related with monsoon rainfall, which is downloaded from 

IITM website for the study period. The connection 

between them is promising. To find out the relationship 

between any two variables used in this study is correlation 

and regression analyses. 


Study of climatological annual cycle 

The mechanisms of interannual variability of circulation 

and climate are related to the functioning of the annual 


cycle and the annual cycle over a region has a significant 

year-to-year variations. Anomaly climatological annual 

cycle of large scale organized convection in 73 pentads 

from 27-year mean is presented in figure 2a. The most 

salient features of the mean annual pentad OLR march of 

convection shows extrema of annual OLR cycle reached 

minimum in August and maximum in February. Out of 

these 73 pentads, P-30 to P-55 pentads show large scale 

monsoon convective activity, which relates to Indian 

monsoon rainfall. Similarly figures 2b and 2c represent 

anomalous annual cycle of winds at U-850 and U-200 hPa 

levels respectively. They depict that trade winds are 

replaced by monsoon westerlies (850 hPa) and got 

strengthened from pentad P-25 to P-55, while easterlies 

(jet stream) at 200 hPa have attained maximum strength 

during the above period. Further OLR field is inversely 

related with the U-850 hPa, while it is positively related 

with U-200 hPa levels for the study period. Both are 

statistically significant. Thus OLR field has profound 

influence on wind fields to relate mean interannual 

variability of monsoon circulation. 

To further examine climatological annual cycle of 

convection and circulation features across India, the OLR 

and wind fields for typical pentads of P-1 (3 rd -7 th 

January), P-26 (8 th -12 th May), P-31 (2 nd -6 th June), P-55 

(30 th Sept.- 4 th Oct.) P-60 (4 th -8 th Nov.) and P-72 (24 th -28 th 

Dec.) are presented (Fig. 3a-c). Details of convection and 

circulation changes are discussed as follows. Pentad (P-1) 

in figure 3a shows a maximum OLR of about 280W/m 2 , 

which is centered over central India. This is the slowly 

emerging part of annual cycle of monsoon circulation. 

Later there is a dramatic change in convective activity in 

the pentad P-26 and this is the beginning of fast intraseasonal 

cycle of Indian summer monsoon, which 

coincides with the Onset of monsoon over Kerala in 

extreme south India. Above intense convective activity 

continued till the pentad P-55 due to planetary scale 

monsoon activity over study region. The value of OLR of 

P-26 to P-55 is negatively correlated with rainfall during 

June through September over India and it amounts to -0.6 

and hence OLR is a good proxy for monsoon rainfall. 

Next pentads, P-56 to P-68 represent the post-monsoon 

season with low OLR field due to seasonal disturbances 

like tropical cyclones and easterly waves etc. In 

December, the climatological annual cycle of OLR fields 

again represents 280 W/m 2 . Thus the convective activity 

is very interesting in climatological annual cycle, which 

reveals both summer and post-monsoon convection across 

India. As OLR is related with zonal wind, climatological 

annual circulation changes at U-850 and U-200 hPa levels 

are discussed. The figure 3b represents pentads of mean 

annual cycle of circulation changes at U-850 hPa. Pentad, 

P-1 of above figure shows a strong anti-cyclonic 

circulation over central India with a dominant easterly 

component over India. There is a spectacular change in 

circulation changes by migrating above anti-cyclone at 

the end of May/early June (P-26) by the appearance of 

strong southwesterlies. Somali jet is also appeared over

the Arabian Sea and maintained its strength till the end of 

September (P-31 to P-55). Later southwesterlies are 

replaced gradually by northeasterlies with a 

commencement of post-monsoon from the Siberian high 

(P-60). At the end of the mean annual cycle these 

easterlies are weakened and conditions are favourable for 

the formation of anti-cyclone over central India (P-72). 

The figure 3c shows corresponding circulation changes at 

U-200 hPa for the above pentads. Pentad P-1 of above 

figure reveals the axis of Sub-Tropical Ridge (STR) at 

10°N and westerly jet stream (28°N). Later easterly jet 

stream is fully established rather abruptly over south Bay 

of Bengal and south India by early June (P-31), while 

existing westerly jet stream disappeared. In September (P- 

55), the axis of STR shifts southwards (15°N) and week 

low level easterlies begin to appear over north Bay, which 

is a beginning of post-monsoon season. Later easterly jet 

stream has disappeared in P-60 and P-72 with the 

weakening of easterlies and the STR is shifted towards 

south. Thus above studies conclude that the climatological 

interannual variability in the tropics is dominated by 

changes in cloudiness and circulation changes caused by 

the Indian monsoon. 

Fig. 1. Study region (0°-38°N and 50°-100°E). 

Anomaly annual circulation changes over India 

In tropics extreme atmospheric circulation changes are 

generally attributed with El Nino and its counter part La 

Nina episodes through Walker circulation. So it is of 

interest to know how monsoon convective activity and its 

related circulation changes are varying during anomaly 

annual cycles using OLR and wind datasets instead of 

precipitation criteria in this study. Figure 4a depicts 

composite annual cycle of OLR for El Nino (2002) and 

La Nina (1998) years, when monsoon rainfall amounts 

received were 81% and 108% respectively over India. 

Negative OLR anomalies were highly dominant over the 

most parts of Arabian Sea, Indian sub-continent and the 

Bay of Bengal in La Nina period, while reverse 

Kumar et al. 

1201 

convection was observed in El Nino period during June 

through September in composite annual cycle (not 

shown). Thus there is a striking contrast in monsoon 

convection in El Nino and La Nina periods, but there is no 

change noticed in the pre-monsoon period in the above 

two cases. During active phase of the Indian monsoon, 

typically there is more rain over central India and a 

stronger monsoon trough. Thus the variations of Indian 

summer monsoon convection are better seen in the 

anomaly annual cycles of above contrasting years. 

Fig. 2. Anomaly annual cycles of (a) large scale organized 

cumulus convention (OLR); (b) zonal wind at 850 hPa; 

(c) Zonal wind at 200 hPa over the study region during 

1979 -2005 in 73 pentads. 

Similarly interannual monsoon variability in El Nino and 

La Nina years are also examined through a composite 

anomaly annual cycle of zonal wind at 850 and 200 hPa 

levels (Fig. 4b) and curves indicate variation of zonal 

wind in terms of direction and speed. At the time of 

Indian summer monsoon though both the low level 

westerlies and upper level easterlies are considerably 

strong in both contrasting years. However there is a 

striking difference in strength of winds at 850 and 200 

hPa levels in the above years. This figure also explains 

that the signal associated with El Nino and La Nina years 

extends backward some seasons before an anomalous 

monsoon. Thus this study explains that significant 

variations may be due to ISOs during Indian monsoon 

season. 

Above interannual monsoon variability studies have 

clearly indicated that OLR and circulation changes are 

more predominant during the Indian summer monsoon 

due to ISOs. And the variations in convective activity and 

circulation changes for the monsoon period of above El

1202 

Nino and La Nina periods are also discussed in detail 

(Figs. 5a & b). The large scale phenomenon are often 

associated with changes with atmospheric circulation that 

encompass areas for larger than a particular affected 

region. There is a distinct pattern of low level and upperlevel 

atmospheric anomalies, which accompany ISO 

related pattern of monsoon rainfall. In this study figure 5 


a b c 

Fig. 3. Mean annual cycles of (a) OLR and wind at (b) U-850 hPa (c) U-200 hPa levels in typical pentads. 

a & b shows composite anomaly OLR and wind fields at 

U-850 hPa and U-200 hPa levels relative in 1998 and 

2002 respectively for the monsoon period. The strength of 

OLR is different in 1998 and 2002 in terms of large scale 

cumulus convection. Negative OLR anomalies lie over 

most of the Indian sub-continent, central Arabian Sea and 

southeast Bay of Bengal, which extends across the

Kumar et al. 

Fig. 4a. Interannual variation of the anomalous OLR relative to El Nino and La Nina years. 

Fig. 4b. Same as above figure except for zonal wind at 850 hPa and 200 hPa levels. 

equator in 1998, while in 2002 reverse situation is shown. 

Thus there is a spectacular contrast in the enhanced and 

depressed convection during La Nina and El Nino periods 

respectively. Next the wind strength at U-850 hPa shows 

much stronger (2 m/s) in 1998, while the wind strength 

was relatively weak in 2002. Similar differences are also 

observed at the anomaly wind at U-200 hPa in terms of 

strength of easterly jet stream. Anomalous easterly wind 

speed of -1.5 m/s is observed in the region of tropical 

easterly jet in 1998, while relatively weak wind appeared 

in the year 2002. Over the monsoon region, U-850 hPa is 

almost a mirror image of U-200 hPa distribution of wind 

fields. Thus these circulation changes clearly indicate that 

stronger monsoon westerlies in the La Nina period 

1203 

influence Indian monsoon rainfall through Walker 

circulation. 

Influence of ISOs on monsoon circulation 

Above studies indicate that OLR and wind fields are very 

potential to examine interannual circulation variability 

and spectacular changes due to ISOs in monsoon season. 

The strength of ISO is determined in terms of MJO index, 

which is positively related with OLR (r = 0.54) and MJO 

index is also statistically related (r = -0.5) with monsoon 

rainfall in this study. Year-to-year variation of mean MJO 

index (June-September) clearly shows El Nino, La Nina 

and neutral periods (Fig. 6a). There are six El Nino (1979, 

1982, 1987, 1992, 1997 and 2002) and five La Nina

1204 

periods (1981, 1984, 1991, 1998 and 2005). Similarly 

monsoon Hadley index is also evaluated to define the 

strength of ISO, which also represents the same El Nino 

and La Nina periods (Fig. 6b). The relationship between 

MHI and monsoon rainfall is 0.6. In the year 2002, MJO 

and MHI are 0.31 and -1.2 respectively, while in 1998 

they are -0.91 and 0.26. Thus year-to-year variability of 

Indian summer monsoon activity (rainfall) can be 

identified using above indices. 


Fig. 5. a & b. Composite anomaly OLR and zonal wind fields for (a) La Nina (1998) and (b) El Nino (2002) 

monsoon years (shaded regions represent negative anomalies; percentage denote all India rainfall). 

CONCLUSIONS 

The analysis of datasets of satellite derived OLR and 

NCEP winds over the study region for 27-years reveals 

some interesting facts of variability of Indian monsoon. 

The mean annual cycle of OLR in the tropics is 

dominated by changes in cloudiness caused by the Indian 

monsoon. Out of 73 pentads, P-30 to P-55 pentads show 

marked large scale monsoon convective activity. Mean

annual cycle of wind field supported above monsoon 

variability. The Interannual variability of ISO is partly 

linked to ENSO cycle. Strong ISO activity is often 

observed with La Nina. 

Indices of MJO and monsoon indicate year-to-year 

variation of summer monsoon rainfall. 


The authors are thankful to Ministry of Earth System 

Sciences, Government of India, New Delhi for financial 

support through a research project (11/MRDF/1/41/P/08). 

The authors are also thankful to NCEP/NCAR and NOAA 

team for providing data. 

Kumar et al. 

Fig. 6a. Inter annual variation of MJO Index during 1979-2005. 

Fig. 6b. Inter annual variation of monsoon Hadley index during 1979-2005. 

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Received: Aug 5, 2009; Revised: April 14, 2010; Accepted: 

May 26, 2010


Vol. 4, No. 2, pp. 1207-1216, June 2010 

ISSN: 1715-9997 

ENZYMATIC SURFACE HYDROLYSIS OF 

POLY(ETHYLENETEREPHTHALATE) BY LIPASE ENZYME 

AND ITS PRODUCTION 

* 1 2 1 

Magda A El-Bendary , Samiha M Abo El-Ola and Maysa E. Moharam 

1 

Microbial Chemistry Department, National Research Center, Dokki, Giza 

2 

Proteinic and Synthetic Fiber Department National Research Center, Dokki, Giza, Egypt 

ABSTRACT 

In this study, hydrolysis of the surface of poly(ethyleneterephthalate) (PET) fabric by Bacillus isolate 5W and 6C lipases 

was confirmed by improved hydrophilic property which tested by wetability and staining with basic dyes, scanning 

electron microscope, and FTIR measurements. Lipase treated PET fabrics showed good physical and mechanical 

properties. It was observed that the retained strength of lipase treated PET was 96-98% and pilling and static charges 

were reduced. Bacillus isolate 5W was chosen for economic production of PET surface modifying lipase enzyme in 14 

agro-industrial by-products. The most suitable substrate for the highest lipase production was cotton seed meal at 6% and 

initial pH 7.5 (90U/ml). Effect of volume of the medium in the flask, inoculums size and incubation period were 

investigated and 25 ml of the medium, 25 x 10 6 CFU/ml, and 24h, respectively were the optimal. Addition of peptone to 

cotton seed meal medium enhanced the lipase production 22.5% (130.7 U/ml). The results of this article focuses on 

production of lipase enzyme that hydrolyze PET under feasible and economic conditions for replacing harsh chemical 

processes currently used for hydrophilization. 

Keywords: Lipase production, Poly(ethyleneterephthalate), surface modification, Bacillus, agro-industrial by-products. 

INTRODUCTION 

In the last few years there has been increasing interest in 

enzymatic surface modification of poly(ethyleneterephthalate) 

(PET), a synthetic polymer which widely used 

in the textile industry with an annual production of 36 

million tons (Alisch-Mark et al., 2004; Fischer-Colbrie et 

al., 2004; Vertommen et al., 2005; Alisch-Mark et al., 

2006; Heumann et al., 2006; Kim and Song, 2006; Silva 

et al., 2007; Guebitz and Cavaco-Paulo, 2008). PET 

produced from purified terephthalic acid or alternatively 

dimethyl terephthalate and ethylene glycol. PET has 

become very successful within the fashion industry due to 

its chemical resistance, wrinkle resistance and quickdrying 

properties. However it has a moisture regain of 

0.4% (measured at 20°C and 65% relative humidity), this 

means that PET is extremely hydrophobic. Therefore, the 

use of PET in textile applications such as sportswear, 

underwear and bedding is restricted due to a relatively 

low level of comfort, as moisture is not absorbed nor 

drawn away from the skin. Additionally, PET exhibits 

static problems such as cling during wear and difficulty in 

cutting and sewing. 

Consequently, increasing its hydrophilicity is essential for 

many applications ranging from textiles to medical and 

electronics (Guebitz and Cavaco-Paulo, 2008). Alkaline 

treatment is used to increase hydrophilicity of PET based 

textile materials (Shalaby et al., 2007). However, it leads 

*Corresponding author email: tasnim41@.yahoo.com 

to the deterioration of the fiber properties such as 

formation of pit-like structure as a result of high weight 

loss and reduction of fiber strength (Shalaby et al., 2007). 

Recent studies clearly indicate that the modification of 

synthetic and natural polymers with enzymes is an 

environmentally friendly alternative to chemical methods 

using harsh conditions. Potential of microbial enzymes for 

the targeted surface functionalization of synthetic fibers 

has recently been assessed (Fischer-Colbrie et al., 2003). 

The major advantages of enzymes in polymer 

modification compared to the chemical methods are 

milder reaction conditions and easier control, 

environmental friendlier process, and specific nondestructive 

transformations on polymer surfaces (Matama 

et al., 2006). 

Enzymes like lipases (EC. 3.1.1) and cutinase (EC. 

3.1.1.74) may potentially hydrolyze the surface ester 

bonds of PET resulting in superficial formation of 

hydroxyl and carboxyl groups (Killis et al., 2001; Gubitz 

and Cavaco-Paulo, 2003; Silva and Cavaco-Paulo, 2004; 

Hasan et al., 2006). Due to the size of enzymes and the 

insoluble nature of PET fiber in an aqueous medium, the 

enzymes are merely active at the surface so that the bulk 

characteristics of fibers remain unchanged. 

Recently attempts to isolate microorganisms that produce 

lipase enzyme which used for surface modification of 

textile gain a special interest.

1208 

The purpose of the present study is selection of a novel 

Bacillus isolate that is capable of producing lipase 

enzyme that has the ability to modify PET surface without 

losing its strength. In addition, production of this enzyme 

by using industrial by-products as cost effective method 

for feasible application in textile industry. The results of 

this work support the research for potential utilization of 

lipase as a hydrolytic source for modification of PET. 


MATERIALS 

p-nitrophenyl-acetate (P-NPA) was from Sigma, sodium 

dihydrogen phosphate, disodium hydrogen phosphate, 

non-ionic detergent Hostpal CVL-EL from Clarient, 

glacial acetic acid purchased from El-Naser 

Pharmaceutical Chemical Company. 

Basic dyes (C.I Basic Red 18 and C.I Basic Yellow 28) 

kindly supplied by European Colour Plc, Stockport, 

England, however, Methylene Blue was from Merck. All 

other used chemicals were of analytical grade. 

Polyester 100% fabric was provided by Hosam Textile 

Company, Bourge El-Arab, Alexandria, Egypt. 

Specification of woven fabric is 22 ends/inch, 20 

picks/inch and 177g/m 2 fabric weight. 

Agro-industrial byproducts were from Oil Extraction Unit 

and Animal Nutrition Department at National Research 

Center. These Agro-industrial byproducts were sugar beet 

pulp, potato peels, orange peels, pea peels, beans peels, 

banana peels, carrots pomace, jojoba meal, cress meal 

coconut meal, linen meal, sesame meal, wheat germ meal, 

and cotton seed meal. 

METHODS 

Screening of lypolytic bacilli 

Ninety one Bacillus isolates were screened for lipase 

production by plating on tributyrin agar medium. These 

isolates were isolated from underground sand of 

underground spring, sands carried by winds during spring 

season in Cairo, sands and plant roots from the Egyptian 

desert, and sugar cane roots from upper Egypt. The 

bacterial isolates producing maximum clear zone were 

selected, subcultured, and stored on nutrient yeast agar 

slants at 4°C. 

Media used 

Nutrient yeast broth (NYB) contains per liter, 5g peptone, 

3g beef extract and 5g yeast extract. For solidification, 

20g agar is added. 

Tributyrin agar medium (TBA) contains per liter, 5g 

peptone, 3g yeast extract, 10ml tributyrin, and 20g agar. 

Agro-industrial by-products media contained 60g of agroindustrial 

wastes in 1 liter tap water. 


Growth conditions and Preparation of cell free 

extracts 

The isolates were grown in 25ml NYB medium in 

Erlenmeyer conical flask (250ml) for 3 days at 30°C and 

150rpm on an orbital shaker. The cultures were harvested 

by centrifugation at 10000 x g for 10min. The supernatant 

was examined for lipase activity. 

Lipase activity assay 

Lipase activity was assayed according to Meghwanshi et 

al. (2006) and Chen et al. (2007) with some 

modifications. Lipase activity was determined using pnitrophenyl 

acetate (p-NPA) as substrate. The substrate 

solution was prepared by dissolving p-NPA in 

isopropanol. The mixture of 880µl of 50mM phosphate 

buffer (pH 8), 20µl of substrate solution and 100µl of 

suitably diluted enzyme solution was incubated at the 

tested temperature for 30min. The reaction was 

terminated by the addition of 0.2ml of 100mM CaCl2 

solution (at 0°C) and keeping it on ice. The reaction 

mixture was centrifuged to clarify the solution and the 

absorbance of the yellow color of the supernatant was 

read at 410nm. 

One unit of enzyme activity was defined as a 0.1 increase 

in OD410 under the standard assay conditions. 

Enzymatic treatments of textile 

Enzymatic treatments of textile were according to (O 

'Neil, 2007). All fabric samples were washed to remove 

the impurities. The fabric was washed with 10g/l hostapal 

at 65°C for 1 h, followed by rinsing several times with tap 

water then the fabric was washed in aqueous solution 

contain 2g/l sodium carbonate for 1h at 65 °C, followed 

by rinsing several times with tap water. Finally, the 

fabrics gently squeezed and air dried. Two sets of 

experiments were performed. One gram of the fabric was 

incubated in a glass vessel containing a solution of 0.05 

M sodium phosphate buffer (pH 8) using the same 

amount of crude enzyme (4%), the liquor ratio was 50:1. 

One set of experiment was incubated at 50°C for 8 h and 

the other set was incubated at 37°C for 24h on an orbital 

shaker at 150rpm. After enzymatic treatments, all samples 

were washed several times with tap water then with 2g/l 

sodium carbonate for 1h at 50°C followed by washing 

with distilled water for 1h at 50°C. Finally, the samples 

were washed with running tap water for 5 min and 

allowed to dry in open air. 

Staining the fabrics 

The staining was carried out below the glass temperature 

(Tg) of PET (at 50°C for 90 min) as described by O 'Neil 

(2007). After enzymatic treatments, the fabric samples 

were stained together in the same sealed glass vessel 

(500ml) on shaking water bath at proper pH. The dyeing 

was performed with 0.5 % shade owf, liquor ratio 50:1 

and at 200rpm agitation. After dyeing process, samples

were washed with aqueous solution containing 2g/l non 

ionic detergent at 55°C for 1h then, washed with tap water 

and dried in oven at 50°C for 6h. 

Wetability test 

A water drop test was applied to lipase treated fabric and 

control samples according to AATCC standard method. 

Colour Measurement 

The K⁄S percentage increments after the enzymatic 

treatments and staining of the fabric samples were 

measured in order to detect differences in reactive groups 

formed on the surface according to Matama et al. (2006) 

and Silva et al. (2007). The colourimetric data K⁄S of the 

dyed samples was collected using Mini Scan XE 

integrated with Hunter lab universal software at 

maximum absorption, as an average of three readings. 

The relative K⁄S of enzymatic treated samples was 

calculated as follow: 

% Relative K/S = {(K/ S treated -K/S control)/ (K/ S) control}*100 

Scanning electron microscope (SEM) 

Changes of the fiber surface morphology due to 

enzymatic treatment were followed by SEM model JEOL 

JXA-84OH Electron Prope Microanalyzer, Japan 

operating at 19 KV. A thin coating film (~10 nm) of gold 

was deposited onto the samples, before examination by 

SEM. 

FTIR measurements 

To investigate the effect of enzymatic treatment on 

structure of the PET fabrics surface, the fabrics were 

analyzed by FTIR spectroscopy using Nicolet 380 

spectrophotometer in the spectral range 4000-650 cm -1 , 

with a resolution of 4 cm -1 and a number of scans of 128. 

Fabrics were measured by "Smart Performer ATR" unit 

accessory with zinc selenide crystal. 

Transfer printing 

Transfer printing was done by using two disperse dyes, 

C.I Disperse blue 56 and C.I Disperse Red 60. 

Weight loss 

Weight loss, was evaluated according to the following 

equation 

Weight loss % = (W1-W2)/W1* 100 

Where W1, W2 are the weight before and weight after 

enzymatic hydrolysis, respectively. 

Oily Stain resistance 

Oily stain release was measured according to AATCC 

Test method 130-1992. 

El-Bendary et al. 

1209 

Pilling resistance 

Pilling resistance of PET fabrics was measured according 

to ASTM 4966/4970 (2002) using Martindale Abrasion 

and Piling Testers (M235 Martindale SDL ATLAB, 

England). 

Air permeability 

Air permeability was measured according to ASTM D 

737 (96). 

Static charge 

Static charge was measured using electricity collect type 

Potentiometer Type KS- 525 KASUGA, DENKJ, Inc., 

Tokyo, Japan. 

Tensile strength 

Tensile strength of PET fabrics was measured according 

to ASTM D3822-01 by INSTRON, USA. Tensile strength 

results are the arithmetic means of three tests per sample. 

Morphological and biochemical identification of 

isolated Bacillus 

Bacillus isolate which produce high lipase enzyme, and 

showed good PET surface modification was identified 

using standard morphological and biochemical procedures 

according to Gordan (1974), Claus and Berkeley (1986) 

and Stukus (1997). 

Optimization of the medium conditions for lipase 

production 

1. A group of locally available agro-industrial byproducts 

were tested as possible media for lipase 

production. 

2. Effect of the best agro-industrial byproduct 

concentrations on lipase production was studied. 

3. Effect of initial pH of the medium was studied by 

buffering the medium with 0.05M phosphate buffer 

at pH values ranging between 5.5 and 9.0. 

4. Effect of aeration level was tested by varying the 

volume of the medium in the experimental flasks 

between 6-100ml. 

5. Effect of inoculum size was studied by varying the 

inoculum size between 5 x10 6 and 25x10 7 CFU/ml. 

6. Effect of incubation period was investigated by 

incubation for varying periods up to five days. 

7. Effect of supplementation of the medium with 

additional nutrient sources at 2% final concentration 

was studied. 


Screening of Bacillus isolates for lipase production. 

Thirty one of the tested Bacillus isolates showed high 

lypolytic activity as clear zone around their colonies on 

TBA medium.

1210 

Quantitative screening for lipase activity by P-NPA 

showed that 19 Bacillus isolates (2U, 1W, 2W, 5W, 6C, 

8C, 12C, 14C, 11P, 16P, 18P, 19P, 20P, 21P, 22P, 36P, 

38P, 41P and 45P) were the most lypolytic isolates at 

37°C. Most of them showed promising activities at 50°C 

(data not shown). 

The literature reported that several Bacillus species 

isolated from several diverse environments produced 

lipase enzyme (Sharma et al., 2001; Kumar et al., 2005; 

Hasan et al., 2006; Deive et al., 2007). 

Lipase enzymes produced by the above 19 Bacillus 

isolates were examined for hydrolysis of PET fabrics. 

Wetability of lipase treated PET fabrics 

The results of wetability of PET fabrics treated with 

lipase of Bacillus isolates 2U, 5W, 6C, 16P and 22P at 

37°C for 24h were more effective than at 50°C for 8h as 

proved by the decrement of the time for water drop 

absorption as shown in table 1. This revealed that they are 

promising lipase producers for improving surface 

wetability of PET fabrics. These results indicated that the 

amount of hydrophilic groups (OH and COOH) at the 

surface of PET increased as a result of lipase treatment. 

Hsieh and Cram (1998) reported that, the surface 

wetability of enzyme treated substrates is associated with 

hydrolytic action rate. 

Cationic Dye Binding 

Significant increase in K⁄S values were observed for 


Table 1. Wetability of PET fabrics treated with lipase produced by Bacillus isolates 

stained lipase treated PET fabrics. The results supported 

that the enzymatic treatment increased adhesion of 

cationic compounds probably by creating carboxyl groups 

on the surface of the treated fabrics. The staining degree 

of lipase treated PET fabrics varies according to the basic 

dye used which may be attributed to the size of dye 

molecule and the pore size created on the fabric surface as 

a result of enzymatic hydrolysis. PET fabrics treated with 

lipase enzyme produced by Bacillus isolates 5W and 6C 

at 37°C for 24 h showed promising increase in K/S 

percentage after staining by Methylene Blue, C.I. Basic 

Yellow 28 and C.I. Basic Red 18 as shown in table 2. 

Transfer Printing 

The impact of lipase treatment on transfer printing of PET 

using two different disperse dyes, C.I Disperse Blue 56 

and C.I Disperse Red 60 was shown in table 3. The results 

revealed that a significant improvement of color strength 

of printed modified PET fabrics. Lipase enzyme produced 

by Bacillus isolate 5W enhanced the relative color 

strength to large extent. 

Physical and Mechanical properties 

Table 4 illustrates the effect of lipase treatment on some 

physical and mechanical properties of PET fabrics. Based 

on these data, it was observed that breaking strength of 

PET decreased with treatment of the fabrics with lipase 

enzymes produced by Bacillus isolates 5W and 6C to a 

limited extent and 96-98% of the strength of the fabric 

was retained. The decrease in breaking strength may be 

due to the formation of pits on the surface which probably 

Bacillus isolate 

Lipase activity (U/ml) at 

37°C 50°C 

Wetting time (min) of lipase 

treated PET at 37 °C for 24 h 

Wetting time (min) of lipase 

treated PET at 50°C for 8 h 

2U 39.6 29.4 0.22 1.6 

5W 39.8 32.4 0.23 1.2 

6C 19.2 21.0 0.28 3.8 

16P 30.7 21.2 0.43 5.37 

22P 30.2 20.9 0.33 2.37 

Control (buffer) - - 6.3 3.3 

Table 2. Relative K/S of dyed PET fabric treated with lipase enzyme produced by Bacillus isolates. 

Lipase 

produced by 

Bacillus 

% Relative K/S of PET treated with lipase enzyme at 37 °C for 24h or 50°C for 8h and stained by 

Methylene blue 

λmax 650 pH 9.5 

C.I. Basic yellow 28 

λmax 460 pH 4.5 

C.I. Basic Red 18 

λmax 520 pH 4.5 

isolate 37°C for 24 h 50°C for 8 h 37°C for 24 h 50°C for 8 h 37°C for 24 h 50°C for 8 h 

2U -13.6 5.7 18.4 3.16 6.3 -2.1 

5W 28 9.49 18.4 17.6 84 -27.3 

6C 18.4 5.13 65.9 18.35 56.3 32.87 

16P 12.4 3.8 22.09 -24.05 12.8 2.8 

22P -3.23 5.13 1.58 7.6 27.3 5.87 

-ve value means that K/S of control is more than K/S of treated samples.

act as weak points when the fabric is elongated under 

stress. In addition, the amount of static charge decreased 

as a result of lipase treatment of PET. This may be 

attributed to the creation of hydroxyl and carboxyl groups 

consequently enhanced the moisture regain and the 

amount of static charge decreased. Improvement of pilling 

resistance and dissipation of electrostatic charge soil 

release resistance were noticed. Lipase pretreatment of 

PET also reduced the susceptibility of the fabric to stain 

by corn oil in comparison to the control. Presumably 

enzymatic hydrolysis of PET and formation of hydroxyl 

and carboxyl groups decrease hydrophobicity and 

attraction for oily soil thereby enabling the easier removal 

of the corn oil stain. 

Scanning Electron Microscope 

The physical modification of lipase treated PET fabrics 

was examined by SEM. As shown in figure 1, a 

significant modification of the fiber surface was observed. 


Table 3. Effect of Lipase treatment of PET fabric on transfer printing. 

1211 

The surface of PET fabrics treated with buffer was 

smooth however, the enzymatic treated fabrics gained 

heterogeneous appearance such as cracks, pits, pores, and 

rough surfaces. 

FTIR measurements 

Infrared spectra for PET fabric samples are shown in 

figure 2. Different spectra were obtained for control 

(denaturated enzyme) and lipase treated samples. There is 

intensity differences in some bands between lipase treated 

and untreated fabrics. The selected region used for 

comparison is from 1350-1150 cm -1 , where the peak of C- 

O is located at 1241cm -1 . The spectra reveal an increase 

in the absorbance of C-O vibration for the lipase treated 

samples as compared to the control. This may be 

attributed to the formation of carboxyl groups (COOH) 

due to lipase hydrolysis of the ester linkages in PET 

producing polar carboxyl and hydroxyl groups. 

Lipase produced by 

Relative K/S % Increase in K/S 

Bacillus isolate C.I Disperse Blue 56 C.I Disperse Red 60 C.I Disperse Blue 56 C.I Disperse Red 60 

5W 0.82 4.331 148.5 305.5 

6C 0.4 1.1242 21.2 5.24 

Control (buffer) 0.33 1.068 - - 

Table 4. Effect of lipase treatment of PET fabric on physical and mechanical properties. 

Lipase 

produced 

by Bacillus 

isolate 

Weight loss 

(%) 

Breaking 

strength 

(Kg) 

Retained 

strength 

(%) 

Elongation 

(%) 

Static 

charge 

(KV) 

Pilling 

resistance 

Soil release 

5W 0.35 108 95.57 24.1 6.6 3-4 3 

6C 0.3 111 98.2 26.1 7.66 3-4 2-3 

Control 

(buffer) 

0.31 113 100 23.3 8 2 1 

A 

B 

C 

Fig.1. SEM of PET fabric treated with buffer solution (A) and lipases of Bacillus isolate 5W and 6C (B and C, 

respectively).

1212 

A bsorbance 

0.70 

0.65 

0.60 

0.55 

0.50 

0.45 

0.40 

0.35 

0.30 

0.25 

0.20 

0.15 

0.10 

0.05 

1350 

1300 

1250 

Wavenumbers (cm-1) 

Fig. 2. FTIR spectra of PET fabric treated with (from 

bottom to top) denaturated enzyme of Bacillus isolate 

5W, denaturated enzyme of Bacillus isolate 6C, lipase 

enzyme of Bacillus isolate 6C and lipase enzyme of 

Bacillus isolate 5W showing increase in the absorbance of 

the C-O vibrations (1241 cm -1 ) as a result of lipase 

treatment. 

Production of lipase by Bacillus isolate 5W on agroindustrial 

by-products. 

Bacillus isolate 5W produced higher lipase activity and 

PET fabrics treated with this lipase showed good 

wetability, staining, physical and mechanical properties. 

These results supported by SEM and FTIR studies, which 

proved the modification of PET surfaces. Therefore, this 

isolate was selected for further studies. It was identified 

according to the morphological and biochemical tests as 

Bacillus subtilis. 

Fourteen agro-industrial residues were studied for 

production of lipase by Bacillus isolate 5W without 

pretreatment under submerged fermentation conditions. 

As shown in table 5 nine substrates enhanced the lipase 

production by the tested isolate as or more than NYB 

medium. The most efficient media were cotton seed meal, 

banana peels, wheat germ meal, and cress meal. The 

maximum production (52.8U/ml) was obtained with 

cotton seed meal (CSM) medium. Concentrations of CSM 

was tested and maximum lipase yield was at 6%. 

Haba et al. (2000) used frying oils wastes for production 

of lipase by 47 strains of bacteria and yeasts. They 

reported that the genera Pseudomonas, Bacillus, Candida, 

Rhodococcus and Staphylococcus grew on the waste oil 

and produced high lypolytic activity. Rohit et al. (2001) 

and Mohan et al. (2008) reported that high levels of lipase 

activity were obtained by Bacillus strains grown in olive 

oil as a medium. Ertugrul et al. (2007) found that 20% 

whey with 1% triolein gave the highest lipase activity by 

Bacillus sp. Souissi et al. (2009) produced lipase by 

Staphylococcus simulans grown on sardinella 

hydrolyzates and peptone. Lin et al. (1996) reported an 

extracellular alkaline lipase produced by Pseudomonas 

alcaligenes F-111 in a medium that contained soybean 


1200 

meal, peptone and yeast extract. Kumar et al. (2005) 

produced lipase by Bacillus coagulans BTS-3 with 

refined mustard oil, peptone and yeast extract. 

Table 5. Production of lipase by Bacillus isolate 5W on 

agro-industrial by-products. 

Industrial byproducts 

Final pH 

Lipase activity 

(U/ml) 

Sugar beet pulp 6.16 5.25 

Potato peels 7.25 24.6 

Orange peels 5.74 0.0 

Pea peels 7.47 36.0 

Bean peels 6.25 30.8 

Banana peels 6.65 44.6 

Carrot pomace 7.65 33.1 

Jojoba meal 7.32 17.8 

Cress meal 8.4 40.1 

Coconut meal 5.94 5.25 

Linen meal 8.32 32.4 

Sesame meal 7.25 31.4 

Wheat germ meal 8.16 43.1 

Cotton seed meal 8.41 52.8 

NYB 8.18 34.5 

Effect of initial pH of the medium 

Effect of initial pH of the medium on lipase production by 

Bacillus isolate 5W is shown in figure 3. There is an 

increase in the lipase production with increasing the pH 

between 5.5 and 7.5. Increasing the pH of the medium 

more than 7.5 has destructive effect on the lipase 

production by the tested organism. These results are in 

consistence with that reported by Achamma et al. (2003) 

and Deive et al. (2007). They found that lipase production 

by Bacillus sp. and Pseudomonas aeruginosa MB was the 

maximum at neutral pH. However Ertugrul et al. (2007) 

reported that the optimal pH for lipase production by 

Bacillus sp was 6. Although bacteria prefer pH around 7 

for best lipase production, maximum activity at higher pH 

values were also reported in the literature (Kumar et al., 

2005). 

Effect of aeration level on lipase production 

The enzyme levels formed at different aeration levels 

under shaking conditions are shown in figure 4. The 

maximum lipase production (91.2U/ml) was at 25ml 

medium in 250ml flask (10% of the flask volume). 

Effect of inoculum size 

Lipase activity of Bacillus isolate 5W reached its 

maximum value (109.3 U/ml) upon using approximately 

25x10 6 CFU/ml as shown in figure 5. However, further 

increase in inoculum size lead to a dramatic decrease in 

lipase production.


Fig. 3. Effect of initial pH of the medium on lipase production by Bacillus isolate 5W. 

Fig. 4. Effect of aeration level on lipase production by Bacillus isolate 5W. 

Effect of incubation period 

Lipase production by Bacillus isolate 5W grown for 

extended incubation periods up to 96h. The highest lipase 

production was obtained after 24h of incubation and no 

significant variations could be observed in activity with 

1213 

the increment of the incubation period up to 96h. Kumar 

et al. (2005) and Ertugrul et al. (2007) reported that the 

time course for the maximum intracellular lipase 

production by Bacillus was 48 and 63h, respectively.

1214 

Effect of supplementation of CSM with additional 

nutritional sources 

Selected nutritional sources were incorporated as 

supplements to CSM medium for further enrichment of 

the growth medium. As shown in figure 6. Peptone and 

corn steep liquor at 2% enhanced the lipase production 


Fig. 5. Effect of inoculum size on lipase production by Bacillus isolate 5W. 

Fig. 6. Effect of additives on the lipase production by Bacillus isolate 5W. 

about 22.5% (130.7 U/ml) and 9% (121.1U/ml), 

respectively. Lin et al. (1996); Kumar et al. (2005) and 

Souissi et al. (2009) added peptone to their media for 

production of lipase by Pseudomonas alcaligenes F-111, 

Bacillus coagulans BTS-3 and Staphylococcus simulans, 

respectively. Corn steep liquor was an optimal nitrogen

source for intracellular and extracellular lipase production 

by the fungus Rhizopium oryzae as reported by Essamri et 

al. (1998) and Hiol et al. (2000), respectively. 

Effect of metal ions 

A group of metal ions (Ca 2+ , Mg 2+ , Na + , Co 2+ , Cu 2+ , Fe 2+ , 

K + , Mn 2+ , Mo 2+ , and Zn 2+ ) were added to the medium 

separately as lipase enhancer but they did not enhance the 

lipase production with exception of Zn 2+ which enhanced 

its production by 8%. 

REFERENCES 

Achamma, T., Manoj, MK., Valsa, A., Mohan, S. and 

Manjula, R. 2003. Optimization of growth condition for 

the production of extracellular lipase by Bacillus 

mycoides. Indian Journal of Microbiology. 43:67-69. 

Alisch-Mark, M., Feuerhack, A., Muller, H., Mensak, B., 

Andreaus, J. and Zimmermann, W. 2004. Biocatalytic 

modification of polyethylene terephthalate fibers by 

esterases from actinomycete isolates. Biocatalysis and 

Biotransformation. 22:347-351. 

Alisch-Mark, M., Herrmann, A. and Zimmermann,W. 

2006. Increase of the hydrophilicity of polyethylene 

terephthalate fibers by hydrolases from 

Thermomonospora fusca and Fusarium solani f sp pisi. 

Biotechnology Letter. 28: 681-685. 

Chen, A., Qian, L. and Shi, B. 2007. Purification and 

properties of enantioselective lipase from a newly isolated 

Bacillus cereus C71. Process Biochemistry. 42: 988-994. 

Claus, D. and Berkeley, RCW. 1986. Genus Bacillus 

Cohn 1872. In Bergey's Manual of Systematic 

Bacteriology (vol. 2 nd ) Ed. Sneath, PHA. Baltimore, 

Williams and Wilkins. Pp. 1105-1141. 

Deive, FJ., Sanroman, MA., and Longo, MA. 2007. 

Lipase production by a newly isolated Bacillus 

thermomylovorans strain. Journal of Biotechnology. 

131:S238-S239. 

Ertugrul, S., Donmez, G. and Takac, S. 2007. Isolation of 

lipase producing Bacillus sp from olive mill wastewater 

and improving its enzyme activity. Hazardous Materials. 

149: 720-724. 

Essamri, M., Valerie, D. and Louis, C. 1998. 

Optimization of lipase production by Rhizopus oryzae and 

study on the stability of lipase activity in organic solvents. 

Journal of Biotechnology. 60:97-103. 

Fischer-Colbrie, G., Heumann, S., Cavaco-Paulo, A. and 

Gubitz, GM. 2003. Enzymatic modification of synthetic 

fiber. 225th ACS National Meeting, Mar 23-27, New 

Orleans, USA. 

Fischer-Colbrie, G., Heumann, S., Liebminger, S., 

Almansa, E., Cavaco-Paulo, A. and Gubitz, GM. 2004. 


1215 

New enzymes with potential for PET surface 

modification. Biocatalysis and Biotransformation. 22:341- 

346. 

Gordon, RE. 1974. The genus Bacillus: In handbook of 

microbiology (vol. 1) (organisms microbiology). Eds. 

Laskin, AI and Lechevalie, HA. Rutgers University, New 

Brunswick, New Jersey. 

Guebitz, GM and Cavaco-Paulo, A. 2008. Enzymes go 

big: surface hydrolysis and functionalization of synthetic 

polymers. Trends in Biotechnology. 26:32-38. 

Guebitz, GM. and Cavaco-Paulo, A. 2003. New substrates 

for reliable enzymes: enzymatic modification of 

polymers. Current Opinion in Biotechnology. 14:577-582. 

Haba, E., Bresco, O., Ferrer, C., Marques, A., Busquets, 

M. and Manresa, A. 2000. Isolation of lipase-secreting 

bacteria by deploying used frying oil as selective 

substrate. Enzyme and Microbial Technology. 26:40-44. 

Hasan, F., Shah, AA. and Hameed, A. 2006. Industrial 

applications of microbial lipases. Enzyme and Microbial 

Technology. 39: 235-251. 

Heumann, S., Eberl, A., Pobeheim, H., Liebminger, S., 

Fischer-Colbrie, G., Almansa, E., Cavaco-Paulo,A. and 

Gubitz, GM. 2006. New model substrates for enzymes 

hydrolyzing polyethyleneterephthalate and polyamide 

fibers. Journal of Biochemical and Biophysical Methods. 

69:89-99. 

Hiol, A., Jonzo, MD., Rugani, N., Druet, D., Sarda, L. and 

Comeau, LC. 2000. Purification and characterization of 

an extracellular lipase from a thermophilic Rhizopus oriza 

strain isolated from fruit. Enzyme and Microbial 

Technology. 26:421-430. 

Hsieh, Y-L. and Cram, LA. 1998. Enzymatic hydrolysis 

to improved wetabilitty and absorbency of polyester 

fabrics, Textile Research Journal. 68:311-319. 

Killis, J., James, T., Ayrookran, PJ. and Yoon, M-Y. 

2001. Enzymatic modification of the surface of a 

polyester fiber. US patent 6254645. 

Kim, HR. and Song, WS. 2006. Lipase treatment of 

polyester fabrics. Fibers Polymers 7:339-343. 

Kumar, S., Kikon, K., Upadhyay, A., Kanwar, SS. and 

Gupta, R. 2005. Production, purification and 

characterization of lipase from thermophilic and 

alkalophilic Bacillus Coagulans BTS-3. Protein 

Expression and Purification. 41:38-44. 

Lin, SF., Chiou, CM., Yeh, C. and Tsai, YC. 1996. 

Purification and partial characterization of an alkaline 

lipase from Pseudomonas pseudoalkaligenes F-111. 

Applied and Environmental Microbiology. 62:1093-1095. 

Matama, T., Vaz, F., Gubitz, GM. and Cavaco-Paulo, A. 

2006. The effect of additives and mechanical agitation in

1216 

surface modification of acrylic fibers by cutinase and 

esterase. Biotechnology Journal. 7:842-879. 

Meghwanshi, GK., Agarwal, L., Dutt, K. and Saxena, RK. 

2006. Characterization of 1,3-regiospecific lipase from 

new Pseudomonas and Bacillus isolates. Journal of 

Molecular Catalysis. B: Enzyme 40:127-131. 

Mohan, ST., Palavesan, A. and Immanvel, G. 2008. 

Isolation and characterization of lipase-producing 

Bacillus strains from oil mill waste. African Journal of 


O'Neill, A., Araújo, R., Casal, M., Guebitz, G. and 

Cavaco Paulo, A. 2007. Effect of the agitation on the 

adsorption and hydrolytic efficiency of cutinase on 

polyethylene terephthalate fibers. Enzyme and Microbial 

Technology. 40:1801-1805. 

Rohit, S., Yusuf, C. and Ullamchand, B. 2001. 

Production, purification, characterization and application 

of lipases. Biotechnology Advances. 19:627-662. 

Shalaby, SE., El-Balacosy NG. and Abo El Ola, SM. 

2007. Alkaline treatment of polyethylene glycol modified 

poly(ethyleneterephthalate) fabrics. Journal of Textile 

Association. May-June 31-38. 

Sharma, R., Chisti, Y. and Banerjee, UC. 2001. 

Production, purification, characterization, and 

applications of lipase. Biotechnology Advances. 19:627- 

662. 

Silva, C. and Cavaco-Paulo, A. 2004. Monitoring 

biotransformations in polyamide fibers. Biocatalysis and 

Biotransformation. 22:357-360. 

Silva, C., Araujo, R., Casal, M., Gubitz, GM. and Cavaco- 

Paulo, A. 2007. Influence of mechanical agitation on 

cutinases and protease activity towards polyamide 

substrates. Enzyme and Microbial Technology. 40:1678- 

1685. 

Souissi, N., Bougatat, A., Triki-ellouz, Y. and Nasri, M. 

2009. production of lipase and biomass by 

Staphylococcus simulans grown on sardinella (Sardinella 

aurita) hydrolysates and peptone. African Journal of 


Stukus, PE. 1997. Investigating microbiology: A 

laboratory manual for general microbiology. Saunders 

College Publishing, New York. pp. 143-209. 

Vertommen, MAME., Nierstrasz, VA., van der Veer, M. 

and Warmoeskerken, MMCG. 2005. Enzymatic surface 

modification of poly(ethyleneterephthalate). Journal of 


Received: April 3, 2010; Accepted: May 26, 2010 

Canadian Journal of Pure and Applied Sciences


Vol. 4, No. 2, pp. 1217-1219, June 2010 

ISSN: 1715-9997 

ON SUBMANIFOLDS OF INDEFINITE COMPLEX SPACE FORM 

Augustus Nzomo Wali 

Department of Applied Mathematics 

Kigali Institute of Science and Technology, B. P. 3900, Kigali, Rwanda 

ABSTRACT 

n 

Sun (1994) showed that if M is a maximal spacelike submanifold of M n () c then either M is totally geodesic 

( n≥2, c≥ 

0) or c 

0≤ S ≤− n( n−1 ) ,( n≥ 2, c< 

0) . The purpose of this paper is to study the geometry of an n- 

4 

dimensional compact totally real maximal spacelike submanifold M immersed in an indefinite complex space form 

n+ p 

M p () c . In this manuscript we have shown that either the square of the length of second fundamental form S=0, 

implying M is totally geodesic for c≥ 0, n> 

1or 

(1 − n)( n+ 2 p) 

S ≤ c for c< 0, n> 

1and 

thus generalized Sun (1994) 

4 

result. 

Keywords: Totally real submanifold, complex space form, totally geodesic. 

INTRODUCTION 

Among all submanifolds of a Kaehler manifold there are 

two classes; the class of totally real submanifolds and the 

class of holomorphic submanifolds. A submanifold of a 

Kaehler manifold is called totally real (resp. holomorphic) 

if each tangent space of the submanifold is mapped into 

the normal space (resp. itself) by the almost complex 

structure of the Kaehler manifold, Chen and Ogiue 

(1974). A Kaehler manifold of constant holomorphic 

sectional curvature is called a complex space form, Wali 

(2005). 

Let M be an n-dimensional totally real maximal spacelike 

submanifold isometrically immersed in a 2(n+p)- 

n+ p 

dimensional indefinite complex space form M p () c of 

holomorphic sectional curvature c and index 2p. We call 

M a spacelike submanifold if the induced metric on M 

from that of the ambient space is positive definite, 

Ishihara (1988). Let J be the almost complex structure of 

n+ p 

M p () c . An n-dimensional Riemannian manifold M 

n+ p 

isometrically immersed in M p () c is called a totally 

n+ p 

real submanifold of M p () c if each tangent space of M 

is mapped into the normal space by the almost complex 

structure, Yano and Kon (1976). 

n+ p 

Let h be the second fundamental form of M in M () c 

*Corresponding author email: tabnzo@yahoo.co.in 

p 

and denote by S the square of the length of the second 

fundamental form h. 

Sun (1994), proved that if M is a maximal spacelike 

n 

submanifold of M n () c then either M is totally geodesic 

( n≥2, c≥ 

0) or c 

0≤S ≤− n( n−1 ) ,( n≥ 2, c< 

0) . The 

4 

purpose of this paper is to study an n-dimensional 

compact totally real maximal spacelike submanifold M 

immersed 

n+ p 

M () c . 

in an indefinite complex space form 

p 

Our main result is: 

Theorem: Let M be an n-dimensional compact totally 

n+ p 

real maximal spacelike submanifold of M p () c . Then 

either S=0, implying M is totally geodesic for 

c≥ 0, n> 

1 or (1 − n)( n+ 2 p) 

S ≤ c for c< 0, n> 

1. 

4 

LOCAL FORMULAS 

n+ p 

Let M p () c be an indefinite complex space form of 

holomorphic sectional curvature c, dimension 2(n+p), 

p ≠ 0 and index 2p. Let M be an n-dimensional totally 

real maximal spacelike submanifold isometrically 

n+ p 

immersed in M p () c . We choose a local field of 

orthonormal frames

1218 

{ e1,..., en; en+ 1,..., en+ p; e1* = Je1 ,..., en* = Jen; e( 1* ) = Jen 1,..., 

n + e( n p) 

= Je 

+ + * n+ p} 

n+ p 

in M () c 

p 


such that restricted to M, the vectors 

{ 1 } ,..., e e n are tangent to M and the rest are normal to 

n+ p 

M. With respect to this frame field of M p () c , let 

1 n n+ 1 n+ p 1* n* 

( n+ ) ( n+ p) 

ω ,..., ω ; ω ,..., ω ; ω ,..., ω ; ω ,..., ω 

be the field of dual frames. 

1* * 

Unless otherwise stated, we shall make use of the 

following convention on the ranges of indices: 

1 ≤ ABCD , , , ≤ n+ p; 

1 ≤i, j, k, l, m≤ n; 

n+ 1 ≤α, βγ , ≤ n+ p; 

and when a letter appears in 

any term as a subscript or a superscript, it is understood 

that this letter is summed over its range. Besides 

ε = g e, e = g Je, Je = 1, 

when 1 ≤i≤ n 

( ) ( ) 

( ) ( ) 

i i i i i 

ε = g e , e = g Je , Je =− 1, 

when 

α α α α α 

n+ 1≤α≤ 

n+ p . 

n+ p 

Then the structure equations of M () c are; 

p 

A B 

B A 

A A B 

i i* 

dω +∑ε ω ∧ ω = 0 , ω + ω = 0 , ω = ω , 

B B 

j j* 

i* j* 

ω j = ωi 

, 

A 

dωB + 

A C 1 

εω C C ∧ ωB = 

2 

C D 

εε C DRABCDω∧ω ∑ ∑ , 

C CD 

c 

R = εε δ δ − δ δ + J J − J J + J J 

4 

( 2 ) 

ABCD C D AC BD AD BC AC BD AD BC AB CD 

where R ABCD denote the components of the curvature 

n+ p 

tensor R on M p () c . 

Restricting these forms to M we have; 

α 

ω = 0, α α i 

α α i i j 

h = h , dω =−∑ω ∧ ω , 

ω = ∑ h ω , 

i ij 

i 

ij ji 

i j 

ωj + ωi 

= 0 , 

i i k 1 

k l 

dωj =−∑ωk ∧ ωj + ∑ Rijklω 

∧ω 

, 

2 kl 

Rijkl = Rijkl − 

α α α α 

( hikhjl−hilhjk), ∑ 

∑ 

α 

α α 

dω =− ω ∧ω 

, 

β 

β β 

α α γ 1 i j 

dωβ =−∑ ωγ ∧ ωβ + Rαβijω 

∧ω 

, 

2 

γ 

j 

α β α β 

( ) 

Rαβ ij = ∑ 

k 

hh ik jl −hh 

il jk 

(2.1) 

From the condition on the dimensions of M and 

n+ p 

M () c it follows that e1* ,..., e n* 

is a frame for 

p 

⊥ 

T ( M) 

. Noticing this, we see that 

c 

α α α α 

Rijkl = ( δikδ jl −δilδ jk ) −∑ ( hh ik jl −hh 

il jk ) (2.2) 

4 

α 

1 α 

We call H = ∑ trh the mean curvature of M and 

n α 

S h α 

= ∑ the square of the length of the second 

ijα 

( ) 2 

ij 

fundamental form. If H is identically zero then M is said 

to be maximal. M is totally geodesic if h=0. 

From (2.2) we have the Ricci tensor R ij given by 

( n −1) 

α α 

Rij = ∑Rikjk = cδij + ∑ hik hkj 

(2.3) 

k 4 

αk 

Thus the Ricci curvature R is 

c 

R = Rii = ( n− 1) 

+ S 

(2.4) 

4 

From (2.3) the scalar curvature is given by 

n( n−1) 

ρ = ∑ R jj = c+ S 

(2.5) 

j 4 

Let hijk α denote the covariant derivative of ij 

hα . Then we 

define hijk α by 

∑h ω = dh + ∑h ω + ∑h ω + ∑ h ω (2.6) 

α k α α k α k β β 

ijk ij kj i ik j ij α 

k k k 

β 

α 

and hijk α 

hikj 

= . Taking the exterior derivative of (2.6) we 

define the second covariant derivative of ij 

hα by 

h ω = dh + h ω + h ω + h ω + h ω (2.7) 

∑ ∑ ∑ ∑ ∑ 

α l α α l α l α l β β 

ijkl ijk ljk i ilk j ijl k ijk α 

l l l l 

β 

Using (2.7) we obtain the Ricci formula 

∑ ∑ ∑ (2.8) 

h − h = h R + h R + h R 

α α α α β 

ijkl ijlk mj mikl im mjkl ij βαkl 

m m 

β 

The Laplacian ij 

hα ∆ of the second fundamental form ij 

hα 

∆ h =∑ h . 

is defined as 

α 

ij 

α 

ijkk 

k 

Therefore, 

∆ h 

c 

= ( n− 1) 

4 

h + h h h + h h h 

∑ ∑ ∑ (2.9) 

α α α β β α β β 

ij ij mi mk kj km mk ij 

βmk βmk 

∑ ∑ 

+ hh h −2hhh 

β α β β α β 

ki jm mk ki mk mj 

βmk βmk

1 

2 

1 

∆ = + − + 

2 4 

From α 

2 

α 

2 

α α 

∆ ∑( hij) = ∑( hijk ) + ∑ hij ∆h 

we obtain, 

ij 

αij αijk αij 

α 

2 

∑( hij ) α 

2 

∑( hijk ) 

c 

( n 

α 

2 

1) 

∑( hij) α α β β 

∑ hijhklhlk hij 

αijαijk αij αβijkl 

α β β α α β β α 

∑ ( hh li lj hh li lj )( hh ki kj hh ki kj ) 

+ − − 

αβijkl 

PROOF OF THE THEOREM 

(2.10) 

Let M be an n-dimensional compact totally real maximal 

spacelike submanifold isometrically immersed in 

n+ p 

M () c . For each α let Hα denote the symmetric 

p 

matrix ( hij ) α and let 

S h h 

α β 

αβ ij ij 

ij 

= ∑ 

. Then the 

(n+2p)×(n+2p)-matrix ( Sαβ ) is symmetric and can be 

assumed to be diagonal for a suitable choice of 

e ,..., e . 

S = S 

2 

= trH and 

n+ 1 n+ p Setting 

α αα α 

S = ∑ Sα, equation (2.10) can be rewritten as 

α 

1 

α 

2 c 

2 

2 

∆ S = ∑( hijk ) + ( n− 1) 

S+ ∑Sα + ∑tr( 

HαHβ −HβHα) 

2 αijk 4 

α αβ 

(3.1) 

α 

2 c 

2 1 2 

= ∑( hijk ) + ( n− 1) 

S+ ∑Sα+ 

S 

4 n+ 2p 

αijk α 

1 

+ − + − 

n+ 2 p∑ 

∑ 

( Sα Sβ) tr( HαHβ HβHα) α> β αβ 

2 2 

⎛c1 ⎞ 1 

= + − + + − 

αijk ⎝4 n+ 2p ⎠ n+ 2p 

α> β 

α 

∑( hijk ) ⎜ ( n 1) 

S⎟S ∑( 

Sα Sβ 

) 

∑ 

2 2 

( ) 2 

α β β α 

+ tr H H −HH 

αβ 

From (3.1) we see that 

1 α 

2 ⎛c1 ⎞ 

∫ ∆Sdv ≥ ( hijk ) dv ( n 1) 

S Sdv 

M 2 ∫ ∑ + 

M ∫ M⎜ 

− + ⎟ 

αijk 

⎝4 n+ 2p 

⎠ 

where dv is the volume element of M. 

By the well known theorem of Hopf (1950), ∆ S = 0 . 

Therefore, 

α 

2 ⎛c1⎞ 0≥ ∫ ∑( 

hijk ) dv+ ( n 1) 

S Sdv 

M ∫M⎜ 

− + ⎟ 

αijk 

⎝4 n+ 2p 

⎠ 

which implies that 

Wali 1219 

⎛c1⎞ ( n 1) S Sdv 0 

M ⎜ − + ⎟ ≤ 

(3.2) 

∫ 

⎝4 n+ 2p 

⎠ 

Thus either S=0 implying M is totally geodesic or 

( 1− n)( n+ 2p) 

S ≤ c. 

This shows that M is totally 

4 

geodesic for c≥ 0, n> 

1 or ( 1− n)( n+ 2p) 

0 ≤ S ≤ 

c 

4 

for c< 0, n> 

1. 

This proves our theorem. 

CONCLUSION 

In this paper we studied the geometry of an n-dimensional 

compact totally real maximal spacelike submanifold M 

n+ p 

immersed in an indefinite complex space form M p () c 

by computing the square of the length of the second 

fundamental form. In conclusion, we have shown that 

either the square of the length of second fundamental 

form S=0, implying M is totally geodesic for 

c≥ 0, n> 

1 or 

(1 − n)( n+ 2 p) 

S ≤ c for c< 0, n> 

1. 

4 

This generalizes the result by Sun (1994). 

REFERENCES 

Chen, BY. and Ogiue, K. 1974. On totally real 

submanifolds. Transactions of the American 

Mathematical Society. 193:257-266. 

Hopf, E. 1950. A theorem on the accessibility of 

boundary parts of an open point set. Proceedings of the 

American Mathematical Society. 1:76-79. 

Ishihara, T. 1988. Maximal spacelike submanifolds of a 

Pseudoriemannian space of constant curvature. Michigan 

Mathematical Journal. 35:345-352. 

Sun, H. 1994. Totally real maximal spacelike 

submanifolds in indefinite complex space form. Journal of 

Northeastern University. 15:547-550. 

Wali, AN. 2005. On bounds of holomorphic sectional 

curvature. East African Journal of Physical Sciences. 

6(1):49-53. 

Yano, K. and Kon, M. 1976. Totally real submanifolds of 

complex space forms II. Kodai Mathematical Seminar 

Reports. 27:385-399. 

Received: Nov 13, 2010; Revised: Feb 15, 2010; May 14, 2010


Vol. 4, No. 2, pp. 1221-1225, June 2010 

ISSN: 1715-9997 

EXPERIMENTAL STUDY ON THE REDUCTION OF PRESSURE DROP OF FLOWING 

WATER IN HORIZONTAL PIPES USING PADDY HUSK FIBERS 

*Hayder A Abdul Bari, Mohd Azimie Ahmad and Rosli Bin Mohd Yunus 

Faculty of Chemical and Natural Resources Engineering, University Malaysia Pahang 

Lebuhraya Tun Razak-26300 Gambang, Kuantan, Pahang Darul Makmur, Malaysia 

ABSTRACT 

Abundant of waste from rice production in Malaysia have become the trigger for this investigation. Paddy husk are the 

waste from the rice production. Utilization of this waste in transportation of fluid can reduce the pressure drop in 

pipelines. Several studies have shown that addition of minute quantities of fiber additives can reduce the drag in pipe and 

maintain the pressure drop along the pipelines. Experimental works have been conducted in the laboratory in order to test 

paddy husk fibers in a closed loop of turbulence water flowing system. Flow tests were conducted using water as the 

transport liquid. The experimental work starts by pumping water from reservoir tank that had mixed with paddy husk 

fibers was pumped with six different flow rates in same pipe diameters a (0.025m ID). The types of pipe used are 

galvanized iron pipe. The testing length of this flow system is 1.0m. The pressure drop and drag reduction were 

measured in the flow varying concentrations of fiber (paddy husk). After adding the fibers to the water, the results have 

shown that the percentage drag reduction (Dr%) of 32% at the first flow rate setting and become depleted in term of the 

reading after travel at larger flow rate setting, but still there was drag reduction involve during transportation of the 

water.The results have shown that the fibers (paddy husk) have an effect on the pressure drop. In addition to this, we 

point out that the paddy husk fiber is available as drag reducing agent. 

Keywords: Waste, paddy husk fiber, closed loop, galvanized iron pipe, reduce drag. 

INTRODUCTION 

The addition of minute amounts of additives such as 

surfactants, polymers or rigid particles can result in 

important drag reduction effects in many types of flows. 

Most of the existing literature dealing with drag reduction 

by additives has focused on wall-bounded flows such as 

pipe flows, due to their importance in many technological 

processes. For this class of flows, there was a special 

interest in the use of high molecular weight surfactants to 

reduce pressure drops and friction effects, and there are a 

large number of experimental and numerical studies that 

document the effects of surfactant additives on such flows 

(Pinho and Whitelaw, 1990; Tiederman, 1990). On the 

other hand, the use of fibre additives as drag-reducing 

agents remains limited. There are, however, few 

experimental studies that show the great potential of these 

additives, and drag reduction effects of up to 60% in pipe 

flows have been reported by Arranaga (1970) and other 

authors. Depending on the flow geometry, the particle 

size and the importance of viscous effects versus inertial 

effects, the addition of fibres to a flow can have either a 

stabilizing (Vaseleski and Metzner, 1974) or a 

destabilizing effect (Pilipenko et al., 1981). In general, 

where particle additives tend to stabilize the flow, it has 

been observed that the stabilizing effect increases with the 

*Corresponding author email: mohdazimie@gmail.com 

particle aspect-ratio and concentration (Vaseleski and 

Metzner, 1974). 

There are much less studies devoted to the effects of fibre 

additives on the mechanisms of instability and transition 

to turbulence in free shear flows. The flow visualizations 

reported by Filipsson et al. (1977) represent one of the 

few available experiments on this subject. In this study, 

the authors presented results for a jet flow of viscoelastic 

(Polyox WSR-301), fibre suspension (chrysotile fibres), 

and Newtonian (water) fluids at high Reynolds numbers. 

The addition of a small amount of either surfactant or 

fibres led to similar trends towards an enhancement of the 

large-scale turbulent structures and a modulation of the 

turbulence by the suppression of small-scale structures. In 

spite of the well-documented experimental and theoretical 

evidence for drag reduction by surfactant and solid 

particle additives, the physical mechanisms responsible 

for these phenomena are still not well understood and are 

subject to debate. 

Later, the usage of the suspended solid (insoluble in liquid 

media) as Drag Reducer opened the wide door for more 

research to examine the availability of the solubility 

condition in the drag reduction phenomena (Hideo et al., 

2000; Toorman, 2002; Mawla and Naderi, 2006).

1222 

The aim of this study is to formulate and to test the 

efficiency of paddy husk fiber as drag reducer agent on 

transport of water in pipes; different sizes of fiber (500 

µm and 800µm) were used. Six different concentrations 

were used in the purpose to investigate the concentration 

effect. The efficiency of fiber was tested using clear 

water. 

MATERIALS AND METHOD 

Liquid Circulation System 

Figure 1 shows a schematic diagram of a build up liquid 

circulation system used in the present investigation. 

Generally, this system consists of reservoir tank, pipes, 

valves, pumps, flow meter and pressure gauge. The 

reservoir tank was supported with two exit pipes 

connected to centrifugal pumps. The first exit pipe with 

was connected to the main centrifugal pump which 

delivers the fluid to the testing sections. The other exit is 

connected to the other centrifugal pump for deliver excess 

solvent to reservoir tank. 

Three galvanized iron pipes of various inside diameters 

0.015 (C), 0.025 (B) and 0.038 (A) m ID were used in 

constructing the flow system. A complete closed loop 

piping system was build. Piping starts from the reservoir 

tank through the pump, reaching a connection that splits 

the pipe into two sections. The first section returns to the 

reservoir tank, build up as bypass and the other splits into 

three sections with different pipe diameters at testing 

section. The testing sections were 1.0m long, 0.025m ID 

and it was located about 50 times of pipe diameter to 

ensure the turbulent flows are fully developed before the 

testing point. Two sets of Baumer Differential Pressure 

Gauge were used to detect the pressure drop in pipelines 

with maximum differential pressure reading up to 0.10 

and 0.20 bars for both. In order to measure the flow rate 

of fluid in pipelines, Ultraflux Portable Flow Meter 

Minisonic P has been used. This ultrasonic flow meter 

measurement was sensitive with small changes in flow 

rate as low as 0.001 ms -1 can be detected. 

Fig. 1. Schematic of the flow system. 


Materials Investigated 

Paddy Husk Fibers 

Paddy husk is obtained from Bernas Sdn. Bhd. at Jitra, 

Kedah. The paddy husk was dried by the oven overnight 

at temperature of 100°C. Once dry, the fibers is graded 

into fibers by using grinder then sample was sieve using a 

screen into two sizes which are 500 and 800µm. Physical 

properties of the paddy husk were determined by using 

pycnometer. The results are as shown in table 1. 

Table 1. Physical properties of paddy husk fibers. 

Paddy husk size (µm) Fibers Density (gcm -3 ) 

500 2.0811 

800 1.2538 

Transported Liquid 

The transported liquid used in the present investigation 

was water. The physical properties of water are shown in 

table 2. 

Table 2. Physical properties of water. 

Water properties @ 25 C 

Viscosity (µ water @ 25C ) 0.8973 x 10 -3 Pa.s 

Density ( water @ 25C ) 997.08 kg/m 3 

Experimental Procedure 

All the experiments were carried in a constructed liquid 

circulation system, testing different variables, which are: 

Paddy husk fibers concentration (100, 300 

and 500ppm) 

Paddy husk sizes (500µm and 800µm) 

Pipe diameter (0.025m) 

Solution flow rates (2.5, 3.0, 3.5, 4.0, 4.5 

and 5.0 m 3 /hr) 

The experimental procedure starts by testing every 

additive concentration and pipe diameter, the operation 

begins when the pump starts delivering the solution 

through the testing section. The solution flow rate is fixed 

at the certain value by controlling it from the bypass 

section. Pressure readings are taken to this flow rate. By 

changing the solution flow rate to another fixed point, 

pressure readings are taken again until finishing the six 

desired values of flow rates. This procedure is repeated 

for each fibers concentrations and sizes to test its effect on 

the drag reduction operation. 

Experimental Calculation 

(a) Velocity and Reynolds number calculations 

The average velocity (V) and Reynolds number (Re) were 

calculated using the solution volumetric flow rate

eadings (Q), density (), viscosity (µ) and pipe diameter 

(D), for each run as follows: 

V. . D 

Re (1) 

 

(b) Percentage Drag Reduction calculations 

Pressure drop readings through testing sections before and 

after drag reducer addition, were needed to calculate the 

percentage drag reduction %Dr as follows (Virk, 1975) 

% Dr 

 

 

Pb 

P 

Pb 


a 

Effect of Fluid Velocity (Re) 

Figures 2 and 3 shows the behaviour of the transported 

water velocity on the percentage drag reduction (Dr%). 

The velocity (V) was represented by the Reynolds number 

(Re). Figures 2 and 3 shows the effect of (Re) on Dr% for 

water transported with paddy husk fibers (500 and 800 

µm) with different addition concentrations. 

% Drag Reduction 

35 

30 

25 

20 

15 

10 

5 

0 

35363 42436 49508 56581 63654 70726 

Reynold Number 

(2) 

100 ppm 

300 ppm 

500 ppm 

Fig. 2. Effect of Re on Dr% for transported water with 

Paddy husk fibers (500µm) with different addition 

concentrations. 


35 

30 

25 

20 

15 

10 

5 

0 

0 100 200 300 400 500 600 

Addition Concentration (ppm) 

Re=35363 

Re=42436 

Re=49508 

Re=56581 

Re=63654 

Re=70726 

Fig. 4. Effect of fibers concentration on Dr% for 

transported water with Paddy husk fibers (500µm) with 

different flowrates (Re). 

Bari et al. 

1223 

From figure 2, it can be noticed that the Dr% reaching 

maximum value 32% power saving with 500ppm addition 

concentration of 500µm in Re number ranges (35363 to 

70726) in the 0.025m ID pipe. Further increase of Re 

resulted decreasing in the Dr% compared with maximum 

value. 

Meanwhile, in figure 3, it was shown that the Dr% 

reaching maximum value of 25% power saving with 

100ppm addition concentration of 800µm fibers size 

before decreasing slowly after Re are above 40000. 

These phenomenons may be caused by the particle 

momentum generated from paddy husk fibers drive the 

eddy to flow straight along the pipe because the 

momentum of the particle is larger from the energy to 

form turbulence eddies. 

Effect of Solid Particles Concentration 

The results of analysis of paddy husks drag reduction 

performances as a function of fibers concentration and Re 

which is from 100 to 500ppm in the range of 35363 to 

70726 are shown in figure 4. Note that the profile pattern 

of each concentration is similar but varies in its value. As 

shown in figure 4, the maximum drag reduction values of 

32 % in 500ppm of concentration occur when the Re at 


30 

25 

20 

15 

10 

5 

0 

35363 42436 49508 56581 63654 70726 


100 ppm 

300 ppm 

500 ppm 

Fig. 3. Effect of Re on Dr% for transported water with 

Paddy husk fibers (800µm) with different addition 

concentrations. 


30 

25 

20 

15 

10 

5 

0 

100 300 

Addition Concentration 

500 

Re=35363 

Re=42436 

Re=49508 

Re=56581 

Re=63654 

Re=70726 

Fig. 5. Effect of fibers concentration on Dr% for 

transported water with Paddy husk fibers (800µm) with 

different flowrates (Re).

1224 

35363. In general based on the experiments conducted, all 

concentration showed a drastic reduction of drag 

reduction at 35363 to 70726 of Re which the drag 

reduction values ranged between 3 and 27%. These 

results showed that the optimum performance of the fibers 

additive investigated is limited to the degree of 

turbulence, that by increasing the flow the degree off 

turbulence will increase also which will provide more 

suitable environment for the drag reducer to perform. 

Further increase in the flow will cause the reduction in the 

additive efficiency due to the decrease of the additive 

concentration to degree of turbulence ration. In another 

words, the reduction of drag as obtained in this 

experiment shows that the paddy husk capable to act as 

drag reducing agent at 500µm size of fibers. 

Figure 5 shows the performance of drag reduction as a 

function of fibers concentration and Re which is from 100 

to 500ppm in the range of 35363 to 70726. The profile 

pattern of each concentration is similar but different in 

values. As shown, the highest drag reduction occurs at 

100 ppm concentration of 800µm fibers size with 25%. 

Further increase of fibers concentration shows decrease in 

drag reduction but still reduction of flow still occur. The 

lowest drag reduction was obtained in 500ppm of paddy 

husk fibers that is 2%. These phenomena probably can be 

defined as the result of the diameter effect towards the 

flexibility of the fibers to acts as drag reducer. By 

increasing the diameter of the fibers lead to the decrease 

of flexibility. In order fibers to act as drag reducer agent, 

basic criteria such as flexibility and surface roughness 

should be fulfilled (Singh, 1990). Singh proven that drag 

reduction of fibers with small diameter is higher than drag 

reduction cause by the fibers with larger diameter because 

fibers with small diameter can improved the flexibility of 

fibers in flow system. These results are consistent with the 

findings in a few literatures, in which the drag reduction 

increase as addition concentration is increases. 

Effect of Particle Size 

There were two sizes of paddy husk fibers that were used 

to investigate drag reduction in this present research (500 

and 800µm). Figure 6 shows a selected sample of fibers 

size effect data. These results clearly shows that the DR% 

of the paddy husk with the size 500 µm is larger than the 

paddy husk with the size of 800µm. 

This may be due to the small momentum needed in order 

to transport smaller particles that make these particles 

easily been drag by the turbulent flow in pipe. Small 

diameter of fiber material increases the flexibility. 

Flexibility is the important for fibers to act as drag 

reducer. From this condition, there was a decreasing 

effect in the spectrum of turbulence causing the drag 

reduction higher at this smaller particles size. 


Normally, during fibers are travelled in pipe, the fibers 

was torn into pieces due to an eddy during turbulence 

flow. In this case, smaller size of fibers has become great 

impact to make larger eddies to break up to smaller eddies 

which as the result, an increment of the Dr%. 


35 

30 

25 

20 

15 

10 

5 

0 

35363 42436 49508 56581 63654 70726 


500 µm 

800 µm 

Fig. 6. Effect of changing the particle diameter (500 to 

800µm) on the Dr% for transported water with paddy 

husk fibers with different Re at 500ppm. 

CONCLUSIONS 

Paddy husk fibers were found to behave as good drag 

reducing agent at 500µm of sizes and 500ppm of 

concentrations. Drag reduction for fibers materials was 

found to increase by smaller fibers size used as drag 

reducer. 

ACKNOWLEDGMENTS 

I wish to express my deepest gratitude to my supervisor 

Dr. Hayder A. Abdul Bari and co-supervisor Prof. Dr. 

Rosli Bin Mohd Yunus for their guidance, advice, 

criticism, encouragements and insight throughout the 

research. 

REFERENCES 

Arranaga, AB. 1970. Friction reduction characteristics of 

fibrous and colloidal substances. Nature. 225:447-449. 

Filipsson, LGR., Torgny, JH., Lagerstedt. and Bark, FH. 

1977. A note on the analogous behaviour of turbulent jets 

of dilute surfactant solutions and fibre suspensions, J. 

non-Newtonian Fluid Mech. 3:97-103. 

Hideo, I., Naoto, H. and Akihiko, H. 2000. Flow drag and 

heat transfer reduction of flowing water containing 

fibrous material in straight pipe. Int. J. Thermal Sci. 

39:18-29. 

Mowla, D. and Naderi, A. 2006. Experimental study of 

drag reduction by a polymeric additive in slug two-phase

flow of crude oil and air in horizontal pipes. Chem. Eng. 

Sci. 61:1549-1554. 

Pinho, FT. and Whitelaw, JH. 1990. Flow of non- 

Newtonian fluids in a pipe, J. non-Newtonian Fluid Mech. 

34:129-144. 

Pilipenko, VN., Kalinichenko, NM. and Lemak, AS. 

1981. Stability of the flow of a fibre suspension in the 

gap between coaxial cylinders, Sov. Phys. Dokl. 26:646- 

648. 

Singh, RD. 1990. Encyclopedia of Fluid Dynamics, Gulf 

Publishing Co., Houston, Texas, Chapt. 9. 14:425-480. 

Tiederman, WG. 1990. The Effect of Dilute Surfactant 

Solution on Viscous Drag and Turbulent Structure. In: A. 

Bari et al. 

1225 

Gyr (Ed.), Structure of Turbulence and Drag Reduction, 

IUTAM Symp. Springer, Berlin. 187-200. 

Toorman, EA. 2002, Modelling of turbulent flow with 

suspended cohesive sediment. Proc. Mar. Sci. 5:155-169. 

Vaseleski, RC. and Metzner, AB. 1974. Drag reduction in 

the turbulent flow of fibre suspensions, AICHE J. 20: 

301-306. 

Vaseleski, RC. and Metzner, AB. 1974. Drag reduction in 

the turbulent flow of fibre suspensions. AICHE J. 20:301– 

306. 

Received: Dec 29, 2009; Accepted: April 20, 2010.


Vol. 4, No. 2, pp. 1227-1231, June 2010 

ISSN: 1715-9997 

EFFECTIVENESS OF ACCESSIBILITY AND USABILITY OF GOVERNMENT 

WEBSITES IN SAUDI ARABIA 

Muhammad Asif Khan and Khalid Abdulmohsen Buragga 

Computer Sciences and Information Technology, King Faisal University, Al Ahsa, Saudi Arabia 

ABSTRACT 

In today’s rapidly changing world, information technology has significant impact in almost all sectors of daily life. With 

greater internet penetration among individuals and organizations use of online services is becoming indispensable. The 

increasing demand of electronic services has forced governments all around the world to provide online services to their 

citizens and residents. The government of Saudi Arabia has also realized the need of such services and it is working on 

an accelerating pace to provide excellent electronic services infrastructure to its citizens. Internet is an exciting 

technological tool that requires innovative design in order to be accessible to everyone. The purpose of this paper is to 

examine and evaluate accessibility and usability of e-government websites of Saudi Arabia. A subjective assessments 

method has been used to carryout the study. The results of the study have been compared with web accessibility tools. 

This study investigates the issues that are required to make a website accessible and examines accessibility guidelines. It 

also evaluates the Saudi government websites in context of W3C Web Content Accessibility Guidelines. The present 

study also presents some suggestions for improving e-government web sites to be more effective and beneficial to 

general public. 

Keywords: E-government, usability, Saudi e-government, accessibility, Saudi Arabia. 

INTRODUCTION 

Information and Communication Technology (ICT) has 

revolutionized the way individuals and organizations used 

to work. Now internet has become an essential tool that is 

used for information dissemination. People around the 

world use Internet mainly for email, product or 

organization information and health information 

(Goodman et al., 2003). With greater Internet penetration 

in society organizations started offering their services 

online so that customers access them whenever and 

wherever they want. People would like to use Internet as a 

transaction tool and now more people are using electronic 

transactions in different areas of their daily life (e.g. 

banking, shopping) due to less effort and quick service. 

Governments also have realized the significance of 

Internet and seem to be undergoing transformation to use 

Internet to deliver services and information according to 

these time and effort expectations (Abdulkarim, 2003). 

More than 160 countries worldwide have started egovernment 

project, creating a major market for IT 

vendors and service providers that facilitate public 

organizations in adoption of technology (Qureshi, 2005). 

E-government has been defined by some researchers in 

terms of specific actions (e.g. obtaining documents, 

accessing information, creating a sharing database) or 

simply as the automation of services. E-government 

concepts introduced in public administration in late 1990s 

*Corresponding author email: asifkhan@kfu.edu.sa 

1127 

(Moon, 2002). There are issues involve in e-government 

such as security and privacy, diverse educational 

background of users, accessibility issues, prioritization of 

e-government over basic functions of government, 

building citizen confidence in e-government whether 

certain forms of government do better with e-government 

than others (Jagear, 2003). E-government has been 

thought as a best tool for serving citizens in a country and 

almost all countries have been trying to design and 

implement e-government. The government of Saudi 

Arabia has realized the effects of information technology 

in the economy and therefore special attention has been 

given to information technology that has brought a huge 

change in the last forty years. 

Governments around the world especially in Gulf region 

are providing funds to the e-government projects in order 

to meet their society’s increasing cyber skills. In every 

part of world from industrialized countries to developing 

ones governments is putting information online to provide 

better services for citizens (Chircu et al., 2005). Currently 

Saudi ARAMCO, Saudi Arabia Basic Industries 

(SABIC), Saudi Telecommunication (STC), Saudi 

Arabian Airlines and banks are using stat-of-the-art 

technologies in their applications. The government of 

Saudi Arabia has also realized the significance of egovernment 

and to achieve the aim of e-government a 

program called YESSER has been initiated that is a usercentric 

and it focuses on governments services to be 

provided to individuals and businesses. The user-centric 

vision for Saudi Arabia’s e-government initiative is

1128 

summarized as ‘By the end of 2010, everyone in the 

Kingdom will be able to enjoy- from anywhere and at 

anytime – world class government services offered in a 

seamless, user friendly and secure way by utilizing a 

variety of electronic means (www.yesser.gov.sa/english). 

As the Internet is fast becoming a major source of 

information and services, a well designed e-government 

website has become an essential so that citizens can 

access public information and improve their participation. 

Government websites can serve as a tool for both 

communication and public relations for the general 

public. Information and data can easily be shared with and 

transferred to external stakeholders (Moon, 2002). 

The Web Accessibility Initiative (WAI) was established 

by World Web Consortium (W3C) in 1997. The W3C 

publishes Web Content Accessibility Guidelines 

(WCAG), which provide a series of checkpoints for web 

content development. These checkpoints are broken down 

into three priorities depending on their impact on 

accessibilities. Table 1 shows each priority with its 

description 

Table 1. Web Content Accessibility Guidelines (WCAG) 

Priorities 

Priority Description 

Priority 1 A web content developer must satisfy 

this checkpoint. Satisfying this 

checkpoint is a basic requirement for 

some groups to be able to use web 

documents 

Priority 2 A web content developer should satisfy 

this checkpoint. 

Satisfying this checkpoint will remove 

significant barriers to accessing web 

documents 

Priority 3 A web content developer may address 

this checkpoint. 

Satisfying this checkpoint will improve 

access to web documents 

The W3C has been promoting web accessibility and 

according to it a web accessibility in general term is 

defined as “people with disabilities can use the Web… 

more specifically [they] can perceive, understand, 

navigate and interact with the Web” (Henry, 2006). 

Accessibility is considered as a subset of usability where 

problems related to usability may affect to all users 

equally apart from ability or disability (Thatcher et al., 

2003). Accessibility can be dealt with as a part of 

usability evaluations process. Usability is defined by the 

International Standards Organizations (ISO) as ‘the 

effectiveness, efficiency and satisfaction with which 

specified users achieve specified goals in particular 

environment’ (ISO, 2000; ISO, 1998). There is a 


possibility that some accessibility problems may affect 

non-disabled users. However, all usability problems are 

within the scope of accessibility meaning that people with 

disabilities encounter all the same problems that people 

without disabilities encounter. The ISO has defined 

accessibility as ‘the usability of a product, service, 

environment or facility by people with the widest range of 

capabilities’ (ISO, 2003). 

Methodology 

There are various methods to evaluate web accessibility 

that include standards review, user testing, subjective 

assessments and barrier walkthrough. These methods 

differ in terms of their usefulness, efficiency and 

effectiveness (Branjik, 2006). Standards review also 

called expert or conformance review is an analytic 

method based on evaluator’s opinions. This method 

depends on chosen checklist that range from standards 

issued by international bodies such as W3C, IBM or 

SUN. This method is costly and requires experienced 

evaluators. Several studies of usability evaluation 

methods have shown that user testing methods may fail in 

yielding consistent results when performed by different 

evaluators (Hertzum et al., 2001). 

Subjective assessments method has various benefits that 

include low cost and its ability to be performed remotely. 

This method does not require experienced evaluators and 

any specific criteria of pages being tested. In the present 

study we have assessed the usability of the government 

web sites that include effectiveness and efficiency and 

satisfaction thoroughly by users without any disability 

even such as color vision. 

In the present study subjective assessments method has 

provided an opportunity to gain experience of general 

evaluators who had no experience of website evaluation. 

We selected two websites for evaluation purpose i.e. 

Saudi Railways (www.saudirailways.org) and Saudi Post 

(www.sp.com.sa). The reason for selecting both of these 

websites was an increasing accessibility and usefulness 

among citizens (www.arabnews.com). Another reason for 

selecting the two websites was being in complete dual 

versions (i.e Arabic and English) which we could not find 

in other websites at the time of decision. It was confirmed 

by the participants that they never had visited the websites 

before this study. Participants were given common tasks 

for evaluation purpose in different places. All participants 

were asked to rate the severity of problems on four point 

scale based on Nielsen’s heuristic evaluation method i.e. 

Cosmetic, Minor, Major or Catastrophic problem 

(Nielsen, 1994). 

We asked 250 evaluators and provided them with 12 

features to be evaluated manually in the websites that 

include logo of the company, office phone, address, 

email, contents, foreign language version, other link,

online payment option, links to other government sites, 

search option, subject index and audio/video clips. We 

received positive and complete response from 173 

participants. Among the participants were 102 males and 

71 females with a median age of 32 and varied education 

background from high school to research 

institutions/universities. Table 2 shows participants’ 

computer literacy, use of internet, website knowledge and 

time consumption on computer. 

Table 2. Participants Computer and Internet Skills. 

Skill 

Median 

Male (1-5) Female (1-5) 

Computer Literate 5 4 

Internet User 5 5 

Website 

Knowledge 

5 3 

Internet Usage 3 2 

Computer Literate - 1 = never; 2 = somehow; 3 = average; 4 = 

often; 5 = very often 

Internet User - 1 = never; 2 = somehow; 3 = average; 4 = often; 

5 = very often 

Web Site knowledge - 1 = never; 2 = somehow; 3 = average; 4 = 

good; 5 = very good 

Hours per day on internet - 1 = up-to 1; 2 = 2 to 4; 3 = 5 to 7; 4 

= 8 to 10; 5 = more than 10 

The results obtained by the evaluators were analyzed 

carefully and then it was decided to use well known tools 

available online to compare the outcomes of this manual 

evaluation with the available tools. We used EvalAccess 

2.0 and CynthiaSays tools for evaluation of the websites. 

We also used W3C MarkUp Validator, Link Checker and 

CSS Validator tools to compare the conformance of web 

pages with the guidelines provided by W3C. EvalAccess 

evaluates web pages based on WAI and WCAG. The 

implemented web service for accessibility evaluation can 

process a web page from its URL or its HTML mark-up. 

The result of the evaluation process is formatted in XML 

following a predefined XML schema. CynthiaSays is a 

web content accessibility validation tool that has been 

designed based on WCAG guidelines. The MarkUp 

Validator is also known as HTML Validator that assists in 

checking web documents in formats like HTML, 

XHTML, SVG and MathML. The Link Checker checks 

the anchors (hyperlinks) in HTML/XHTML documents 

while CSS Validator tool validates CSS stylesheets or 

documents using CSS Validator. 


All participants were provided with the clear instructions 

about the 12 features to be evaluated and they were told to 

use their internet browsing experience and aesthetic 

features during the evaluation of the websites. There was 

a significant difference of evaluation of the websites 

Khan and Buragga 1129 

between male and female participants. We found the 

participants rated the severity of problems differently, for 

example, 81% male participants considered a page ‘not 

found’ as a catastrophic problem while 53% female 

participants rated the same page ‘not found’ as a general 

problem. Likewise, 77% female participants have given 

more consideration to the aesthetic features of the website 

over the contents while 42% male participants focused 

more on contents than aesthetic features. Since the 

website has English language version as well, therefore, 

participants evaluated both the versions. Table 3A shows 

the summary of the results of Saudi railways website. 

Table 3. Web page and Problems Statistics in Saudi 

Railway Website. 

Version Mean Male Female 

Standard 

Deviation 

English Number of pages 

visited 

50.0 48.0 1.414214 

Number of pages 

with problems 

7.0 6.0 0.707107 


with severity 

1.25 0.86 0.275772 

Arabic Number of pages 

visited 

50.0 48.0 1.414214 


with problems 

3.0 2.0 0.707107 


with severity 

0.5 0.15 0.247487 

For the Saudi Post website all the participants evaluated 

the above stated features and their findings are 

summarized in the table 3B: 

Table 3b. Web Pages and Problems Statistics In Saudi 

Post Website. 

Version Mean Male Female Standard 

Deviation 

English Number of 

pages 

visited 

70.0 67.0 2.12132 

Number of 

pages with 

problems 

3.0 4.0 0.707107 

Number of 

pages with 

severity 

0.73 0.82 0.06364 

Arabic Number of 

pages 

visited 

68.0 65.0 2.12132 

Number of 

pages with 

problems 

0.65 0.34 0.219203 

Number of 

pages with 

severity 

0.17 0.04 0.091924

1130 

Evaluation by EvalAccess 2.0 and CynthiaSays Tools 

Following analyzing results of the manual evaluation of 

both websites, we used online tools for accessibility 

evaluation. Following table IVA shows the results for 

Saudi Railways website obtained by using EvalAccess 2.0 

that evaluates automatically the accessibility of web pages 

using the WCAG 1.0 from the W3C: 

Table 4a. Web Pages Accessibility Results for Saudi 

Railways Website. 

Priority 1 Priority 2 Priority 3 

Errors 2 47 23 

Warnings 110 178 208 

General 

Warnings 

5 7 9 

It is evident from the table 4A above that the website has 

two errors of priority 1 that must satisfy the basic 

requirement for some groups to be able to use web 

documents. Likewise, Priority 2 shows that there are some 

significant barriers that should be removed to access the 

web pages. Priority 3 shows that there are some areas 

which require attention for improving the website. 

Following table 4B shows the results for Saudi Post 

website using the same EvalAccess 2.0 tool 

Table 4b. Web Pages Accessibility Results for Saudi Post 

Website. 

Priority 1 Priority 2 Priority 3 

Errors 6 5 2 

Warnings 27 13 4 

General 

Warnings 

5 7 9 

The table 4B above shows there are six errors of Priority 

1 that must satisfy the basic requirements for some groups 

to be able to use web document. However, there are less 

significant barriers and areas of improvement in the 

website as depicted by Priority 2 and Priority 3. 

We also used CynthiaSays tool to evaluate both the 

websites and found that the Saudi Railways website could 

not pass the Priority 1, Priority 2 and Priority 3 

checkpoints. The same tool passed the Saudi Post website 

the Priority 1, Priority 2 with 2 warnings and Priority 3 

with 1 warning. 

Evaluation by MarkUp Validator, Link Checker and 

CSS Validator 

We used different online tools to evaluate the web 

accessibility of the websites and following Table 4C 

shows the results: 


Table 4c. Web Accessibility Evaluation by Different 

Tools. 

Website MarkUp 

Validator 

Saudi 73 Errors, 1 

Railways Warning, 

Doctype: 

XHTML 1.0 

Transitional 

Saudi 6 Errors, 5 

Post Warnings, 

Doctype: 

HTML 4.01 

Transitional 

Link 

Checker 

Invalid 13 

links, valid 

4 anchors 

Valid links, 

Valid 

anchors 

CSS 

Validator 

File not 

found 

No error 

found 

Validates 

as CSS 

Level 2.1 

The above table 4C depicts that Saudi Railways web 

pages have significant number of errors in XHTML pages 

that were evaluated by MarkUp Validator. Also, there are 

various invalid links found by Link Checker. The CSS 

Validator tool could not find any CSS file on this website. 

However, it is evident that Saudi Post website has less 

number of error pages, all valid links and CSS level 2.1 

files evaluated by the above stated tools. 

CONCLUSION 

The present study has investigated the accessibility and 

usability of the two government websites. We have 

evaluated the websites manually and the results obtained 

were compared with the results obtained by using 

different tools. We found that the manual results are more 

or less same as of the results we obtained by using 

different tools. In the research it has been found that 

Saudi Railways (SR) website has more severity problems 

than Saudi Post (SP) website. We found that authorities of 

the websites are not well equipped to provide services 

electronically that are required to the Saudi citizens and 

residents. The authorities of the websites should ensure 

basic compliance with legal HTML, XHTML and CSS 

standards. This would entail regular validating and 

correcting code, but would mean that files could then be 

compatible with different browsers and platforms. To 

make the websites usable to all users the competent 

authorities need to take measures immediately in order to 

provide appropriate services electronically that are in 

compliance with W3C guidelines. Although disabled 

users have not participated in the present study but as 

Thatcher et al. (2003) have proposed that usability 

problems affect all users regardless of ability or disability. 

This study also suggests that government should improve 

their websites in terms of providing necessary information 

in both English and Arabic versions equally so that all 

residents in the country are served effectively and 

efficiently. The evaluation of the two more familiar

websites indicates the plight of government websites that 

need to be developed according to W3C standards. 

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Received: Feb 22, 2010; Revised: May 21, 2010; Accepted: 

May 29, 2010

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