INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 4, Copyright 2010 All rights reserved Integrated Publishing services

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1 Optimization of the medium for the production of cellulase by the Trichoderma viride using submerged fermentation Gautam S.P. 1, Bundela P.S. 2, Pandey A.K. 3, Jamaluddin 4, Awasthi M.K. 2, Sarsaiya S. 2 1 Central Pollution Control Board, New Delhi, India. 2 Regional office, M.P. Pollution Control Board, Vijay Nagar, Jabalpur (M.P.), India 3 Mycological Research Laboratory, Department of Biological Sciences, Rani Durgavati University, Jabalpur (M.P.), India. 4 Yeast and Mycorrhiza Biotechnology Laboratory, Department of Biological Sciences, Rani Durgavati University, Jabalpur (M.P.), India. surendra_sarsaiya@yahoo.co.in ABSTRACT The main purpose of this study to reduced the production cost of cellulase by using alternative carbon source such as municipal solid waste residue and optimized fermentation medium for high yielding. In the present investigation, to isolate the novel cellulase producing fungi, Trichoderma viride from municipal solid waste and to optimize the physicochemical and nutritional parameters for cellulase production. Municipal solid waste residue and yeast extract were found to be the best combination of carbon and nitrogen source for the production of cellulase by Trichoderma sp. Optimal concentration of municipal solid waste residue and yeast extract for the production of cellulase were 4 5% (w/v) and 1.0% (w/v), respectively. Optimum temperature and ph of the medium for the cellulase production by Trichoderma viride was 45 C and 6.5. Cellulase production from Trichoderma viride can be an advantage as the enzyme production rate is normally higher as compared to other fungi. Keywords: Fermentation, optimization, biodegradation, cellulase, municipal solid waste 1. Introduction Globally the estimated quantity of the wastes generation was 12 billion tones in a year 2002 of which 11 billion tones were industrial wastes and 1.6 billion tones were municipal solid waste. About 90 billion tones solid wastes are expected to be generated annually by the year Annually, Asia alone generates 4.4 billion tones of solid wastes and municipal solid waste comprises 790 million tones of which about 48 million tones are generated in India. Unscientific disposal of waste causes an adverse impact on all components of the environment and human health (Lee et al., 2007). Agricultural waste is also caused environmental pollution. Their conversion into useful products may ameliorate the problems they cause. Municipal solid waste (MSW) is composed of 40 50% cellulose, 9 12% hemicellulose and 10 15% lignin on a dry weight basis. Complete cellulose hydrolysis to glucose demands the action of exoglucanases (also cellobiohydrolyses), endoglucanases and β glucosidases. Exoglucanases (1,4 β D glucan cellobio hydrolase, EC ) are usually active on crystalline cellulose and are lacking from incomplete cellulase systems. Endogluconases (1,4 β D glucan 4 glucanohydrolase, EC ) are more active against the amorphous regions of cellulose and they can also hydrolyse substituted celluloses, such as carboxymethylcellulose (CMC) and hydrxyethyl cellulose (HEC). Cellobiohydrolases cleave disaccharide (cellobiose) units either from non reducing or reducing ends, whereas 656

2 endoglucanases hydrolyse the cellulose chain internally. β glucosidase (EC ) are needed to cleave cellobiose and other soluble oligosaccharides to glucose (Beguin, 1990). In the recent years, one of the most important biotechnological applications is the conversion of agricultural wastes and all lignocellulosics into products of commercial interest such as ethanol, glucose and single cell products (Ojumu et al., 2003). The key element in bioconversion process of lignocellulosics to these useful products is the hydrolytic enzymes mainly cellulases (Ojumu et al., 2003; Fan et al., 1987; Immanuel et al., 2007). The bioconversions of cellulosic materials are now a subject of intensive research as a contribution to the development of a large scale conversion process beneficial to mankind. Such process would help alleviate shortages of food and animal feeds, solve modern waste disposal problem and diminish man s dependence on fossil fuels by providing a convenient and renewable source of energy in the form of glucose. A diverse spectrum of cellulolytic microorganism mainly fungi (Falcon et al., 1995) and Bacteria (McCarthy, 1987) have been isolated and identified over the years and this still continue to grow rapidly. One of the most extensively studied fungi is Trichoderma reesei, which converts native as well as derived cellulose to glucose. Most commonly studied cellulolytic organism including fungal species Trichoderma sp., Humicola sp., Penicillium sp. and Aspergillus sp. In this investigation, a cellulase producing strain of Trichoderma viride, isolated from municipal solid waste, were subjected to optimization of media and cultivation parameters for cellulase production. 2. Materials and Methods 2.1. Materials Municipal solid waste was collected from different dumping sites of Jabalpur (M.P.), India. Fungal culture of Trichoderma viride was isolated from composted municipal solid waste in June It was grown at 30± 0 C, maintained on potato dextrose agar containing potato 2.5% (w/v), dextrose 2.0% and agar 1.5 % at 4±2 0 C and then identified by a previous study (Barnett and Hunter 1972) Enzyme production The culture was grown in 150 ml Erlenmeyer flask that contained 50 ml of basal salt medium. The ph of the medium was adjusted to 6.5 prior to sterilization. The flask were inoculated with 2 agar discs (2 mm in diameter) of 7 days old culture from PDA plates and were incubated under stationary condition at 40±2 0 C upto 7 days. The crude enzyme were filtered and centrifuged at x g for 10 min Enzyme assay Filter paper activity (FPase) for total cellulase activity in the culture filtrate was determined according to the standard method (Hankin and Anagnostakis 1975). Aliquots of appropriately diluted cultured filtrate as enzyme source was added to whatman no. 1 filter paper strip (1 x 6 cm; 50 mg) immersed in one milliliter of 0.05 M Sodium citrate buffer of ph 5.0. After incubation at 50±2 0 C for 1 hrs, the reducing sugar released was estimated by dinitrosalicylic acid (DNS) method (Miller 1959). One unit of filter paper (FPU) activity was defined as the 657

3 amount of enzyme releasing 1 µmole of reducing sugar from filter paper per ml per min. Endoglucanase activity (CMCase) was measured using a reaction mixture containing 1 ml of 1% carboxymethyl cellulose (CMC) in 0.5 M citrate acetate buffer (ph 5.0) and aliquots of suitably diluted filtrate. The reaction mixture was incubated at 50±2 0 C for 1 h and the reducing sugar produced was determined by DNS method. β glucosidase activity was assayed by the method of Pointing (1999). One unit (IU) of endoglucanase activity was defined as the amount of enzyme releasing 1 µmole of reducing sugar per min Optimization of culture conditions for enzyme production Effect of incubation period in enzyme production Fermentation period is important parameter for enzyme production by A. niger. In this study, experiments fermentation was carried out up to 7 days and production rate measured at 24 h intervals Effect of ph and temperature on enzyme production The most suitable ph of the fermentation medium was determined by adjusting the ph of the culture medium at different levels in the range of ph 3 to 9 using different buffers. In order to determine the effective temperature for cellulase production by the A. niger, fermentation was carried out at 10 0 C intervals in the range of 20 to 80±2 0 C Effect of carbon sources on enzyme production Effect of various carbon compounds viz., cellulase, CMC, glucose, sucrose and maltose were used for studying. The broth was distributed into different flasks and 0.5 to 3.0 % of each carbon sources were then added before inoculation of the strain and after culture inoculation, the flasks were incubated for 7 days at 45±2 0 C Effect of nitrogen sources on enzyme production In the present study, to detect the appropriate nitrogen source for cellulase production by the Trichoderma viride. The influence of peptone, beef extract, ammonium nitrate, sodium nitrate and yeast extract were studied. The fermentation medium was supplemented with organic and inorganic compounds at 0.5 to 3.0% level, replacing the prescribed nitrogen source of the fermentation medium Effect of cations on enzyme production Effect of cations on cellulase activity were determined by using different cations (Na +, K +, Ca ++, Mg ++ & EDTA). The broth was distributed into different flask and 10 mm to 80 mm of each cations were then added and after culture inoculation, the flask were incubated at 45±2 0 C for 7 days. 658

4 Effect of municipal solid waste residue in enzyme production In the present study, to detect the appropriate municipal solid waste residue for cellulase production by Trichoderma viride. The fermentation medium was supplemented with municipal solid waste residue at 1.0 to 6.0 % level, replacing the prescribed carbon source of the fermentation medium Statistical analysis Data presented on the average of three replicates (±SE) obtained from there independent experiments. 3. Results and Discussion 3.1. Effect of incubation period on enzyme production The incubation period is directly related with the production of enzyme and other metabolic upto a certain extent. Trichoderma sp. showed the most active cellulolytic species along different incubation period. The incubation period to achieve peak cellulase activity by the isolate Trichoderma sp. was 6 th days (Figure 3) which suitable for commercial point of view (Kang et al., 2004). It might be due to the depletion of nutrients in the medium which stressed the fungal physiology resulting in the inactivation of secretary machinery of the enzymes (Nochure et al., 1993) Effect of ph on enzyme production Cellulase yield by Trichoderma viride appear to depend on ph value. Results illustrated by Figure 1 clearly show that cellulase production, expressed as enzyme activity, gradually increased as the ph value increased from 5 6 and reached its maximum at ph of 5.5 being exoglucanase (2.16 U/ml), endoglucanase (1.94 U/ml) and β glucosidase (1.71 U/ml). The enzyme activity gradually increased when increasing the ph up to the optimum followed by a gradual full in activity. It was also noted that the enzyme activity was stable at ph range of Effect of ph on cellulase production by these fungi supports the findings of Lee et al. (2002) who reported that CMCase, Avicelase and FPase activities exhibit a ph optimum of approximately 4, while the ph optimum of β glucosidase was between ph 5 and Effect of temperature on enzyme production Like ph, temperature is also an important factor that influences the cellulase yield. Maximum enzyme production by Trichoderma viride was found to be exoglucanase (1.95 U/ml) and endoglucanase activity (1.88 U/ml) and β glucosidase (1.88 U/ml) activities between C (Figure 2). Many workers have reported different temperatures for maximum cellulase production either in flask or in fermentor studies using Trichoderma sp. suggesting that the optimal temperature for cellulase production also depends on the strain variation of the microorganism (Murao et al., 1988, Lu et al., 2003) Effect of carbon sources on enzyme production 659

5 Data presented in table 1 shown that cellulase production by Trichoderma viride was significantly influenced by the type of carbon source in the basal salt medium. Sucrose was the most effective as a sole carbon source for cellulase enzyme production, results increased in enzyme activity, being exoglucanase (2.68 U/ml), endoglucanase (2.17 U/ml) and β glucosidase (2.06 U/ml) were obtained in culture medium containing 1.0% Sucrose followed by glucose, cellulose maltose and CMC. Cellulase production increased with increases in initial sugar concentration from 1.0 to 1.5% while further increases in sugar concentration slightly reduced the yield. Mendals and Reese (1957) also reported that maximum yields of cellulase were obtained on 1% different carbon substrate using T. viride. Cellulase production commended on reaching nitrogen limiting conditions and the yield of cellulase decreased when excess peptone was presented, various inorganic nitrogen sources have been optimized by different workers for cellulase production (Sherief et al. 2010; Solomon et al. 1997; Lee et al. 2010). Table 1: Effect of carbon sources on cellulases production (U/ml) by Trichoderma viride Different carbon sources Glucose Maltose ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.03 Different carbon sources Cellulose Sucrose ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.04 Different carbon sources Carboxymethyl cellulose ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6 Table 2: Effect of nitrogen sources on cellulases production (U/ml) by Trichoderma viride Different nitrogen sources Peptone Yeast extract ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.05 Different nitrogen sources Ammonium nitrate Sodium nitrate ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.03 Table 3. Effect of cations (mm) on cellulases production (U/ml) by Trichoderma viride Different nitrogen sources Beef extract ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.05 Different cation sources (mm) Na + K ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7 Table 3. Continued. Different cation sources (mm) Ca ++ EDTA ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.03 (mm) Different cation sources Mg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Effect of nitrogen sources on enzyme production To evaluate the effect of nitrogen source on cellulase formation, the nitrogen sources in the basal medium was replaced by different nitrogen sources. Data revealed that the supplementation of organic and inorganic nitrogen sources (ranged from 0.5 to 3.0 % (w/v) peptone, beef extract, yeast extract, ammonium nitrate and sodium nitrate) stimulated the cellulase yield and activity. The maximum enzyme activities were obtained with yeast extract (1.0%) which brought about an improvement in all the three cellulase components, including exoglucanase (2.40 U/ml), endoglucanase (2.28 U/ml) and β glucosidase (1.99 U/ml). where peptone also produce second most cellulase producing nitrogen source by Trichoderma sp. (table 2) It was reported that good cellulase yield can be obtained with ammonium compound as the nitrogen source. Though the addition of organic nitrogen sources such as beef extract and peptone resulted in increased growth and enzyme production, was reported before, they were not an effective replacement for inorganic nitrogen sources because of their higher cost (Sun et al., 1999) Effect of cations on enzyme production In the presented study, it was found that all of these cations leaded to reduction in enzyme activity with increase the concentration (10 mm to 80 mm). Most ions such as Na + and K + 662

8 did not significantly reduce the expression of maximum activity. It was found that the addition of 10 mm of Ca ++ in the production medium was requirement for the expression of full enzymatic activity (table 3). EDTA has been cause reduction of enzyme activity with increase the concentration in the production medium. Mg ++ was also strongly affecting the cellulase production by Trichoderma viride when the concentration was increased. Enzymatic activity (U /ml) ph Figure 1 Effect of ph on cellulase production (U/ml) by Trichoderma viride Enzym atic activity (U /ml) Incubation period E nzym atic activity (U /m l) Temperature Figure 2 Effect of temperature on cellulase production (U/ml) by Trichoderma viride E nzym atic activity (U /m l) MSW residue Figure 3 Effect of incubation period on cellulase production (U/ml) by 3.7. Effect of municipal solid waste residue in enzyme production Figure 4 Effect of MSW residue on cellulase production (U/ml) by Trichoderma The results revealed (Figure 4) that MSW residue (5.0%) was the best carbon substrate for FPase, CMCase and β glucosidase by Trichoderma sp. 4.0% MSW residue was best for cellulase [exoglucanase (1.77 U/ml), endoglucanase (1.95 U/ml) and β glucosidase (1.66 U/ml)] enzyme production. these results were confirmed by the results of Abo State et al. (2010). This variation may be attributed to the chemical nature and nutrient availability of the used substrates. 4. ACKNOWLEDGEMENTS The authors are thankful to Madhya Pradesh Pollution Control Board Bhopal and Head, Department of Biological Sciences, R.D. University, Jabalpur, for laboratory facilities. Also 663

9 thanks to Municipal Corporation of Jabalpur for his support. Ministry of Environment and Forest, New Delhi is also thankfully acknowledged for financial support. 5. REFERENCES 1. Lee, Y.B., Lee, B., Jo, K., Lee, N., Chang, C., Lee, Y. and Lee, J. 2007, Purification and characterization of cellulase by Bacillus amyoliquefaciens DL 3, utilizing rice hull. Bioresource Technology, 98(2), pp Beguin, P., 1990, Molecular biology of cellulose degradation. Annual Review of Microbiology, 44, pp Ojumu, T.V., Solomon, B.O., Betiku, E., Layokun, S.K. and Amigun, B., 2003, Cellulase production by Aspergillus flavus Linn isolate NSPR 101 fermented in sawdust, bagasse and corncorb. African Journal Biotechnology, 2, pp Fan, L.T., Gharpuray, M.M., Lee, Y.H., 1987, Cellulose hydrolysis. Berlin, Germany. Springer Verlag, 3, pp Immanuel, G., Dhanusha, R., Prema, P. and Palavesam, A. (2006). Effect of different growth parameters on endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment. International Journal Environmental Science Technology, 3, pp Falcon, M.A., Rodriguez, A., Carnicero, A., 1995, Isolation of microorganism with lignin transformation potential from soil of Tenerife Island. Soil Biology Biochemistry, 27(2), pp McCarthy, A. J., 1987, Lignocellulose degrading Actinomycetes. FEMS Microbiology, 46, pp Barnett, H.L. and Hunter, B.B., 1972, Illustrated genera of imperfecti fungi. Burgess Publishing Co. 9. Hankin, L. and Anagnostakis, S.L., 1975, The use of solid media for detection of enzyme production by fungi. Mycologia, 47, pp Miller, G.L., 1959, Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chemistry, 31, pp Pointing, S.P., 1999, Qualitative methods for the determination of lignocellulolytic enzyme production by tropical fungi. Fungal Diversity, 2, pp Kang, S.W., Park, Y.S., Lee, J.S., Hong, S.I. and Kim, S.W., 2004, Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresource Technology, 91, pp

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