Nutritional and Environmental Factors Affecting ...

A us tralian Journal of Bas ic and A pplied Sciences , 3(3): 2429-2436, 2009 ISSN 1991-8178 © 2009, INSInet Publication

Nutritional and Environmental Factors Affecting Cellulase Production by Two Strains of Cellulolytic Bacilli 1

Abou-Taleb, Khadiga A.A., 1Mashhoor, W.A., 1Nasr, Sohair A., 1Sharaf, M.S. and 2 Abdel-Azeem, Hoda H.M.

1

Agricultural Microbiology Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. 2 Soil Microbiology Unit, Desert Research Center, Cairo, Egypt.

Abs tract: Effect of s ome nutritional and environmental factors on g ro w t h and cellulas e production by Bacillus alcalophilus S39 and Bacillus amyloliquefaciens C23 was inves tigated. Res ults indicated t h a t 1% carboxymethylcellulos e (CM C) and 0.7% yeas t extract were mos t effective as the carbon an d nitro g e n s o urces res pectively. Initial pH 7 and 3% inoculum s ize found to be optimal for growth and cellu la s e production. Incubation temperature at 30 and 45ºC achieved the highes t activity of cellulas e for Bacillus alcalophilus S39 and Bacillus amyloliquefaciens C23 res pectively, and the s uitable s haking rate was 150 and 200 rpm. Key words : N u t rit ional & environmental factors , Cellulas e production, B. alcalophilus S39, B . amyloliquefaciens C23 . INTRODUCTION Cellulos e is t h e mos t abundant biomas s on the earth (Tomme et al., 1995). It is the primary product of photos ynthes is in terres trial environments , and the mos t abundant renewable biores ource produced in the bios phere (100 b illion dry tons /year) (Jarvis , 2003 and Zhang & Lynd, 2004). Cellulos e is commonly degraded by an enzyme called cellulas e. This enzyme is produced b y s e v e ra l microorganis ms , commonly by bacteria and fungi (Bahkali, 1996; M angelli & Forchias s in, 1999; Shin et al., 2000 and Immanuel et al., 2006). Complete enzymatic h y d ro lys is of enzyme requires s ynergis tic action of 3 types of enzymes , namely cellobiohydrolas e, endoglucanas e or carboxymethycellulas e (CM Cas e) and â-glucos idas es (Bhat, 2000). Cellulas es are us ed in the textile indus try for cotton s often in g a n d d e nim finis hing; in laundry detergents for color care, cleaning, and anti-depos ition; in the food indus try for mas hing; in the pulp and paper indus tries for deinking, drainage improvement, and fiber modification and they are even us ed for pharmaceutical applications (Kirk et al., 2002 and Cherry & Fidants ef, 2003). Bacteria, which has high growth rate as compared to fungi has good potential to be us e d in c e llulas e production. However, the application of bacteria in producing cellulas e is not widely us ed. Celluloytic property of s ome bacterial genera s uch as Cellulomonas, Cellovibrio, Pseudomonas, Sporosphytophaga s pp. (Nakamura and Kappamura, 1982); Bacillus and Micrococcus (Immanue l e t a l ., 2006) were als o reported. Enzyme production is clos ely controlled in microorganis ms and for improving its productivity thes e cont ro ls can be a me liorated. Cellulas e yields appear to depend on a complex relations hip involving a variety of factors like inoculum s ize, pH value, temperature , p re s ence of inducers , medium additives , aeration, growth time, etc. (Immanuel et al., 2006). The pres ent work was carried out to optimize the nutritional and environmental pa ra me t e rs for improving cellulas e production by the two cellulolytic bacterial s trains . MATERIALS AND METHODS M icroorganisms Used: The two bacterial s trains , Bacillus alcalophilus S39 and Bacillus amyloliquefaciens C23 us ed in this s tudy were is olated form s oil and compos t res pectively and were dis tinguis hed as potent cellulas e producers . The purified bacilli is olates were identified according to their cultural, morphological and biochemical characteris tics bas ed on Bergey's M anual of Sys tematic Bacteriolo g y (Claus and Berkeley, 1986) and Biolog A utomated Sys tem was us ed. Corresponding Author: Hoda Hassan Abdel-Azeem, Soil M icrobiology Unit, Desert Research Center, Cairo, Egypt. E-mail: [email protected] 2429

Aust. J. Basic & Appl. Sci., 3(3): 2429-2436, 2009 M edia used: M edium (1): Nutrient agar (Difco M anual, 1984) was us ed for the maintenance of Bacillus s trains . M edium (2): Carboxymethyl cellulos e medium recommended by Ray et al. (2007) was u s e d for the production of cellulas e by Bacillus s p. It has the following c o mp o s ition (g/l): Carboxymethylcellulos e (CM C), 10; Tryptone, 2; KH2 PO4 , 4; Na 2 HPO4 , 4; M gSO4 .7H2 O, 0.2; CaCl2 .2H2 O, 0.001; FeSO4 .7H2 O, 0.004; A gar, 15 and pH adjus ted to 7. Inoculum Preparation and Fermentation Process: For preparation of s tandard inoculum, both s t ra ins were cultured in nutrient broth individually at 30 ºC for 24 h where an average viable count of 3.5 - 4.3 ×10 6 c e ll/ml culture broth was obtained. This was us ed as the inoculum for the production medium. Fermentation was carrie d out in 250 ml plugged Erlenmeyer flas ks , each containing 50 ml s terile prod u c t ion medium and inoculated with 3% of s tandard inoculum (containing about 3.5 ×106 and 4.3 ×106 cell/ml for Bacillus amyloliquefaciens C23 and Bacillus alcalophilus S39, res pectively). The inoculat e d flas ks were incubated at 30 ºC and 45 ºC for Bacillus alc a l o p h i lus S39 and Bacillus amyloliquefaciens C23 , res pectively on rotary s haker at 150 rpm for 72h. Preparation of Crude Enzyme: A ft e r incubation, cultures were centrifuged at 1600 g for 15 min at 4°C and s upernatants were us ed as s ource of cru d e e n zy mes . The crude enzyme s olution was utilized for determination of enzyme activities (Kotchoni et al., 2003). Enzyme Assays Procedures: Carboxymethyl-cellulase(CM Case) activity: CM Cas e act iv ity was as s ayed us ing a method described by M andels and W eber (1969). The activity was es timated us ing 1 % s olution of carboxymethlycellulos e (CM C) in 0.05 M cit ra t e b u ffer (pH 4.8) as s ubs trate. The reaction mixture contained 1 ml citrate buffer, 0.5 ml of s ubs trate s olution and 0.5 ml of s uitably diluted enzyme s o lu t io n . T h e reaction was carried out at 50°C for 30 min. The amount of reducing s ugar releas ed in the hydrolys is was mea s u re d . One unit of CM Cas e activity was expres s ed as 1 ì mol of glucos e liberated per ml enzyme per minute. Filter-paperase (FPase) Activity: The activity of FPas e was as s ayed according to the method explained by M andels and W eber (1969). This me t h o d is s imila r to the CM Cas e as s ay method, but the s ubs trate was W hatman No. 1 filter paper s trip (1 x 6 cm) s oaked in 1 ml 0.05 M s o d ium citrate buffer (pH 4.8). The s amples were incubated with 0.5 ml enzyme s olution at 50°C for 1 h, the reducin g s u g a rs lib erated during growth were determined. One unit of FPas e activity was determined as 1 ì mol of glucos e liberated per ml enzyme per minute. â-Glucosidase Activity: One-tenth ml of the culture s upernatant was incu b a t e d w it h 0.5 ml of 0.05 M acetate buffer (pH 5) containing 2.5 mg cellobios e. A fter incubation at 50 ºC for 10 min, the glucos e rele a s e d w a s meas ured by the glucos e oxidas e peroxidas e method (Zaldívar et al., 2001). Determination of Reducing Sugars: T h e t o t a l amount of reducing s ugars was determined us ing potas s ium ferricyanide method, as des cribed by Park and Johns on (1949). Carbon Sources: The appropriate carbon s ource was s elected by replacing the original carbon s ubs trate of the bas al medium with equivalent carbon amount of each of the tes ted carbon s ources (Glucos e, Carboxyme t h y c e llulos e, Cellobios e and Cellulos e). Nitrogen Sources: To detect the adequate nitrogen s ource for cellulas e production by s elected s trains , the pres cribed nitrogen s ource of the fermentation medium was replaced by equivalent nitrogen amount of each of the tes te d o rg a nic [Beef extract, Cas ein, M alt, Peptone, Tryptone, Urea & Ye a s t e xtract] and inorganic [KNO3 , (NH4 )3 PO4 , NaNO3 , NH4 NO3 , NH4 Cl & (NH4 )2 SO4 ] nitrogen s ources . 2430

Aust. J. Basic & Appl. Sci., 3(3): 2429-2436, 2009 pH: Seven values of pH ranged between 5.5 and 8.5 were chos en for s tudying their effects on cellulas e enzyme to s elect the mos t s uitable pH of the production medium. Incubation Temperature: To d e t ermine the optimum temperature for cellulas e production, fermentation was carried out at various temperatures in the range of 5, 20, 25, 30, 35, 40, 45, 50 and 55 ºC. Shaking Rate: Erlenmeyer flas ks (250 ml) containing production medium were inocu la t e d with the s elected s trains and placed onto a rotary s haker at different rpm (i.e. 0, 50, 100, 150 and 200 rpm) to obtain proper aeration for maximal cellulas e production. Inoculum Size: T h e inoculum s ize was optimized for maximal enzyme production. The fermentation mediu m w a s inoculated with 1, 2, 3, 4, 5, 6 and 7 % of s tandard inoculum. Statistical Analysis: The collected data were s tatis tically analyzed us ing SPSS Computer A nalys is Programs (Fos ter, 2001). RES ULTS AND DIS CUS S ION Effect of Different Carbon Sources: Data pres ented in Table (1) s how that cellulas e production with both two Bacillus s trains was s ignificantly influe n c e d b y t h e type of carbon s ource in the bas al medium. Carboxymethylcellulos e (CM C) was mos t effective as a s ole carbon s ource for cellulas e enzyme production by Bacil l u s a l c a l o philus S39 and Bacillus amyloliquefaciens C23 , res ults in increas e in enzyme activity, being 1.81 & 1.88 U/ml of CM Cas e activity, 0.87 & 0.86 U/ml of FPas e activity and 1.31 & 1.41 U/ml of â-glucos idas es , res pectively. Thes e values were followed, in des cending order by cellobios e and cellulos e. Thes e res ults are in agreement with thos e of Naras imha et al. (2006) a n d N ira n ja n e e t al. (2007) who found that carboxymethyl cellu los e was the bes t carbon s ource followed by cellulos e for cellulas e production. A higher production of cellulas e when CM C s erved as s ubs trate may be as a res ult of induction of the enzyme, s ince cellulos e is known to be a univers al inducer of cellu la s e s y n thes is . Paul and Varma (1993) had reported the induction of endocellulas e by CM C. M edium containing glucos e as the growth carbon s ource pres ented the minimum cellulas e a c t iv ity (expres s ed by CM Cas e, FPas e an d â -g lu cos idas e). Muthuvelayudham and Viruthagiri (2006) obtained s imilar res ults which s howed that the cellulas e activity was les s when glucos e was us ed as carbon s ource becaus e of inhibition. A nother experiment was carried out to s t u d y t he effect of different concentrations of carboxymethylcellulos e (CM C) which exhibited s uperiority among other tes ted carbon s ources for Bacillus s t ra in s . Data in Fig. (1) clearly s how that 1% carboxymethylcellulos e (CM C) gave the highes t activity of cellulas e being 1.85 & 1.88 U/ml of CM Cas e; 0.87 & 0.87 U/ml of FPas e a nd 1.35 & 1.40 U/ml of âglucos idas es by B. alcalophilus S39 and B. amyloliquefaciens C23 , res pectively. This is s imilar with p re v ious inves t ig a t io n s (F u ku mori et al., 1985; Kawai et al., 1988 and Shikata et al., 1990) where the CM Cas e activity in Bacillus s p. was detected in cultures that contained 1% (w/v) CM C as the growth s ubs trate. Effect of Different Nitrogen Sources: To evaluate the effect of nitrogen s ource on cellulas e formation, the nitrogen s ource in the bas al me d ium w a s replaced by different nitrogen s ources . Data revealed that the s upplementation of organic and in o rg a n ic nitrogen s ources s timula t e d t h e c e llulas e yield and activity. Us ing of organic N s ources res ponded in the pos itive cellulas e activity more than the inorganic ones . A mong the tes ted complex N s ources , the effectivenes s in s upporting cellulas e production and cellulolytic activity by both Bacillus s trains s ignificantly de c re a s e d in the following order: yeas t extract > peptone > beef extract > NH 4 Cl. Res ults recorded in Table (2) clearly s how that yeas t extract was the bes t nitrogen s ource for both s trains giving 2.07 & 2.17 U/ml of CM Cas e, 0.99 & 1.01 U/ml of FPas e an d 2.18 & 2.55 U/ml of â-glucos idas es for Bacillus alcalophilus S39 and Bacillus amyloliquefaciens C23 , res pectively. Data are in accordance with th e re s ults of Ray et al. (2007) who reported t h a t organic nitrogen s ources were found to be more s uitable for optimizing cellulas e production by B a c illu s subtilis and Bacillus circulans than inorganic s ources . 2431

Aust. J. Basic & Appl. Sci., 3(3): 2429-2436, 2009 Table 1: Effect of carbon sources on the production of cellulase enzyme by the two Bacillus strains. Different carbon sources Bacillus alcalophilus S39 Bacillus amyloliquefaciens C23 ----------------------------------------------------------------------------------------------------------------------------------Biomass Cellulase Activity (U/ml) Biomass Cellulase Activity (U/ml) g/100ml ----------------------------------------------g/100ml ---------------------------------------------------CMCase FPase â- glucosidases CMCase FPase â- glucosidases Glucose 0.501 a 0.23 e 0.25 f 0.05 f 0.484 b 0.30 e 0.10 e 0.04 f Carboxymethycellulose (CMC) (Control) 0.361 f 1.81 a 0.87 a 1.31 b 0.382 e 1.88 a 0.86 a 1.41 a Cellobiose 0.403 c 0.71 d 0.19 d 0.13 e 0.392 d 1.32 b 0.34 c 0.06 f Cellulose 0.157 h 1.00 c 0.41 c 1.27 c 0.295 g 1.43 b 0.51 b 1.24 d Values in the same column followed by the same letter do not significantly differ from each other, acco rd i n g t o D u ncan’s at 5 % level. Table 2: Effect of nitrogen sources on the production of cellulase enzyme by Bacillus strains. Nitrogen sources Bacillus alcalophilus S39 Bacillus amyloliquefaciens C23 -----------------------------------------------------------------------------------------------------------------------------------Biomass Cellulase Activity (U/ml) Biomass Cellulase Activity (U/ml) g/100ml -----------------------------------------------g/100ml ---------------------------------------------------CMCase FPase â- glucosidases CMCase FPase â- glucosidases Beef extract 0.417 c 2.05 b 0.98 c 2.04 c 0.420 b 1.99 c 0.99 c 2.25 b gh l n lm n d n o Casein 0.395 0.41 0.04 0.67 0.413 0.03 0.01 0.64 m n Malt 0.356 p 1.18 j 0.01 o 0.73 lm 0.380 k 0.31 m 0.04 m n 0.68 l m n Peptone 0.407 e 2.05 b 0.99 c 2.16 b 0.381 k 2.14 a 1.00 b 2.47 a T ryptone (Control) 0.360 o 1.83 f 0.87 i 1.34 h 0.387 i 1.88 e f 0.89 i 1.41 gh Urea 0.375 lm 1.50 h 0.47 k 0.70 lm n 0.385 ij 1.64 g 0.64 j 0.75 kl Yeast extract 0.404 e f 2.07 b 0.99 c 2.18 b 0.417 c 2.17 a 1.01 a 2.55 a KNO3 0.407 e 1.24 i 0.06 l 1.06 j 0.420 b 0.81 k 0.05 m 0.81 k (NH4 )3PO4 0.378 l 0.41 l 0.05 m 0.64 n 0.395 gh 1.14 j 0.02 o 0.98 j NaNO3 0.374 l m 1.88 e f 0.89 h 1.62 e 0.370 l m n 1.90 e f 0.90 g 1.10 i NH4 NO3 0.397 g 1.88 e f 0.90 gh 1.42 fg 0.371 l m n 1.89 e f 0.92 f 1.55 f NH4 Cl 0.359 o 1.97 c d 0.96 e 1.77 d 0.361 o 1.93 c de 0.98 c 1.80 d (NH4 )2SO4 0.424 a 1.91 de 0.92 f 1.49 f 0.417 c 1.91 de 0.97 d 1.55 f Values in the same column followed by the same letter do not significantly differ fro m each o t h er, according to Duncan’s at 5 % level.

Data illus t rated in Fig. (2) obvious ly indicates that s uitable concentration of yeas t extract was found to be 0.7% which gave the highes t CM Cas e being 2.35 & 2.30 U/ml; FPas e being 1.15 & 1.19 U/ml and âglucos idas es being 3.56 & 3.49 U/ml of B. alcalophilus S39 and B. amyloliquefaciens C23 , res pectively. It is notable at all experiments to s tate t h a t t here was no relations hip between the production of cellulas e enzyme and biomas s yield. Effect of initial pH: Cellulas e y ie ld by both s trains appear to depend on pH value. Res ults illus trated by Fig. (3) clearly s how that cellu la s e p ro d u c tion, expres s ed as enzyme activity, gradually increas ed as the pH values increas ed from 6.5 to 7.5 and reached its maximum at initial pH of 7 b e in g 2.41 & 2.40 U/ml of CM Cas e, 1.19 & 1.19 U/ml of FPas e and 3.55 and 3.49 U/ml of â-glucos idas es by B. alcalophilus S39 and B. a m y l o l i q u e fa c i e ns C23 , res pectively. The cellulas e activity was les s in other tes ted pH levels , where enzyme activity was minimal at pH 5.5 and it indicates a marginal increa s e a t pH 6.5 and 7. Further, this activity was greatly reduced to reach the lowes t at pH 8.5 (where 2.06 & 2.07 U/ml of CM Ca s e; 1.07 & 1.04 U/ml of FPas e and 0.72 and 0.56 U/ml of â-glucos idas es was obtained by B. alcalophilus S39 and B. amyloliquefaciens C23 , res pectively. Obtained data confirmed the findings reported by Ray e t a l., (2007) who mentioned that pH 7 – 7.5 more s uitable for optimization of cellulas e produc t ion by Bacillus subtilis and B. circulans. Furthermore, the cellulolytic enzyme, endogluconas e obtained from Cellulomonas, Bacil l u s , a nd Micrococcus s pp. hydrolyzed s ubs trate in the pH range of 4.0 to 9.0, with maximum activ it y t ra n s p iring at pH 7 (Immanuel et al., 2006). Incubation Temperature: Like pH, temperature is als o an important factor that influences the cellulas e yield. It is o bvious from Fig. (4) that the highes t cellulas e activity was obtained at temperatures 30 to 45ºC for B. alcaloph i l u s S 39 a n d B. amyloliquefaciens C23 res pectively, whereas it was les s at other tes ted degrees for each s train. Thes e res ults are clos ed to thos e of Bakare et al. (2005) who found that the cellulas e e n zy me p ro d u c e d b y Pseudomonas fluorescence was activated at 30 to 35 ºC s howing the optimum temperature at 35 ºC. Ray et al. (2007) reported that minimum cellulas e yield was obs erved when fermentation was carried out at 45°C, while maximum yield was obtained at 40°C by Bacillus subtilis and Bacillus cir c u l a n s . Immanuel et al. (2006) als o recorded maximum endoglucanas e activity in Cellulomonas, Bacillus and Micrococcus s p. at 40°C and neutral pH. 2432

Aust. J. Basic & Appl. Sci., 3(3): 2429-2436, 2009

Fig. 1: Effe ct of different concentrations of carboxymethylcellulos e (CM C) on the production of cellu la s e enzyme by Bacillus s trains .

Fig. 2: Effect of different concentrations of yeas t extract on the production of cellu la s e enzyme by Bacillus s trains .

Fig. 3: Effect of initial pH on the production of cellulas e enzyme by Bacillus s trains .

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Fig. 4: Effect of incubation temperature on the production of cellulas e enzyme by Bacillus s trains . Effect of Shaking Rate: Concerning the effect of s haking rate on cellulas e yield , it w a s fo u n d from the current data (Fig. 5) that the maximum activity was obtained at the range of s haking rate of 150-200 rpm for B. alcalophilus S39 and B. amyloliquefaciens C23 . No s ignificantly different was noticed in enzyme activit y p ro d uced at rate of 150 and 200 rpm. Similar data was found by Bin A mwarali Khan a nd Hus aini (2006) who noticed a remarkable increas e of cellulas e activity in fermentation medium under s haking condition compared to s tatic condition. It was obs erved more than 2 fold higher cellulas e enzyme a c t ivity in s haking condition (2.97 IU/ml) compare to non s haking condition (1.38 IU/ml) for Bacillus amylolique fa c i ens UM A S 1002 s train. They als o reported that the highes t cellulas e enzyme production by Bacillus amyloliquefaciens UM A S 1002 s train were 2.97 and 2.89 IU / ml at agitation s peed of 100 and 200 rpm res pectively. Inoculum Size: Fig. (6) s hows that the inoculum s ize of 3.0 % achieved t h e highes t cellulas e enzyme production being 2.40 & 2.39 U/ml of CM Cas e; 1.20 & 1.18 U/ml of FPas e and 3.61 & 3.53 U/ml o f â -g lu c o s id a s e s b y B. alcalophilus S39 and B. amyloliquefaciens C23 , res pectively. Thes e re s u lt s were almos t s imilar with findings collected by Ray et al. (2007) elucidated the enzyme production increas ed gradually up to 3% inoculum s ize, but decreas ed thereafter. The enzyme pro d u c t io n b y both s trains Bacillus subtilis and Bacillus circulans in 3% inoculum s ize was n o t s ig n ificantly different (P < 0.05) from that in 2% inoculum s ize. In the pres ent s tudy, it could be concluded that carbon and nitrogen s o u rc e s , p H value, temperature, inoculum s ize and aeration play a mos t crucial role in cellulas e production by B . a l calophilus and B. amyloliquefaciens.

Fig. 5: Effect of s haking rate on cellulas e production of by Bacillus s trains .

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Fig. 6: Effect of inoculum s ize on the production of cellulas e production of by Bacillus s trains . REFERENCES Ba h ka li, A .H., 1996. Influence of various carbohydrates on xylanas e production by V. tricorpus. Biores ource Technol., 33(3): 265 - 268. Bakare, M .K., I.O. A dewale, A . A jayi and O.O. Shonukan, 2005. Purification and characterization of cellulas e from the wild-type and two improved mutants of Pseudomonas fluorescens. A frican J. o f Biotech., 4(9): 898-904. Bhat, M .K., 2000. Cellulas es and related enzymes in biotechnology. Biotech. A dv., 18: 355-383. Bin A mwarali Khan, F.A . and A .A .S.A . Hus aini, 2006. Enhancing á – a mylas e and cellulas e in vivo enzyme expres s ions on s ago pith res idue us ing Bacillus amyloliquefaciens UM A S 1002. Biotechnology, 5(3): 391- 403. Cherry, J.R. and A .L. Fidants ef, 2003. Directed evolution of indus trial enzy me s : a n u p date. Curr. Opin. Biotechnol., 14: 438–443. Claus , D. and R.C.W . Berkeley, 1986. Genus Bacillus. In Bergey's M anual of Sys tematic Bacteriology, vol. 2, pp. 1105–1139. Sn e a t h , P . H .; N . M air; M .E. Sharpe & J.G. Holt (Eds .). W illiams & W ilkins Co., Baltimore, USA . DIFCO M anual 1984. Dehydrated culture M edia and reagents fo r M icrobiology. 10 th Ed. Difco Laboratories , Detroit, M . (ed.) U.S.A . Fos ter, J.J., 2001. D a ta analys is us ing SPSS for W indows Vers ions 8 –10. Fos ter, J.J. (ed.), Sage publication Ltd. London. Fukumori, F., T. Kudo and K. Horikos hi, 1985. Purification and properties of a cellulas e fro m a lka lo p h ilic Bacillus s p. No. 1139. J. Gen. M icrobiol., 131: 129-135. Immanuel, G., R. Dhanus a, P. Prema and A . Palaves am, 2006. Effect of d iffe re n t g ro wth parameters on endo g lucanas e enzyme activity by bacteria is olated from coir retting effluents of es tuarine environment. Int. J. Environ. Sci. Tech., 3(1): 25-34. Jarvis , M ., 2003. Cellulos e s tacks up. Nature, 426: 611-612. Kawai, S., H. Okos hi, K. Ozaki, S. Shikata, K. A ra and S. Ito., 1988. Neutrophilic Bacillu s s t ra in, KSM 522 that produces an alkaline carboxymethyl cellulas e. Agri. Biol. Chem., 52: 1425-1431. Kirk, O., T .V. Borchert and C.C. Fugls ang, 2002. Indus trial enzyme applications . Curr. Opin. Biotechnol., 13: 345-351. Kotchoni, O.S., O.O. Shonukan and W .E. Gachomo, 2003. Bacillus pumilus BpCRI 6, a promis ing candidate for cellulas e production under conditions of catabolite repres s ion. A frican Journal of Biotechnology, 2(6):140-146. M andels , M . and J. W eber, 1969. The production of celluas es . In: Cellulas es and their applications . Hajny, G.J. and E.T. Res s e (ed.), A mer. Chem. Soc. A dv. Ser., 95: 391-414. M angelli, P. and F. Forchias s in, 1999. Regulation of the cellulas e comp le x production by Saccobolus saccaboloides, induction and repres s ion by carbohydrates . M ycologia., 91(2): 359 - 364.

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Aust. J. Basic & Appl. Sci., 3(3): 2429-2436, 2009 M uthuvelayudham, R. and T. Viruthagiri, 2006. Fermentative production and kinetics of cellulas e protein on Trichoderma reesei us ing s ugarcane bagas s e and rice s traw. A frican Journal of Biotechnology, 5(20): 18731881. Nakamura, K. and K. Kppamura, 1982. Is olation and identification of crys talline c e llulos e hydrolyzing bacterium and its enzymatic properties . J. Ferment. Technol., 60(4): 343 - 348. Naras imha, G., A . Sridevi, B. Vis w a n a th, S.M . Chandra and R.B. Reddy, 2006. Nutrient effects on production of cellulolytic enzymes by Aspergillus niger. A frican Journal of Biotechnology, 5(5): 472-476. Niranjane, A .P., P. M adhou and T.W . Ste v e n s on, 2007. The effect of carbohydrate carbon s ources on the production of cellulas e by Phlebia gigantea. Enzyme and M icrobial Technology, 40(1): 1464-1468. Park, J.T. and M .J. Johns on, 1949. A s ubmicro-determination of glucos e. J. Biol. Chem., 181: 149-151. Paul, J., A .K. Varma, 1993. Hydrolytic enzymes production in Micrococcus roseus growing o n d ifferent cellulos ic s ubs trates . Lett. A ppl. M icrobiol., 16: 167- 169. Ray, A .K., A . Bairagi, K.S. Ghos h and S.K. Sen, 2007. Optimization of fermentation conditions for cellulas e production by Bacil l u s s u btilis CY5 and Bacillus circulans TP3 is olated from fis h gut. A cta Ichthyologica ET Pis catoria, 37(1): 47-53. Shikata, S., K. Saeki, H. Okos hi, T. Yos himats u, K . O za ki, S. Kawai and S. Ito, 1990. A lkaline cellulas e for laundry detergents : production by alkalophilic s t ra in s of Bacillus and s ome properties of crude enzymes . A gri. Biol. Chem., 52: 91-96. Shin, C.S., J.P . Le e , P.S. Lee and S.C. Park, 2000. Enzyme production of Trichoderma reesi Rut C-30 on a various lingocellulos ic s ubs trates . A ppl. Biochem and Biotechnol., 84-86: 237-245. Tomme, P., R.A . W arren and N.R. Gilke s , 1995. Cellulos e hydrolys is by bacteria and fungi. A dv. M icrob. Phys iol., 37: 1-81. Zaldívar, M ., J.C. Velás quez, I. Contreras and L.M . P é re z, 2001. Trichoderma aureoviride 7-121, a mutant with enhanced production of lytic en zy mes : its potential us e in was te cellulos e degradation and/or biocontrol. EJB Electronic Journal of Biotechnology, 4(3):1-9. http://www.ejb.org/content/vol4/is s ue3/full/7 Zhang, Y-HP. and L.R. Lynd, 2004. Toward an agg re g a t e d unders tanding of enzymatic hydrolys is of cellulos e: noncomplexed cellulos e s ys tems . Biotechnol. Bioeng., 88: 797-824.

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