Journal of Multidisciplinary Scientific Research , 2014,2(3):01-05 ISSN: 2307-6976 Available Online: http://jmsr.rstpublishers.com/ CULTURE CONDITIONS FOR GROWTH AND PIGMENT PRODUCTION OF A MANGROVE PENICILLIUM SPECIES. Lathadevi Karuna Chintapenta1*, Chandi Charan Rath2, Bapuji Maringinti3 and Gulnihal Ozbay1 1)Delaware State University, Department of Agriculture & Natural Resources, Dover, DE 19901, USA. 2)DLR College of PG Courses, Gollala Mamidada, Andhra Pradesh, India. 3)North Orissa University,Baripada, Orissa,India. Email: kchintapenta@desu.edu Received:03,Feb,2014 Accepted:02,May,2014. Abstract Penicillium strain (DLR-7) producing red extracellular pigment was isolated from the mangrove soils of Andhra Pradesh, India. A multifactorial and step-wise experiment was designed to study the physical and nutritional conditions that favor red pigment and biomass production. Potato extract prepared in the laboratory produced more pigment than the commercial potato dextrose broth and was therefore used as the basal medium. Culture conditions such as xylose 2% (w/v), glycine 1% (w/v), pH and temperature of 3.0 and 25 ˚C, were observed to be the optimal conditions producing 1050 mg/L of red pigment and 3.1 g/L of mycelia biomass. At pH 2.0, yellow fluorescent pigment was observed instead of red and spores were completely absent. Pigment was not produced when basic amino acids like arginine or lysine were supplemented to the medium, but acidic amino acids such as aspartic acid and glutamic acid enhanced pigment production. Also simple amino acids such as glucose maximized the growth of fungus whereas, amino acids such as xylose, mannose and glycine stimulated pigment yield. Therefore, this study demonstrates the significance of carbon-nitrogen combination in pigment production. Several other interesting observations from this study have been discussed in this paper. Keywords: Culture conditions, mangroves, mycelia biomass, optimization, Penicillium and pigment yield. INTRODUCTION There is a great demand for pigments in food industry; pigments are used as additives, color intensifiers and antioxidants. The use of synthetic colors in food industry is controversial because of their potential negative impacts on the consumer health [10]. This has generated a strong interest in the production of natural coloring alternatives; nature is rich in pigment producing microorganisms including fungi, yeasts and bacteria. Microbial pigments are more stable and soluble than pigments from plant and animal sources [13] and can be grown rapidly, and obtained throughout the year without limitations of seasonal conditions [17]. Carotenoids, melanins, flavins, quinones, monascins, and violacein or indigo are common microbial pigments that are industrially significant [10]. Microorganisms from the genus Aspergillus and Penicillium are potential producers of natural pigments [15, 22]. Pigments in fungi are not essential for their growth and development and are therefore, classified as secondary metabolites with wide applications. They have been derived from the products of primary metabolism and have been used as anti-fungal, anti-bacterial or anti-tumor agents [25]. Some pigments play a significant role in resistance of their spores to ultraviolet radiation and other harmful environmental conditions. In our previous studies, we reported the isolation of different pigment-producing mangrove fungi from Godavari delta, India [20]. The objective of our study is to improve the culture conditions of the mangrove Penicillium for its red pigment. This study has been performed as fungal pigments are considered to be industrially significant and isolation of novel microbial strains with improved pigment characteristics is highly desirable. Fungal pigment has been extracted and the structure was elucidated, but in this paper we concentrate on describing the culture conditions of the fungus with some interesting observations recorded during the study. MATERIALS AND METHODS Microorganism and inoculum preparation Mangrove fungi were isolated from mangrove sediments of Godavari delta, Andhra Pradesh, India and identified [2]. Fungal identification was performed at the Department of Botany, Osmania University, Hyderabad, India [20]. A fungal isolate (DLR-7) producing deep red pigment, identified as Penicillium sp. was used in this study. Stock cultures of Penicillium were maintained on potato dextrose agar slants prepared with 50% aged seawater, transferred every month and stored at 4 ˚C until needed. Inoculum for the culture studies was prepared by growing the fungus initially at 25 ˚C on potato dextrose agar (PDA) plates for 7 days. Plates having uniform growth and sporulation were selected and a 0.7 cm 2 plug from the outer zone of the colony was punched with a sterile cutter. The plugs were transferred to 100 ml of medium in 250 ml flasks and incubated under static conditions at 25 ˚C until maximum pigment was produced [13]. Media preparation All the media used in this study were procured from (Hi-media Laboratories Pvt. Ltd., Mumbai, India). Chemicals and organic solvents used were of analytical grade and obtained from Qualignes Fine Chemicals Pvt. Ltd., (Mumbai, India). Natural substrates like potatoes, sweet potatoes, yam, sago palm and banana tuber used in this study were purchased from the local market. For natural media preparation, 200 g of each substrate were cleaned and weighed separately. Substrates were sliced, added to five different beakers with 500 ml of deionized water, and cooked for 30 min. The cooked tubers were mashed and the liquid was filtered through a muslin cloth [3], 500 ml of seawater was then added to the medium and autoclaved. Synthetic media (potato dextrose broth, Sabourd’s Lathadevi Karuna Chintapenta et.al 2 dextrose broth, and Czapek Dox broth) were prepared according to the manufacturer’s instructions (Hi-media Laboratories). Further culture conditions were studied using carbon sources (fructose, glucose, lactose, maltose, mannose, ribose, starch, sucrose, and xylose), inorganic nitrogen (ammonium sulfate and ammonium nitrate) and amino acids (alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, lysine, phenyl alanine, tryptophan, and tyrosine). All optimization media were prepared with 50% aged seawater because the fungus was isolated from a marine environment. Analytical methods Fungal cultures were harvested after incubation and passed through filter paper (No. 1; Whatman, India Liasion Office, Mumbai, India), and mycelia were washed with deionized water. Yield of mycelia biomass was calculated by drying at 50 ˚C for 48 h [7]. The red pigment was extracted with chloroform and the solvent was removed using a rotary vacuum evaporator. Red pigment was analyzed by scanning its absorbance between 400 to 600 nm using a UV-visible spectrophotometer (model 117, Systronics, India). Maximum absorbance was determined at 510 nm; 1 mg of the pigment gave an optical density (OD) of 1.81. Thus OD values obtained during this study were converted to weight of the pigment in mg/L. Pigment production was monitored every 48 h under aseptic conditions. At each monitoring interval 3 ml of sample was carefully removed from the flasks and centrifuged at 11,200 g for 5 min to remove suspended cells. The supernatant was filtered using a 0.45 µm sterile membrane and its absorbance was measured at 510 nm [22]. EXPERIMENTAL DESIGN Optimization of biomass and pigment production from mangrove Penicillium sp. was designed in a step-wise manner (Fig 1), and statistically analyzed using the linear regression for contrasts. Five factors or variables were optimized at different levels. The factors studied were basal media selection, pH, carbon, nitrogen, and incubation time. Basal medium was selected from five different natural sources (banana tuber, potatoes, sago palm, sweet potato, and yam) and three synthetic media (Potato dextrose broth, Czapek Dox broth, and Sabourd’s dextrose broth). Media which produced more pigment was selected as the optimal basal medium and further checked during pH studies. Basal medium with optimized pH was supplemented with different carbon sources. Medium with a selective carbon source producing high pigment yield was supplemented with different nitrogen sources. Finally, incubation time for pigment yield and fungal growth was studied using the optimized medium. All experiments were conducted in stationary flasks incubated at 25 ˚C, performed in triplicates, and monitored every 48 h. Statistical analysis Data analysis for the treatments (basal media, pH, carbon, and nitrogen) was performed sequentially to determine conditions for maximum growth and pigment productivity. Stepwise linear regression analysis for contrasts was performed using R version 2.15.3 for Statistical Computing (http://wwww.r-project.org/). Linear regression function “lm ()” in R was used to determine if there was significant difference between the treatments using {lm (formula = y ~ x)}. Statistically significant (p < 0.05) factors were considered to be the optimal conditions for growth and pigment production. Regression coefficients and p values of significant treatment comparisons for growth and pigment were presented (Table 1 and Table 2). RESULTS Basal medium is the medium that supports the growth of major number of microorganisms. Potatoes, yam, banana tuber, and other natural substrates used for media preparation are comparatively inexpensive and readily available compared to synthetic media. Basal medium selection was performed to determine if natural media supports fungal growth equal to or more than synthetic media. Media which supports maximum fungal growth and pigment formation was used as the basal media for the entire experiment. Growth of Penicillium was greater in potato dextrose broth (PDB), Sabourd’s dextrose broth (SDB), and natural potato media. Regression analysis between natural and synthetic media shows that fungal biomass was greater in synthetic media, and particularly in SDB. But regression coefficients showed that there was no significant difference between the treatments since p = 0.124, which is > 0.05. Growth and pigment were not detected in natural media prepared with sweet potato and banana tuber. Pigment yield in natural potato medium and synthetic PDB was 450 mg/L and 360 mg/L respectively. Results from statistical analysis shows that natural potato medium is highly significant for pigment yield (p < 0.001) when compared to other treatments. Other natural and synthetic media produced 80 to 100 mg/L of pigment. This experiment shows that natural potato medium was good for pigment yield and growth of Penicillium sp. and further treatment studies were performed using natural potato medium as the basal medium. Effect of pH on pigment production Basal media were cultivated with Penicillium sp. at different initial maximum pH values (2.0 to 9.0) in stationary flask cultures and incubated at 25 ˚C. The optimal pH recorded for mycelial biomass and pigment was 3.0, pigment yield increased to 500 mg/L and growth was 1.8 g/L. Linear regression analysis confirms pH 3.0 as optimum for growth and pigment production, p value < 2 x 10-16 was recorded for the difference between the treatments. At pH 2.0 yellow pigment with maximum absorption at 420 nm was detected, spores were absent at this pH and growth was minimum. Dighton et al [9] demonstrated that microbial cells can only grow within a certain pH range affecting metabolite formation and their stability. Basal media adjusted to pH 3.0 was used for further optimization experiments. Effect of carbon source To study a suitable carbon source for mycelial growth and pigment, Penicillium sp. was cultivated in basal medium containing various carbon sources at 2% (w/v) concentration [13]. Basal media without a carbon source was used as a control and cultivated with the carbon treatments. Multiple comparison analysis was performed on all possible pairs of carbon sources to see if the difference between the treatment pairs was significant. Mycelial biomass was greater in media with glucose, starch, ribose, fructose, maltose and mannose when compared to the control. Maximum growth of 2.8 g/L was recorded in glucose medium and is highly significant (p = 2.6 x 10-14). Alexandra et al. [1] observed that fungi will grow rapidly using glucose and then enter a slower growing phase favoring secondary metabolism whereas pentose sugars like xylose do not favor growth and are thereby used for metabolite production. Pigment yield of 850 mg/L, 752 mg/L and 696 mg/L was detected in media with xylose, mannose and starch, respectively. According to Griffin [12], slowly metabolized compounds, like starch and sucrose, stimulate the production of secondary products when added to the culture medium. Comparison analysis performed between mannose and xylose, mannose and starch, xylose and starch, control and xylose and other 3 Journal of Multidisciplinary Scientific Research , 2014,2(3):01-05 media shows that xylose produces maximum pigment. The results are highly significant because p values for these comparisons are ~ 2.59 x 10-14 (Table 2). As xylose, mannose, and starch produce more pigment, they were supplemented with various nitrogen sources to study the combined effect of carbon and nitrogen on growth and pigment. The combined effect of carbon and nitrogen sources Microorganism growth and metabolite production are influenced by the organism’s utilization of different nitrogen sources during fermentation. Ammonium sulfate and ammonium nitrate at 0.5% (w/v) were also used as inorganic nitrogen sources [4] and various amino acids at 1% (w/v) were added to the basal media containing xylose, sucrose and starch. Basal medium without a nitrogen source served as a control. In this experiment Penicillium growth was high in xylose + alanine media (3.12 g/L) and pigment yield was high in glycine + xylose (1030 mg/L), mannose + alanine (910 mg/L), glycine + starch (900 mg/L), glutamic acid + xylose (900 mg/L) and aspartic acid + xylose (870 mg/L) media, respectively. Pigment was not detected in media containing ammonium sulphate and ammonium nitrate but biomass was 3 g/L and 2.85 g/L respectively when used as nitrogen sources. Pigment was also not detected in media with combinations of arginine and lysine whereas a light yellowish pigment was recorded in the presence of tryptophan, histidine and tyrosine. Various pigment derivatives with color range of orange-red to violet-red were produced by Monascus in the presence of different amino acids [4]. Multiple comparison analysis demonstrates that pigment yield varies with the nature of carbon and nitrogen source in the medium. When mannose and glycine are supplemented together, pigment yield is more and if histidine replaces glycine the pigment yield decreased. Similarly, xylose and glycine proved to be the best combination for pigment by Penicillium sp. but if glycine was replaced with alanine or lysine etc. pigment production decreased. These observations are highly significant and p value for these comparisons is 0. Therefore, this demonstrates that carbon and nitrogen sources affect growth and pigment yield differently depending on their combination in the culture medium and efficient selection is very critical for significant pigment yield. Determination of incubation period Biomass and pigment yield of Penicillium varies with the type of culture medium and incubation time. In order to study optimum incubation time, the optimized medium was inoculated with the fungus; optical density of the medium was recorded every day after the start of pigment production. The optimized medium designed from the results was a basal medium supplemented with xylose 2% (w/v), glycine 1% (w/v), pH adjusted to 3.0 and incubated at 25 ˚C. Basal medium with optimum carbon source (xylose) alone, started pigment production on 3rd day and was maximum (830 mg/L) on the 15th day. In media containing carbon and nitrogen sources (xylose + glycine), pigment started on the 3rd day and was maximum (1050 mg/L) on the 12th day, whereas in the control (basal medium), pigment started on the 6th day and reached a maximum (490 mg/L) on the 21st day (Fig 2). With all optimum nutrients in the medium, incubation time significantly decreased and pigment increased. At the end of incubation pigment concentration remained constant for 8 to 10 days before it slowly started to decrease. Similar results were observed in Fusarium solani [24]. DISCUSSION In this study, pigment yield in optimized medium doubled when compared to commercial potato dextrose broth. Our results confirm that pigment increased dramatically with the addition of nitrogen sources and similar findings were reported [7]. This study also demonstrates that carbon-nitrogen combination is important for metabolite production. When amino acids like lysine and histidine are supplemented with optimum carbon source, the carbon source failed to show a positive effect on pigment yield. Inorganic nitrogen salts supported maximum growth (3 g/L) of the fungus but they did not stimulate pigment production. Among the culture conditions studied, maximum pigment (1050 mg/L) was observed in xylose + glycine medium and growth was high (3.12 g/L) in xylose + alanine and glucose + alanine medium. These results confirm that growth and pigmentation are two independent phenomena operating within the microbial cell, controlled by environmental and physical characteristics. Similar observations were recorded [23]. From these results we also demonstrate that pigment production is proportional to the complexity of medium constituents. We emphasize that basal medium (potato extract) may serve the purpose for fungal growth while xylose and glycine may be utilized slowly for pigment production. This study proves that the potential of a fungus for pigment production can be increased significantly by altering the culture conditions. More detailed experiments are needed to make this industrially significant. This study also shows the feasibility of mangrove Penicillium in producing natural pigments and the possible use of this red pigment in the food industry. More detailed experiments for toxicity tests and pigment production will improve its potential. Acknowledgments We would like to thank Mr. Vivekananda Reddy, DLR College, Gollala Mamidada, India, for the financial support and use of research facilities. 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Omura S (1992) The search for bioactive compounds from microorganisms. Springer Verlag, New York. Table (1). Comparative analysis of significant treatments affecting growth of Penicillium sp. *: indicates comparison; +: indicates that both components are included in the media; P > 0.05 is not significant for the contrasts in treatments. 5 Pigment yield mg/L Table (2). Comparative analysis of significant treatments effecting pigment yield in Penicillium sp. 1200 3.5 1000 3 800 2.5 2 600 1.5 400 1 200 0.5 0 0 Mycelia biomas s g/L Journal of Multidisciplinary Scientific Research , 2014,2(3):01-05 Treatments Fig( 2). Effect of significant parameters on mycelia growth and pigment yield from Penicillium. sp. ♦ (diamond) indicates mycelia growth (g/L); (bars) indicate pigment yield (mg/L). *: indicates comparison; +: indicates that both components are included in the media; P > 0.05 is not significant for the contrast in treatments Fig (1). Experimental design for optimizing the conditions for growth and pigment yield of Penicillium (PDB-potato dextrose broth; CDB- Czapek Dox broth; SDBSabourd’s dextrose broth, text in bold indicates maximum pigment production).