MINISTRY OF EDUCATION and TRAINING CAN t

MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY STUDYING ON CELLULASE PRODUCTION BY ANAEROBIC BACTERIA ON PAPER POWDER SUBSTRATE SUPERVISORS STUDENT MSc DUONG THI HUONG GIANG MSc VO VAN SONG TOAN TRAN NON NUOC Student code: 3064407 Session: 32 (2006-2010) Can Tho, 2010 APPROVAL SUPERVISORS MSc DUONG THI HUONG GIANG STUDENT TRAN NON NUOC MSc VO VAN SONG TOAN Can Tho, November, 2010 PRESIDENT OF EXAMINATION COMMITTEE ABSTRACT Optimal selection of strain, fermentation conditions and substrate, which are very important for the successful production of cellulase by bacteria, were investigated in this study A total of 37 anaerobic bacterial isolates collected from soil, rumen cow, bagasse, rice husk was screened on modified Delafield medium, in which Whatman filter paper powder was used as substrate The result showed that the isolate VK52 adopted highest cellulase productivity among the others, illustrated by the largest haloes in diameter (20.5 mm) on Congo red-containing medium The optimal conditions for cellulase production by VK52 were determined as 300C, pH and 4day-incubation on the medium supplemented with 0.4 % yeast extract On laboratory scale the yield of endoglucanase and exoglucanase activities were 0.107 U/ml and 0.06 U/ml respectively, and 9.025% of substrate degradation There was a decrease of culture pH from to 6.89 after cultivation Paper and cellulose powder were the best substrates for cellulase production by the isolate VK52 in comparision with rice straw, sugarcane bagasse and rice husk Key words: anaerobic bacterial isolates, cellulase, filter paper powder, isolate VK52 i CONTENTS Abstract i Content ii Introduction Materials and methods 3 Results and discussion 3.1 Screening of cellulase activity produced by anaerobic bacterial isolates 3.2 Effect of temperature and pH on cellulase production by VK52 3.3 Effect of yeast extract concentration on cellulase 10 production by VK52 3.4 Effect of incubation time on cellulase production by 12 VK52 3.5 Production of cellulases at laboratory scale by VK52 14 under optimal conditions 3.6 Effect of carbon sources on cellulase production by 15 VK52 Conclusion and Sugestion 18 4.1 Conclusion 18 4.2 Sugestion 18 References 19 ii INTRODUCTION Cellulose is the most abundant organic biomass on the earth (Tomme et al., 1995) It is the primary product of photosynthesis in terrestrial environments, and the most abundant renewable biological resource produced in the biosphere (100 billion dry tons/year) (Jarvis, 2003 and Zhang & Lynd, 2004) Cellulose is commonly degraded by an enzyme called cellulase This enzyme is produced by several microorganisms, but commonly by bacteria and fungi (Bahkali, 1996; Shin et al., 2000 and Immanuel et al., 2006) Complete enzymatic hydrolysis of cellulose requires synergistic action of types of enzyme, namely cellobiohydrolase or exoglucanase, carboxymethycellulase (CMCase) or endoglucanase and ß-glucosidases (Bhat, 2000) Cellulases are used in textile industry for cotton softening and denim finishing; in laundry detergents for color care, cleaning, and anti-deposition; in the food industry for mashing; in the pulp and paper industries for de-inking, drainage improvement, and fiber modification They are also used for several pharmaceutical applications (Kirk et al., 2002) and (Cherry & Fidantsef, 2003) Bacteria, which have significantly high growth rate in comparison to fungi, are well potential to be used for large scale cellulase production However, it has not been widely used Celluloytic property of some bacterial genera such as Cellulomonas, Cellovibrio, Pseudomonas, Sporosphytophaga spp (Nakamura and Kappamura, 1982); Bacillus and Micrococcus (Immanuel et al., 2006) were reported Enzyme production is firmly controlled in microorganisms; and these controls can be ameliorated for improving productivity Cellulase yields appear to depend on a complex interaction of several factors like inoculum size, pH value, temperature, presence of inducers, medium additives, aeration, growth time and so on (Immanuel et al., 2006) Therefore, this study was carried out to optimize the nutritional and environmental parameters for improving cellulase production by the cellulolytic anaerobic bacteria 2 MATERIALS AND METHODS 2.1 Materials - 37 anaerobic bacterial isolates used in this study had been obtained from soil, cow rumen and sugarcane bagasse, and stored at 40C in Biotechnology Research&Development Institute, Cantho University - Media: M1 medium (modified Delafield medium) ( Ryckeoer et al, 2003) was used for the production of cellulose by the isolates It is composed (in g/l) of: Ground Whatman filter paper 10.0, (NH4)2SO4 1.0, K2HPO4 1.0, MgSO4.7H2O 0.5, NaCl 0.001, Agar 20.0 M2 medium was prepared as above without agar - Chemicals: Ammonium sulfate (NH4)2SO4 (Merck), Bovin Serum Albumin (BSA) (Merck), cellulose powder (Himedia, India), yeast extract (India), K2HPO4 (Merck), MgSO4 (Merck), Congo Red (Merck), etc - Facilities: Pbi-international autoclave (Germany), Orion 420A pH meter (USA), Incucell incubator (Germany), Laminar Flow (France), etc 2.2 Methods 2.2.1 Screening of cellulase activity produced by anaerobic bacterial isolates - Objective: To select the isolates which produces highest cellulase activity - Method: Preliminary qualification of enzymatic activity from 37 isolates was done following the method described by Laurent et al (2000) 20µl inoculums of each isolate were spotted on ground filter paper solid (GFPS) medium The plates were incubated at 30°C for days Cellulase activity was detected by staining the plates with Congo red dye (0.1 g/l) for 15 minutes The plates were then washed with a 1M NaCl solution to visualize cellulase activity, which was indicated by a bright orange halo surrounding the colony against a red medium background The diameters of the haloes, which were proportional to cellulose activity, were measured The isolates generating highest cellulase activity would be used for further experiments 2.2.2 Effect of temperature and pH on cellulase production by the selected isolates - Objective: To determine the temperature and pH optimum for cellulase production of the selected bacterial isolate from experiment 2.2.1 - Method: 20µl of primary culture was inoculated on a series of GFPS plates whose pH varying from to 11 The plates were incubated in different temperature: 300C, 350C, 400C, 450C, 500C The cellulolytic enzyme production of the selected isolates was determined according to Laurent et al (2000) after days Obtained data were subjected to two-way ANOVA for significant different 2.2.3 Effect of yeast extract content on cellulase production of selected isolates - Objective: To determine the appropriate content of yeast extract added to cultivation medium for highest cellulase production - Method: 300 µl of bacteria primary culture were inoculated into falcons containing 15 ml M2 medium supplemented with different yeast extract content: 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% The pH of the media was adjusted to pH optimum selected in experiment 2.2.2 After incubation at optimum temperature chosen in experiment 2.2.2, cell-free culture supernatants were obtained by centrifugation at 7,000 rpm for 20 minutes at 40C as crude enzyme mix Enzyme activity and protein content were measured by the methods of Nelson – Somogyi (1944) and Bradford (1976), respectively 2.2.4 Effect of fermentation time on cellulase production of selected bacteria - Objective: To determine the optimal cultivation time for highest cellulase production - Method: 15ml M2 medium (supplemented optimal yeast extract concentration in experiment 2.2.3) inoculated with isolates as previous experiment were incubated at selected optimal temperature Crude enzyme mix was sampled after 0, 1, 2, 3, 4, 5, 6, 7, 8, days; and then was determined for cellulose activity and protein content as described above 2.2.5 Cellulase production by selected bacteria at laboratory scale under determined optimal conditions - Objective: To conduct an effective cellulase production protocol at laboratory scale - Method: Small scale experiments were carried out in conical flasks (1000 ml) Each flask contained 800 ml cellulase production medium prepared as previous experiment After inoculation with 24 ml of selected anaerobic bacterial isolates (at the density of circa approximately (c.a.) 3.5 x 106 CFU/ml), the flasks were incubated at optimal temperature and cultivation time that were selected from previous experiments Crude enzyme mix was obtained and examined for cellulose activity and protein content as previous experiments 2.2.6 Effect of cellulose-containing substrates on cellulase production by seleted bacteria under optimal conditions - Objective: To study the effect of different carbon sources on cellulase production - Method: Various cellulose-abundant substrates including cellulose powder, rice straw, sugarcane bagasse, and rice husk were used as carbon sources in cellulase production medium Falcons supplemented optimal yeast extract concentration were inoculated with 300 µl of the inoculums These falcons were incubated under optimal conditions derived from previous experiments Then, cellulolytic activity of crude enzyme mix and protein content was determined as above 2.3 Experimental Design and Statistical Analysis Raw data were analyzed by Analysis of Variance (ANOVA) in completely randomized design (CRD) mode using Statgraphics 3.0 software All experiments were performed in triplicates Least Significant Difference (LSD) test was used for comparison between the means VK 83 8.7 VK 67 VK 65 9.5 0.0 4.8 VK 62 6.7 VK 37 VK 30 VK 26 5.3 0.0 VK 20 5.3 VK 18 3.2 5.5 VK 17 VK 16 0.0 VK 15 6.0 VK 8.3 8.3 Anaerobic bacterial isolates VK 64 2.2 VK 80 VK 31 12.3 11.2 VK 13.8 VK 44 VK 43 12.5 12.2 VK 85 20.5 VK 52 7.8 VK 66 VK 27 1.3 VK 36 3.8 10.3 VK 49 6.5 VK VK 39 17.7 12.3 VK 13 9.3 VK 63 9.7 VK 75 7.7 VK 32 VK 33 9.5 15.7 VK 76 10.3 VK 72 6.5 VK 38 VK 84 11.3 9.7 VK 0.0 5.0 10.0 15.0 20.0 25.0 Diameters of haloes (mm) Figure 3’ Diameters of haloes (mm) in Congo Red-staining assay generated by 37 anaerobic bacterial isolates 3.2 Effect of temperature and pH on cellulase production by VK52 The cellulase produce by VK52 seemed to be completely inhibited at strong acidic pH (below 5) or alkaline pH (above 10) The activity was also decreased at temperatures higher than 300C, and completely lost at 550C (Figure 5’) High was achieved at neutral of slightly alkaline pH at 30 C Ariffin et al (2006) reported that pH had significant influence on the enzyme biosynthesis of the bacteria, and the stability of the enzyme molecules themselves, which resulted in the change of its activity Diameters of haloes (mm) 35 30 25 30oC 35oC 20 40oC 45oC 15 50oC 10 5 pH value 10 11 Figure 5’ Effect of pH and temperature on cellulose production of VK52 Results from this experiment indicated that pH and incubation temperature of 300C were optimal for cellulase production by VK52 on M1 medium; and thus were applied for further experiments These results are similar to those from Abou–Taleb et al (2009) who reported that the cellulase produced by Bacillus alcalophilus S39 had highest activity at 300C and pH Likewise, Fred (1972) reported that pH was optimal for cellulase production by Thermomonospora curvata 3.3 Effect of yeast extract content on cellulase production of selected anaerobic bacterial isolates The influence of yeast extract supplement was clarified in Figure 6, in which enzyme activity was increased with addition of ≤ 0.4% of yeast extract Maximum activity of 0.067 U/ml was obtained when 0.4% (w/v) yeast extract was added into cellulase production medium (3-fold increase in cellulose activity than the control) 0.08 Cellulase activity (U/ml) 0.07 0.067 0.06 0.05 0.043 0.04 0.039 0.031 0.03 0.02 0.027 0.023 0.021 0.01 0.00 0.0% 0.1% 0.2% 0.3% 0.4% 0.5% 0.6% 0.7% Yeast extract content (% ) Figure Effect of yeast production of VK52 10 extract on cellulase The decrease of cellulase activity (Figure 6) at supplemetary yeast extract of more than 0.4% might be due to the overproduction of reducing sugars (glucose) and cellobiose through product inhibition in enzyme kinetics (Howell and Mangat, 1978) This is supported by the results in Figure 7, in which there was a significant increase of protein content when yeast extract was added to cultivation medium The maximum protein content of 99.35 µg/ml was gained when 0.4% (w/v) yeast extract was supplied into medium Protein content (µg/ml) 120 100 99.35 80 69.26 60 40 56.39 32.41 40.00 49.91 44.44 20 0.0% 0.1% 0.2% 0.3% 0.4% 0.5% 0.6% Yeast extract content (%) Figure Variation of protein content by yeast extract 11 0.7% Yeast extract was reported to act as a stimulator for the producing of cellulase (Amtul, 1989), (Azzaz, 2009), (Abou-Taleb et al., 2009) The appropriate yeast extract content added into medium depends on different cultures Ammut (1989) determined optimum yeast extract for cellulase production by Cellulomonas flavigena was 0.2%, while Abou–Taleb (2009) indicated that 0.7% yeast extract was the best for B alcalophilus S39 and B amyloliquefaciens C23 For most efficient cellulase production by Clostridium sp M7, 0.5% yeast extract was chosen by Lee and Blackburn (1975) 3.4 Effect of fermentation time on cellulase production by VK52 The change of cellulase activity over cultivation time was recorded in Figure 8, in which the activity was rapidly increased during the first days and gradually decreased afterwards The maximum cellulase activity of 0.064 U/ml was obtained after days of cultivation The activity loss might be due to denaturation of the enzyme when it was exposed to the fermentation conditions different than those of physical cellular metabolism (Krishna, 1999), (Liu and Yang, 2007) In addition, the depletion of nutrients and substrates in medium could also cause the reduction in cellulase production (Laurent et al., 2000) Furthermore, the accumulation of cellobiose products might also be as an inhibitor to both endoglucanase and βglucosidase as reported in other studies (Howell and Mangat, 1978) 12 Cellulase activity (U/ml) 0.08 0.07 0.064 0.06 0.05 0.052 0.051 0.04 0.033 0.03 0.042 0.038 0.038 0.034 0.029 0.02 0.01 0 10 Cultivation time (days) Figure Effect of cultivation time on cellulase production by VK52 Protein content (µg/ml) 250 215.37 200 156.11 152.22 150 138.33 136.11 121.67 127.96 115.74 109.63 100 50 37.04 0 10 Cultivation time (days) Figure Variation of protein content by cultivation time 13 On the other hand, the production of protein over time also got similar scheme, in which it increased rapidly in the first days and yielded maximum value of 215.37 µg/ml, and gradually decreased later on (Figure 9) Nutritional depletion could be the cause of this phenomenon Ray et al (2007) claimed the cellulase productivity by Bacillus subtilis CY5 and Bacillus circulan TP3 reached maximum after 96h, with corresponding activities of 30.5 U/ml and 25 U/ml However, optimumal cultivation time is different among species For example, maximum CMCase and Avicelase activities of Cellulomonas flavigena (10 U/ml and 1.2 U/ml, respectively) were achieved after 72h of fermentation (Amtul, 1989); while Abhaykumar (1992) reported that highest CMCase activity of Vibrio agar-liquefaciens was determined at 0.09 IU/ml after days 3.5 Cellulase production VK52 at laboratory scale in selected optimal conditions A laboratory-scale production (c.a 800 ml culture in litter conincal flasks) of cellulose by VK52 was set up under the optimal conditions mentioned above Various parameters of this process were determined and shown in Table 4’ The activity (in U/ml) and protein content (in µg/ml) were higher than those obtained from 15 ml culture described in previous experiments The decrease of pH from to 6.89 after days could be explained by the appearance of acidic secondary products during fermentation, which in turn caused the activity loss of the enzymes later on Similar results were reported in the study of Krishna (1999) 14 Table 4’ Overall assessment of the laboratory scale by VK52 EndoCellular glucana Density se (CFU/ml) activity (U/ml) 6.7 x 108 0.107 Exoglucanas e activity (U/ml) 0.060 Protein pH after content fermenta- (µg/ml) tion 260.56 6.89 Yield (%) 9.025 Substrate dry matter decreased from g at the beginning to 6.853 g after days VK52 appeared to be able to produce types of cellulase enzymes: endoglucanase, exoglucanase and cellobiase The synergetic action of these enzymes caused the degradation of cellulose in the medium and thus resulted in the decrease of substrate matter after fermentation (Knowles et al., 1987), (Wood and GaricaCampayo, 1990), (Teeri, 1997), (Bhat, 2000), (Lynd et al., 2002) The overall cellulolytic yield was determined as 9.025% 3.6 Effect of cellulose-containing substrates on cellulase production by VK52 Various substrates including ground filter paper, cellulose powder, rice straw, sugarcane bagasse and rice husk were investigated for Endoglucanse and exoglucanase activities were determined on CMC and cellulose powder as substrates (Figure 10’) Ground Whatman filter paper and cellulose powder were most efficient for by VK52, as seen in the produced endoglucanase (0.032 U/ml and 0.031 U/ml, respectively) and exoglucanase (0.025 U/ml 15 and 0.024 U/ml, repsectively) Cellulolysis was also observed on 0.040 70 0.035 60 0.030 50 0.025 40 0.020 30 0.015 20 0.010 Protein content (ug/ml) Cellulase activity (U/ml) other substrates with lower enzymatic activity 10 0.005 0.000 Cellulose Grinded Filter Paper Rice straw Sugarcane bagasse Rice husk Endoglucanase 0.031 0.032 0.028 0.026 0.023 Exoglucanase 0.024 0.025 0.010 0.014 0.011 Protein content 22.75 26.99 41.49 35.45 58.97 Carbon sources Figure 10’ Effect of various substrate sources on cellulase production and protein content by VK52 under optimal conditions In contrast, protein content produced on rice straw, sugarcane bagasse, rice husk (41.49, 35.45 and 58.97 µg/ml, respectively) were much dominant against the use of ground filter paper and cellulose (Figure 10’) This might be due to high amount of lignin, hemicellulose, and xylan in these materials, which prevented the 16 exposure of cellulose to the ezymes However, the considerable cellulolytic activities of VK52 on these substrates suggested that the bacteria could produce other enzymes such as ligninase, hemicellulase and xylanase to aid cellulose degradation Such simultaneous appearance of various enzymes probably resulted in the mutual inhibition between them, whereas the protein content was increased (Badhan et al., 2004) 17 CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions Isolate VK52 yielded highest cellulase activity on ground Whatman filter paper-agar medium among 37 anaerobic bacterial isolates Optimal cellulose productivity by VK52 could be achieved through anaerobic fermentation in the paper powder medium supplemented with 0.4% yeast extract at pH and 30oC for days Protein content, endoglucanase and exoglucanase activities of isolate VK52 at laboratory scale were determined as 260.56 µg/ml, 0.107 U/ml and 0.060 U/ml respectively The fermentation process gained the cellulolytic yield of 9.025% and medium pH decrease from to 6.89 Celulose powder and ground filter paper were concluded as the best carbon sources for cellulase production by the VK52 Therefore, isolate VK52 could be the potential bacteria waste paper treatment 4.2 Suggestions Due to the time limitation, further studies should emphasize on:  Studying other conditions such as the effect of the inoculum size and substrate concentration on the cellulase production of the isolate VK52  Idenfication of the scientific name of the selected isolate VK52  Purification and characterization of the cellulases produced by the VK52 using chromatography techniques 18 and electrophoresis REFERENCES Abhaykumar, V.K and H.C Dube, 1992 Cellulases of Vibrio agarliquefaciens isolated from sea mud Microbiol and Biotechl (8): 313-315 Abou-Taleb, A.A Khadiga, W.A Mashhoor, A Sohair, M.S Sharaf and H.M Hoda, 2009 Nutritional and Environmental Factors Affecting Cellulase Production by Two Strains of Cellulolytic Bacilli Australian Journal of Basic and Applied Sciences 3(3): 2429-2436 Amtul, J S., 1989 Purification and Characterization of Microbial Cellulolytic Enzymes Ph.D Thesis, Institute of Chemistry, University of the Punjab Lahore – 1, Pakistan, pp: 176 Ariffin, H, N Abdullah, M.S.U Kalsom, Y Shirai, M.A Hassan, 2006 Production and characterization of cellulase by Bacillus pumilus EB3 Int J Eng Tech 3(1):47-53 Azzaz, H.H., 2009 Effect of cellulolytic enzymes addition to diets on the productive performance of lactating goats M.Sc Thesis, Faculty of Agriculture, Cario University, Egypt, pp: 141 Badhan, A.K., B.S Chadha, K.G Sonia, H.S Saini and M.K Bhat, 2004 Functionally diverse multiple xylanases of thermophilic fungus Myceliophthora sp IMI 387099 Enz and Microbiol Technol 35, 460–466 Bahkali, A.H., 1996 Influence of various carbohydrates on xylanase production by V tricorpus Bioresource Technol 33(3): 265 268 Bhat, M.K., 2000 Cellulases and related enzymes in biotechnology Biotech Adv 18: 355-383 19 Bradford, M.M., 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing principle of protein-dye binding Anal Biochem 72: 142-146, Cherry, J.R and A.L Fidantsef, 2003 Directed evolution of industrial enzymes: an update Curr Opin Biotechnol 14: 438–443 Fred, J S., 1972 Cellulolytic activity of Thermomonospora curvata: Nutritional requirements for cellulase production Journal of American Society for Microbiol 24 (1): 77-82 Howell, J.A and M Mangat, 1978 Enzyme deactivation during cellulose hydrolysis Biotechnol Bioeng 20: 847-863 Immanuel, G., R Dhanusa, P Prema and A Palavesam, 2006 Effect of different growth parameters on endoglucanase enzyme activity by bacteria isolated from coir retting effluents of estuarine environment Int J Environ Sci Tech 3(1): 25-34 Jarvis, M., 2003 Cellulose stacks up Nature 426: 611-612 Kirk, O., T.V Borchert and C.C Fuglsang, 2002 Industrial enzyme applications Curr Opin Biotechnol 13: 345-351 Knowles, J., P Lethtovaara and T Teeri, 1987 Cellulase families and their genes Trends Biotechnol 5: 255-261 Krishna, C., 1999 Production of bacterial cellulases by solid state bioprocessing of banana wastes Bioresour Technol 69, 231239 Laurent P., L Buchon, J.F.G Michel, N Orange, 2000 Production of pectate lyases and cellulases by Chyrseomonas luteola strain MFCL0 depends on the growth temperature and the nature of the culture medium: evidence for two critical temperatures App and Env Micro 66 (4) 1538- 1543 20 Lee B H and T H Blackburn, 1975 Cellulase Production by a Thermophilic Clostridium Species App Micro 30 (3) 346353 Lynd L.R., P.J Weimer, W.H Zyl and I.S Pretorius, 2002 Microbial cellulose ultilization: Fundamentals and Biotechnology Microbiol Mol Biol Rev 66: 506-577 Nakamura K and K Kppamura, 1982 Isolation and identification of crystalline cellulose hydrolyzing bacterium and its enzymatic properties J Ferment Technol 60(4): 343 - 348 Nelson, N (1944) A photometric adaptation of the Somogyi method for the determination of glucose J Biol Chem 153: 375–80 Ray, A.K., A Bairagi, K.S Ghosh and S.K Sen, 2007 Optimization of fermentation conditions for cellulase production by Bacillus subtilis CY5 and Bacillus circulans TP3 isolated from fish gut Acta Ichthyol Piscat 37 (1): 47–53 Ryckeboer J., J Megaert, J Coosemans, K Deprins and J Swings, 2003 Microbiological aspects of biowaste during composting in a monitored compost bin Jour of Appl Microbiol 94: 127 – 137 Shin, C.S., J.P Lee, P.S Lee and S.C Park, 2000 Enzyme production of Trichoderma reesi Rut C-30 on a various lingocellulosic substrates Appl Biochem and Biotechnol 8486: 237-245 Teeri, T T., 1997 Crystalline cellulose degradation: New insights into the function of cellobiohydrolases Trend Biotechnol 15: 160-167 Tomme, P., R.A Warren and N.R Gilkes, 1995 Cellulose hydrolys is by bacteria and fungi Adv Microb Physiol 37: 1-81 21 Wood, T.M and V Garica-Campayo, 1990 Enzymology of cellulose degradation Biodegradation 1: 147-161 Zhang, Y.H.P and L.R Lynd, 2004 Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulose systems Biotechnol Bioeng 88: 797824 22 ... Yeast extract content (% ) Figure Effect of yeast production of VK52 10 extract on cellulase The decrease of cellulase activity (Figure 6) at supplemetary yeast extract of more than 0.4% might... according to Laurent et al (2000) after days Obtained data were subjected to two-way ANOVA for significant different 2.2.3 Effect of yeast extract content on cellulase production of selected isolates... DUONG THI HUONG GIANG STUDENT TRAN NON NUOC MSc VO VAN SONG TOAN Can Tho, November, 2010 PRESIDENT OF EXAMINATION COMMITTEE ABSTRACT Optimal selection of strain, fermentation conditions and substrate,