Screening of Rice Straw Degrading Microorganisms and Their Cellulase Activities

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1 Research 83 KKU Sci. J.37 (Supplement) (2009) Screening of Rice Straw Degrading Microorganisms and Their Cellulase Activities Abstract Atcha Boonmee 1,2* Rice straw is one of the most abundant agricultural by-products in Thailand. It generally contains approximately 39% cellulose, 27% hemicellulose and 12% lignin. Cellulose and hemicellulose, when hydrolyzed by chemical or enzymatic hydrolysis, are converted into glucose and other fermentable sugars for ethanol production. In this study, screening of cellulolytic microorganisms for degrading rice straw has been investigated. Twenty nine bacterial isolates and the 30 fungal isolates were selected for further study on their cellulolytic activity. All isolates were assayed for exoglucanase, endoglucanase and β-glucosidase specific activities, by which following substrates were used: filter paper (Whatman No.1), carboxymethyl cellulose and cellobiose, respectively. Specific activities of those enzymes were determined by measuring reducing sugar released from substrates. From the results, the following isolates showed the highest specific activity of each catagory of cellulases; FR14 for Filter paper cellulase (FPase) (0.032 unit/ mg protein), FR4 for CMCase (0.5 unit/ mg protein) and FC1 for cellobiase (0.6 unit/ mg protein). Two isolates showed nearly equal activitiy of CMCase and cellobiase. The isolate FR3 had 0.22 unit/ mg protein CMCase specific activity and 0.23 unit/ mg protein cellobiase specific activity, while the isolate FR18 showed CMCase specific activity with 0.25 unit/ mg protein and cellobiase specific activity with 0.30 unit/ mg protein. 1 Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand 2 Protein & Proteomics Research Group (PPRG), Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand atcha@kku.ac.th

2 84 Research Introduction Due to global warming problem and continuous decrease of fossil fuel source, alternative energy such as gasohol or bio-diesel has been increasingly consumed. Therefore, bio-ethanol serving as gasohol additive has been consecutively produced and improved as well. At present, bio-ethanol production is based on sugar (from sugar cane) or flour (from maize or cassava) as substrates. Biomass feedstock represents about 40% of bio-ethanol production cost (Hamelinck et al., 2005) and makes the price of ethanol produced from those substrates much higher than those comparing to fossil fuel. Therefore, reducing cost of ethanol production by using lignocellulosic materials such as bagasse, wood scraps, corncob or rice straw as an alternative feedstock becomes a very interesting topic for researchers nowadays. The choice of the best technology for lignocellulose to bio-ethanol conversion should be decided on the basis of overall economics (lowest cost), environment (lowest pollutants) and energy (higher efficiency), i.e. comprehensive process development and optimization are still required to make the process economically viable (Chandel et al., 2007). Rice straw is one of the most abundant agricultural by-products in Thailand. It generally contains approximately 39% cellulose, 27% hemi-cellulose and 12% lignin (Kaur et al., 1998). Cellulose and hemi-cellulose, when hydrolyzed by chemical or enzymatic hydrolysis, are converted by microorganisms into glucose and other fermentable sugars for ethanol production. Lignocellulose-degrading microorganisms such as bacteria, fungi, actinomycetes and protozoa generally exist in nature and yet remain to be discovered by researchers. The mechanism of enzymatic cellulose hydrolysis involves synergistic actions by endoglucanase, exoglucanase or cellobiohydrolase, and β-glucosidase. Endoglucanases hydrolyze accessible intramolecular β-1,4-glucosidic bonds of cellulose chains randomly to produce new chain ends; exoglucanases progressively cleave cellulose chains at their both ends to release soluble cellobiose or glucose; and β-glucosidases hydrolyze cellobiose to glucose in order to eliminate cellobiose inhibition. These three hydrolysis processes occur simultaneously as shown in Figure 1 (Zhang, 2006). Figure 1. Mechanistic scheme of enzymatic cellulose hydrolysis by Trichoderma non-complexed cellulase system (Zhang et al., 2006).

Research 85 In this study, screening of cellulase-producing microorganisms, considering various types of cellulases: endoglucanase, exoglucanase and β-glucosidase, was investigated in order to

Material and methods 1. Screening of cellulose-degrading bacteria Ten grams of each sample was diluted with 90 ml of 0.85% NaCl, resuspended well by using vortex mixer for 5 min.

1% cellulose agar or 2% rice straw agar and incubated 3-5 days at 37 C.

1 crude enzyme preparation One loop-full of each bacteria isolate was inoculated in 25 ml 0.1% cellulose broth, incubated 24 hours at 37 C, 150 rpm.

3 Research 85 In this study, screening of cellulase-producing microorganisms, considering various types of cellulases: endoglucanase, exoglucanase and β-glucosidase, was investigated in order to determine their potential use as agent to degrade rice straw hich contains almost 39% cellulose and to provide preliminary data for further bio-ethanol production from lignocellulosic materials. Material and methods 1. Screening of cellulose-degrading bacteria Ten grams of each sample was diluted with 90 ml of 0.85% NaCl, resuspended well by using vortex mixer for 5 min. Ten-fold dilution series were performed with the suspension and 3 appropriate dilutions were selected for the screening. From each selected dilution, 0.1 ml of the suspension were spread on NA, 0.1% cellulose agar or 2% rice straw agar and incubated 3-5 days at 37 C. Colonies of bacteria and fungi were collected, thereby bacteria were streaked on screening agar and fungi were stabbed in screening agar. 2. Assaying cellulase specific activities 2.1 crude enzyme preparation One loop-full of each bacteria isolate was inoculated in 25 ml 0.1% cellulose broth, incubated 24 hours at 37 C, 150 rpm. Cell suspension was measured by OD 600 and adjusted to OD 600 = 0.1, then 5 ml of the adjusted cell suspension were inoculated in 45 ml 0.1% cellulose broth and incubated for 24 hours at 37 C, 150 rpm. Ten milliliters of cell suspension were centrifuged for 10 minutes at 4 C, 5,000 rpm. Supernatant containing crude enzyme was used to assay enzyme specific activities. 2.2 cellulase specific activity assay Total protein amount of crude enzyme was determined by Lowry method (Lowry et al., 1951) and protein concentration (mg/ ml) was later used to calculate specific activity (unit/ mg protein). Crude enzymes were then tested for their abilities to hydrolyze the following substrates: 1 1 cm filter paper (Whatman No.1), 1% carboxymethyl cellulose (1% CMC) and 0.1% cellobiose. All of these substrates were resuspended in 0.2 M sodium acetate buffer ph 5. Each reaction was prepared in 2 sets for incubation at 30 C and 50 C by mixing 1 ml crude enzyme with 1 ml buffer, incubated for 1 hr when filter paper was used as substrate and for 10 minutes when CMC or cellobiose were used as substrates. Reducing sugar was measured by Somogyi-Nelson method (Nelson, 1944) (for filter paper and CMC) or DNS method (Miller, 1959) (for cellobiose). Enzyme activities were reported in unit/ ml of crude enzyme. Results 1. Screening of cellulose-producing microorganisms From eight samples of soils and waste water, 11 bacterial isolates (BC1-11) and 9 fungal isolates (FC1-9) were found on 0.1% cellulose agar; 19 bacterial isolates (BR1-19) and 20 fungal isolates (FR1-20) could grow on 2% rice straw agar containing pretreated rice straw as carbon and energy source. Thus, total number of isolates are 59, by which 30 of them are bacteria and 29 are fungi. Those isolates were then selected for further study on their cellulolytic activities. Figure 2 shows some of the isolates on screening agar. BC9 FC1 BR8 FR14 Figure 2. Some of the isolates grown on 0.1% cellulose agar (BC9 and FC1), or 2% rice straw agar (BR8 and FR14)

4 86 Research 2. Assaying cellulolytic activities with various substrates All isolates were assayed for exoglucanase, endoglucanase and β-glucosidase specific activity. The following substrates were used: filter paper (Whatman No.1), carboxymethyl cellulose, and cellobiose. Specific activities were determined by measuring reducing amount of sugar released from substrates. All reactions were performed at 30 C and 50 C Filter paper cellulose activity (FPase activity) In this work, we measured activity of enzyme which hydrolyzes filter paper. We referred to those enzymes as exoglucanase. Some isolates showed high FPase specific activity at low temperature (30 C) and some showed higher activity at high temperature (50 C). However, overall activities of their filter paper cellulases were still very low, where the average of specific activity found in bacteria isolates was unit/ mg protein and in fungi isolates was unit/ mg protein. The isolates with high specific activity of filter paper cellulase (FPase) are shown in Figure 3, which are the isolates FR14, BR15, BR11, FR9 and BR4. The isolate FR14 showed the highest FPase specific activity with unit/ mg protein. Specific activity (Unit/mg protein) FPase Specific activity of Top 5 Isolates FR14 BR15 BR11 FR9 BR4 Isolates FPase Figure 3. Summary of bacteria (B) or fungi (F) isolates with high specific activity of filter paper cellulase (FPase) The screenings were done on 0.1% cellulose agar (C) and 2% rice straw agar (R) at 30 C and 50 C. Isolates FR14 and BR4 showed high activity at 30 C, whereas BR15, BR11 and FR9 showed high activity at 50 C CMCase and cellobiase specific activity. The term carboxymethyl cellulase (CMCase) and cellobiase represent endoglucanase and β-glucosidase, respectively. After screening on 0.1% cellulose agar and 2% rice straw agar at various temperatures, some isolates showed high cellulase specific activity at low temperature (30 C) and some showed higher activity at high temperature (50 C). Moreover, bacteria isolates tended to produce more cellulose enzymes hydrolyzing than carboxymethyl cellulose hydrolyzing enzymes (CMC), whereas fungal isolates were able to produce various enzymes comparing to bacterial isolates. Some fungal isolates produced either CMCase or cellobiase, when some could produce both enzymes at the same level. The average of CMCase and cellobiase specific activity found in bacterial and fungi isolates were 0.06 and unit/ mg protein, respectively. Isolates with high specific activity of CMCase and cellobiase are shown in Figure 4 and 5, respectively. The isolates with high CMCase specific activity are FR4, FR19, BC9, FR14, FR18 and FR3, where the isolate FR4 showed the highest CMCase specific activity with 0.5 unit/ mg protein. The isolates with high cellobiase specific activity are FC1, FC2, BR8, FC9 and BR14, where FC1 showed

5 Research 87 the highest cellobiase specific activity with 0.6 unit/ mg protein. Furthermore, several fungi isolates moderately produced both enzymes. For example, FR18 had CMCase and cellobiase specific activity of 0.25 unit/ mg protein and 0.30 unit/ mg protein, respectively and FR3 had CMCase and cellobiase specific activity of 0.22 unit/ mg protein and 0.23 unit/ mg protein, respectively. Specific activity (Unit/mg protein) Specific activity (Unit/mg protein) CMCase Specific activity of Top 6 Isolates FR4 FR19 BC9 FR14 FR18 FR3 Isolates CMCase Cellobiase Figure 4. Summary of bacteria (B) or fungi (F) isolates with high specific activity of carboxymethyl cellulase. The screening were done on 0.1% cellulose agar (C) and 2% rice straw agar (R) at 30 C and 50 C. All isolates shown here performed their high activity at 50 C Cellobiase Specific activity of Top 5 Isolates Figure 5. Summary of bacteria (B) or fungi (F) isolates with high specific activity of cellobiase. The screenings were done on 0.1% cellulose agar (C) and 2% rice straw agar (R) at and 50 C. Isolates FC1, FC2, BR8, FC9 showed their high activity at 30 C, whereas BR14 showed its high activity at 50 C FC1 FC2 BR8 FC9 BR14 Isolates CMCase Cellobiase 0.40

6 88 Research Discussion The application of cellulolytic enzymes in degrading lignocellulosic materials needs synergistic actions of various types of cellulases. Thus, there are 2 feasible ways of making use of the enzymes from the isolates found in this study. The first way is to select the isolates with high specific activity of individual enzymes and use them as mix cultures. The other way is using only one isolate which is able to produce various types of enzymes, where the problem of optimal growth conditions of each isolates by co-fermentation could be avoided. Specific activities of cellulases assayed in this research are still very low comparing to others which have been reported. However, cellulase production from these isolates has to be further optimized under different conditions in order to enhance cellulose degradation and consequently make them useful for further applications. Conclusion Screening of cellulolytic microorganisms for degrading rice straw was investigated. The 29 isolated bacteria and the 30 isolated fungi were studied on their cellulolytic activities. The isolate FR14 showed the highest Filter paper cellulase (FPase) specific activity with unit/ mg protein, while FR4 had the highest CMCase specific activity with 0.5 unit/ mg protein. The highest cellobiase activity was shown by the isolate FC1 with 0.6 unit/ mg protein. Cellulase production from these isolates has to be further optimized under different conditions. Acknowledgment This research has been supported by the Faculty of Science and the Protein & Proteomics Research Group (PPRG), Faculty of Science, Khon Kaen University, Khon Kaen, Thailand. The author also thanks S. Saraihorm for her excellent assistance. References Chandel, A. K., Chan, ES., Rudravaram, R., Narasu, M. L., Venkateswar Rao, L. and Ravindra, P. (2007). Economics and environmental impact of bioethanol production technologies: an appraisal. Biotechnol Mol Biol Rev. 2 (1): Hamelinck, C. N. Hooijdonk, G. V. and Faaij, A. PC. (2005). Ethanol from lignocellulosic biomass: techno-economic performance in short-, mid dle-, and long-term. Biomass Bioenergy. 22: Kaur, P. P. Arneja, J. S. and Singh, J. (1997). Enzymatic Hydrolysis of Rice Straw by Crude Cellulase from Trichoderma reesei. Bioresorce Technology Lowry, O. H. (1951). Protein Measurement with the Folin Phenol Reagent. J.Boil. Chem. 193: 265. Miller, G. L., Blum, R., Glennon, W. E. and Berton A. L. (1959). Measurement of carboxymethyl cellulase activity. Analytical Biochemistry 1(2): Nelson, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem. 153: Zhang, Y-HP. (2006). Outlook for cellulose improvement : Screening and selection strategies. Biotech nology Advances. 24:

Saccharification of corncob using cellulolytic bacteria - Titi Candra Sunarti et al.

Saccharification of corncob using cellulolytic bacteria - Titi Candra Sunarti et al. Figure 2. (a) (b) (c) (d) Microscopic structures of (a) corncob, (b) delignified corncob, (c) cellulose fraction, (d)