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

Abstract

Abs tract: Effect o f s o me n u tritio n al an d en v iro n men tal facto rs o n g r o w t h an d cellulas e p ro d u ctio n 3 by Bacillus alcalophilus S39 and Bacillus amyloliquefaciens C2 was inves tigated. Results indicated th at 1% carboxymethylcellulose (CMC) and 0.7% yeast extract were most effective as the carbon and nitro g en s o urces respectively. Initial pH 7 and 3% inoculum size found to be optimal for growth and cellu lase production. Incubation temperature at 30 and 45ºC achieved the highest activity of cellulase 3 for Bacillus alcalophilus S39 and Bacillus amyloliquefaciens C2 res p ectiv ely , an d th e s u itab le s h akin g rate was 150 an d 200 rp m.

Full text

Aus t ralian Jo urna l of Ba s ic and A pplied Sciences, 3(3): 2429-2436, 2009
ISSN 1991-8178
© 2009, INS In e t Pu b licat io n
Corre spondin g Author: Hoda Hassan Abdel-Azeem, Soil Microbiology Unit, Desert Research Center, Cairo, Egypt .
E-mail: hebamashhoor@hotmail.com
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Nutritional and Environmental Factors Affecting Ce llulase Production by Two
Strains of Cellulolytic Bacilli
Abou-Taleb, Khadiga A.A., Mashhoor, W.A., Nasr, Sohair A., Sharaf, M.S. and
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Abdel-Azeem, Hoda H.M.
2
Agricultural Microbiology Department, Faculty of Agriculture, Ain Shams University,
1
Cairo, Egypt.
Soil Microbiology Unit, Desert Research Center, Cairo, Egypt.
2
Abstract: Effe c t of s o me nutrit io n a l an d e n v iro n menta l fa c tors o n g r o w t h an d c ellula s e p ro d u c tion
3
by Bacillus alcalophilus S39 and Bacillus amyloliquefaciens C2 was inves tigated. Results indicated
th a t 1% carboxymethylcellulose (CMC) and 0.7% yeast extract were most effective as the carbon and
nitrog e n sources respectively. Initial pH 7 and 3% inoculum size found to be optimal for growth and
cellu las e production. Incubation temperature at 30 and 45ºC achieved the highest activity of cellulase
3
for Bacillus alcalophilus S39 and Bacillus amyloliquefaciens C2 re s pe ctiv ely, and th e s u it able shaking
rate was 150 an d 200 rp m.
Ke y wo r ds : N u trit io n al & e n v iro n menta l fa c tors , Ce llu las e pro d uct io n , B. alcalop hilus S39, B.
3
amyloliquefaciens C2 .
INTRODUCTION
Cellulose is t h e most abundant biomass on the earth (Tomme et al., 1995). It is the primary product of
photos ynthes is in terrestrial environments, and the most abundant renewable bioresource produced in the
biosphere (100 b illion dry tons/year) (Jarvis, 2003 and Zhang & Lynd, 2004). Cellulose is commonly degraded
by an e n zy me ca lled ce llu las e. This en zy me is p ro d u c ed b y s e v e r a l micro o rg anis ms , c o mmo n ly by bac teria
an d fungi (Bahkali, 1996; M ange lli & Fo rch ia s s in , 1999; Shin et al., 2000 a n d Immanuel et al., 2006).
Co mp lete enzymat ic h y d ro ly s is of e n zy me requires s y n erg is tic a c tion of 3 types o f en zy me s , n a me ly
cellobiohydrolase, endoglucanase or carboxymethycellulase (CMCase) and â-gluco s idases (Bhat, 2000).
Cellulas es are u s ed in the textile ind ustry for cotton s o ften in g a n d d e nim finis hing; in lau ndry de terge nt s for
co lor care, clean ing , an d anti-depo s ition ; in th e fo od ind ustry for mas hing; in the pu lp and paper ind ustries for
dein king, drainag e imp ro v emen t , a n d fib e r mo d ific atio n a nd th e y a re eve n u s e d fo r p h a rma ceu t ic al ap p lic atio n s
(Kirk et al., 2002 an d Cherry & Fidan ts e f, 2003).
Bacteria, which has high growth rate as compared to fungi has good potential to be used in cellulase
production. However, the application of bacteria in producing cellulase is not widely used. Celluloytic property
of some bacterial genera such as Cellulomonas, Cellovibrio, Pseudomonas, Sporosphytophaga sp p. (Nakamura
and Kappamura, 1982); Bacillus and Micrococcus (Immanuel et al., 2 006) we re als o re p orted . Enzyme
production is closely controlled in microorganis ms and for improving its productivity these controls can be
ameliorated. Cellulase yields appear to depend on a complex relationship involving a variety of factors like
inoculum size, pH value, temperature, p re s ence of inducers, medium additives, aeration, growth time, etc.
(Imma n u el et al., 2006).
Th e p re s e n t wo rk wa s carried out t o o p timize t h e nu t rit io n al an d e n v iro n menta l p a r a m e t e r s for impro v ing
cellulase production by the two cellulolytic bacterial strains.
MATERIALS AND METHODS
Microorganisms Used:
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The two bacterial strains, Bacillus alcalophilus S39 and Bacillus amyloliquefaciens C2 used in this study
were isolated form soil and compost respectively and were distinguished as potent cellulase producers. The
purified bacilli isolates were identified according to their cultural, morphological and biochemical characteris tics
based on Bergey's Manual of Systematic Bacteriology (Claus and Berkeley, 1986) and Biolog Automated
System was u s ed .

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Media used:
Med ium (1): Nutrien t agar (Difco Man ua l, 1984) was us ed fo r th e maintenance o f Bacillu s strains.
Medium (2): Carboxymethyl cellulose medium recommended by Ray et al. (2007) was u s e d for the
production of cellulase by Bacillu s sp. It has the following c o mp o s i tion (g /l): Carbo xymethy lcellulose (CM C),
24 2 4 42 22 42
10; Try pto ne , 2; KH PO , 4; Na HPO , 4; MgSO .7H O, 0.2; CaCl .2H O, 0.001; FeSO .7H O, 0.004; A gar,
15 an d pH adjus ted to 7.
Inoculum Prep ara tio n and Fermentation Process:
For prep aration of s tan dard inoculum, both s t rains were cultured in nutrient broth individually at 30 ºC
for 24 h where an average viable count of 3.5 - 4.3 ×10 ce ll/ml culture broth was obtained. This was used
6
as the ino culum for the prod uction medium.
Fe rme n tat io n wa s c a rr ie d o u t in 250 ml p lu g ged Erlen me y er flas ks , ea c h cont a in in g 50 ml s t erile
pro d u c t ion med ium and ino culated with 3% o f stand ard inoc ulum (co ntaining a bou t 3.5 ×10 an d 4.3 ×10
66
3
cell/ml for Bacillus amyloliquefaciens C2 and Bacillus alcalop hilus S39, res pectively). Th e inoc ulat e d flasks
3
were incu b ated at 30 ºC and 45 ºC for Bacillu s alcalo p h ilus S39 and Ba cillus amyloliquefaciens C2 ,
res p ec tiv ely on ro ta ry sha ker a t 150 rp m for 72h .
Preparation of Crude Enzyme:
Aft er incubation, cultures were centrifuged at 1600 g for 15 min at 4°C and supernatants were used as
source of crude enzymes. The crude enzyme solution was utilized for determination of enzyme activities
(Kotchoni et al., 2003).
Enzyme Assa ys Pro ced ures:
Carboxymethyl-cellulase(CMCase) activity:
CMCase activ ity was assayed us ing a method described by Mandels and W eber (1969). The activity was
estimated using 1 % solution of carboxymethlycellulose (CMC) in 0.05 M citra te b u ffer (pH 4.8) as substrate.
The reaction mixture contained 1 ml citrate buffer, 0.5 ml of subs trate solution and 0.5 ml of suitably diluted
en zy me s o lu t i o n . T h e r e act io n wa s c a rrie d o u t at 50°C fo r 30 min. The a mo u n t of reducin g s u g a r re le as ed in
th e h y drolysis was mea s u r e d . On e u nit of CMCas e activity was expre s s ed as 1 ì mol of glucose liberated per
ml enzyme per minute.
Filter-paperase (FPase) Activity:
Th e a ctiv it y o f FPas e wa s a s s a y ed ac c o rd in g to th e meth o d e xpla in ed by M andels an d We b e r (1969). Th is
me t h o d is s imila r to th e CMCas e as s ay met hod , but th e s u b s tra te was Wha tman No. 1 filter pa per s trip (1 x
6 cm) s o a ke d in 1 ml 0 . 0 5 M s o d ium cit ra te b u ffer (p H 4.8). T h e s a mples we re in cubat e d wit h 0.5 ml e n zy me
solution at 50°C for 1 h, the reducing s u g a rs lib erated during growth were determined. One unit of FPase
activity was d etermined as 1 ì mol of glucose liberated per ml enzyme per minute.
â-Glucosidase Activity:
One-ten th ml of the cu ltu re sup ern atant was incu b a t e d wit h 0.5 ml of 0.05 M ac etate b uffer (p H 5)
containing 2.5 mg cellobiose. After incubation at 50 ºC for 10 min, the glucose releas ed was measured by the
gluco se o xidase p ero xidase metho d (Zaldívar et al., 2001).
Determination o f Red ucing Sugars:
Th e to t a l amount of reducing sugars was determined using potassium ferricyanide method, as described
by Park and Jo hns o n (1949).
Carbon Sources:
Th e a p p ro p ria te c a rb o n s o u rc e wa s s e le cte d b y re plac in g th e o rig in al carbon s u b s t ra te of the bas al med iu m
with equivalent carbon amount of each of the tested carbon s ources (Glucose, Carboxyme thyce llulose,
Cellobios e a nd Cellulos e).
Nitrogen So urces:
To detect the adequate nitrogen s ource for cellulase production by selected strains, the prescribed nitrogen
s o u rc e of the fe rme n t atio n me d ium wa s re p lace d b y equiva le n t nitrog e n a mount o f ea ch of the t es te d o r g a n ic
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[Beef extract, Casein, Malt, Pepto ne, Tryptone, Urea & Ye a s t e xtract] and ino rgan ic [KNO , (NH ) PO ,
3434 424
NaNO , NH NO , NH Cl & (NH ) SO ] nitrogen sources.

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pH:
Se v en valu e s o f p H ranged b e twe en 5.5 an d 8.5 were cho s en fo r s tu d y in g t h eir effects o n c e llulas e en zy me
to s elec t t h e mos t s u it able p H o f t h e pro d u c tion medium.
Incubation Temperature:
To d e t e rmin e the o p t imu m te mp e ra ture for cellu la s e p ro d u ctio n , fermen t a tion w as carried o u t a t va rio u s
temperature s in t he ran g e o f 5, 20, 25, 30, 35, 40, 45, 50 an d 55 ºC.
Shaki ng Ra te :
Erlenmeyer flasks (250 ml) containing production medium were inocu la ted with the selected strains and
placed ont o a rot ary s ha ker at different rpm (i.e. 0, 50, 1 00, 15 0 a nd 200 rpm) to obt ain pro pe r aeration for
maximal cellulase production.
Inoculum Size:
Th e inoculum size was optimized for maximal enzyme production. The fermentation mediu m wa s
inocu la ted w ith 1, 2, 3, 4, 5, 6 and 7 % of s tan d a rd in o c u lum.
Statistical A nalysis:
Th e collected data were s tatis tically an alyzed u s ing SPSS Co mpu te r Ana lys is Prog rams (Foster, 2001).
RESULTS AND DISCUSSION
Effect of Different Carbon Sources:
Data presented in Tab le (1) show that cellulase p rodu ction with b oth two Bacillus s trains was significantly
influ e n c e d b y t h e ty p e o f c arb o n s o u rc e in th e b as al med iu m. Ca rb o xymet h y lcellulo s e (CM C) wa s mo s t
effect iv e as a sole c a rb o n s o u rc e fo r c ellula s e e n zyme pro duct io n b y Bacil l u s a l c a l o philus S39 and Bacillus
3
amyloliquefaciens C2 , res u lts in incre ase in e nzyme activity , being 1.81 & 1.8 8 U/ ml of CM Cas e ac tiv ity , 0.87
& 0.86 U/ml of FPas e a ctivity an d 1.3 1 & 1. 41 U/ ml of â-glucosidas es, res pect ively. Th es e va lues were
followed , in d escend ing o rder b y cellobio s e a nd cellulo se.
These results are in agreement with thos e of Narasimha et al. (2006) and Niranjane et al. (2007) who
fo u n d tha t carboxymeth y l c ellu lo s e wa s t h e bes t ca rb o n s o u rc e fo llo we d b y cellu lo s e fo r ce llu las e product io n .
A higher production of cellulase when CMC served as substrate may be as a result of induction of the enzyme,
since cellulose is known to be a universal inducer of cellula s e s y nthesis. Paul and Varma (1993) had reported
th e in duc tion o f end ocellulas e by CM C.
Medium containing glucose as the growth carbon source presented the minimum cellulase ac t ivity
(expressed by CMCase, FPase and â-g lu cos idase). Muthuvelayudham and Viruthagiri (2006) obtained similar
results which showed that the cellulase activity was less when glucose was used as carbon source because of
inhibition.
A n o ther experimen t wa s c a rrie d o u t to s t u d y t h e effect o f d ifferen t conce n t ra tion s o f
carbo xymeth ylcellulos e (CMC) which exhibited sup eriority among o th er tes ted carbo n so u rces for Bacillus
s train s . Data in Fig. (1) clearly show that 1% carboxymethylcellulose (CM C) gave the highest activity of
cellulase being 1.85 & 1.88 U/ml of CM Case; 0.87 & 0.87 U/ml of FPas e a nd 1.35 & 1.40 U/ml of â-
3
glucosidases by B. alcalophilus S39 and B. amyloliquefaciens C2 , res p ect iv e ly. This is s imilar wit h p r e v io u s
investig at ion s (Fu ku mori et al., 1985; Kawa i et al., 1988 and Shikata et al., 1990) where the CMCase activity
in Bacillus s p . wa s detected in cu ltu res tha t c ont ain ed 1% (w/v ) CM C as the g rowth s ubs t rat e.
Effect of Different Nitrogen Sources:
To evaluate the effect of nitrogen source on cellulase formation, the nitrogen source in the basal me dium
wa s replaced by different nitrogen s ources. Data revealed that the supplementation of organic and in o rg a nic
nitrogen sources stimula ted the cellulase yield and activity. Using of organic N sources responded in the
positive cellulase activity more than the inorganic ones. Among the tested complex N sources, the effectiveness
in supporting cellulase production and cellulolytic activity by both Bacillus strains significantly decreas ed in
4
th e fo llo wing order: ye a s t extra ct > pep t o n e > b eef extract > NH Cl. Re s u lts re corded in T able (2) c learly
sho w t hat ye as t extrac t was th e be s t n itro gen s ourc e fo r bot h strains giving 2.07 & 2.17 U/ml of CM Ca s e, 0.99
& 1.01 U/ml of FPas e a n d 2. 18 & 2.5 5 U/ml of â-gluco s idases for Bacillus alcalo philus S39 and Bacillus
3
amyloliquefaciens C2 , respectively. Data are in accordance with the res ults of Ray et al. (2007) who reported
t h a t o rg a n ic nitro g e n s o u rc e s were fo u n d t o b e more s u itab le fo r o p t imizing ce llu las e pro duct io n b y Bac illu s
subtilis and Bacillus circulans t han ino rgan ic s ourc es .

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Table 1: Effect of carbon sources on the production of cellulase enzyme by the two Bacillus strains.
3
Different carb on s ources Bacillus alcalophilus S39 Bacillus amyloliquefaciens C2
----------------------------------------------------------------- -- - -- - - -- - --- - -- - - -- - - ----------------------------------- -- - - -- - - --
Biomass C ellulase Activity (U/ml) Biomass Cellulase Activity (U/ml)
g/1 00m l ----------------------------------------------- g/1 00m l ----------------------- - -- - - -- - - -- - - -- - - -- - - --------
CMCase FPase â- g lucos idas es CM C ase FP ase â- glu cosi dases
Glucose 0.501 0.23 0.25 0.05 0.484 0.30 0.10 0.04
a eff beef
Carboxymethycellulose
(CMC) (Control) 0.361 1.81 0.87 1.31 0.382 1.88 0.86 1.41
f aab eaaa
Cellobiose 0.403 0.71 0.19 0.13 0.392 1. 32 0.34 0.06
c dde dbcf
Cellulose 0.157 1.00 0.41 1.27 0.295 1.43 0.51 1.24
hc cc gbbd
Values in the same column followed by the same letter do not significantly differ from each other, ac co rd i ng to D uncan’s at 5 % level.
Table 2: Effect of nitrogen sources on the production of cellulase enzyme by Bacillus strains.
3
Nit rogen sources Bacillus alcalophilus S39 Bacillus amyloliquefaciens C2
------------------------------------------------------------------ ----------- -- - - -- - - -- - --- - -- - - -- - - -- - - -- - - -- -----------------------
Biomass C ellulase Activity (U/ml) Biomass Cellulase Activity (U/ml)
g/1 00m l ------------------------------------------------ g/ 100m l ---------------------- - -- - --- - -- - - -- - - -- - - -- - -------
CMCase FPase â- g lucos idas es CM C ase FP ase â- glu cosi dases
Beef extract 0.417 2.05 0.98 2.04 0.420 1.99 0.99 2.25
c bcc bccb
Casein 0.395 0.41 0.04 0.67 0.413 0.03 0.01 0.64
gh l n lmn d n o mn
Malt 0.356 1.18 0.01 0.73 0.380 0.31 0.04 0.68
p j o lm k m mn lmn
Peptone 0.407 2.05 0. 99 2.16 0.381 2.14 1.00 2.47
e bcb kaba
Tryptone (Control) 0.360 1.83 0.87 1.34 0.387 1.88 0.89 1.41
ofih iefigh
Urea 0.375 1. 50 0.47 0.70 0.385 1.64 0.64 0. 75
lm h k lmn ij g j kl
Yeast extract 0. 404 2.07 0.99 2.18 0.417 2. 17 1.01 2.55
efbcb caaa
3
KNO 0.407 1.24 0.06 1.06 0.420 0.81 0.05 0.81
eilj bkmk
43 4
(NH ) PO 0. 378 0. 41 0.05 0.64 0.395 1.14 0.02 0.98
l l mn ghj o j
3
NaNO 0.374 1.88 0.89 1.62 0.370 1.90 0.90 1.10
lm ef h e lmn ef g i
43
NH NO 0.397 1.88 0.90 1.42 0.371 1.89 0.92 1.55
g ef gh fg lmn ef f f
4
NH Cl 0.359 1.97 0.96 1.77 0.361 1.93 0.98 1. 80
o cd e d o cde c d
42 4
(NH ) SO 0. 424 1. 91 0.92 1.49 0.417 1.91 0.97 1.55
a de ff cde d f
Values in the same column followed by the same letter do not significantly differ from e ach ot h er , according to Duncan’s at 5 % level.
Data illus trated in Fig. (2) obviously indicates that suitable concentration of yeast extract was found to
be 0.7% which gave the highest CMCase being 2.35 & 2.30 U/ml; FPase being 1.15 & 1.19 U/ml and â-
3
gluco sidase s be ing 3.56 & 3.49 U/ml of B. alcalop hilus S39 and B. amyloliquefaciens C2 , respectively.
It is notable at all experiments to state th at t here was no relationship between the production of cellulase
enzyme and biomass yield.
Effect of initial pH:
Cellulas e y ie l d by both s train s appe a r t o d epe n d o n p H v a lu e. Re s u lts illus trate d b y Fig . (3) c learly show
th at cellu l a s e p r o d u c tion, expressed as en zyme act ivity , gradu ally increas ed as t he pH values increas ed from
6.5 t o 7.5 an d rea c h ed it s ma ximum a t initia l p H o f 7 b e in g 2 . 4 1 & 2.40 U/ ml o f CM Cas e, 1.19 & 1.19 U /ml
3
of FPas e a nd 3.55 an d 3.49 U/ml of â-glucosidases by B. alcalophilus S39 and B. amyloliquefaciens C2 ,
respectively. The cellulas e activity was less in other tested pH levels, where enzyme activity was minimal at
pH 5.5 and it indicates a marginal increas e a t pH 6.5 and 7. Further, this activity was greatly reduced to reach
th e lo west at pH 8.5 (wh ere 2.06 & 2.07 U/ ml of CM C a s e; 1.07 & 1.04 U/ml of FPas e an d 0.72 an d 0.56
3
U/ml of â-glucosidases was obtained by B. a lca lo ph ilus S39 and B. amyloliquefaciens C2 , res p ectively.
Obtained data confirmed the findings reported by Ray et al. , ( 2007) wh o mentioned th at pH 7 – 7.5 more
suitable for optimization of cellulase production by Bacillus subtilis and B. circulans. Furthermore, the
ce llu lo ly tic e n zy me, endog lu c o n as e obta in ed fro m Cellulomonas, Bacill u s, a nd Micrococcus spp. hydrolyzed
s u b s t ra te in t h e pH ra n g e of 4.0 t o 9.0, wit h maximum act iv i t y t r a n s p i r ing at p H 7 (Immanuel et al., 2006).
Incubation Temperature:
Like pH, t emperat u re is a ls o an importan t fa cto r th a t influen c e s t h e ce llu las e yie ld . It is o b v io u s from Fig .
(4) that the highes t c ellulas e activity was ob ta ined at te mperatu res 30 to 45ºC fo r B. alcalophilus S39 an d B.
3
amyloliquefaciens C2 respe ct ive ly, wh ereas it was less at ot her tes ted de grees for e ac h strain .
These results are clos ed to thos e of Bakare et al. (2005) who found that the cellulase en zy me p roduce d
by Pseudomonas fluorescence was activated at 30 to 35 ºC showing the optimum temperature at 35 ºC. Ray
et al. (2007) repo rte d th at minimum cellulas e yield was ob s erv ed wh en fe rmen ta tion was carried o ut at 45°C,
while maximum yield was obtained at 40°C by Bacillus subtilis and Bacillus circulans. Immanuel et al. (2006)
also recorded maximum endoglucanase activity in Cellulomonas, Bacillus and Micrococcus sp. at 40°C and
neu tral pH.

Aust. J. Basic & Appl. Sci., 3(3): 2429-2436, 2009
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Fig. 1: Effe ct of different concentrations of carboxymethylcellulose (CMC) on the production of cellula s e
enzyme by Bacillus strains.
Fig. 2: Effect of different concentrations of yeast extract on the production of cellula se enzyme by Bacillus
strains.
Fig. 3: Effect of initial pH on the production of cellulase enzyme by Bacillus strains.

Aust. J. Basic & Appl. Sci., 3(3): 2429-2436, 2009
2434
Fig. 4: Effect of incubation temperature on the production of cellulase enzyme by Bacillus strains.
Effect of Shaking Rate:
Concerning the effect of shaking rate on cellulase yield, it w as found from the current data (Fig. 5) that
th e maximum activ ity was obtained at t he ran ge of shaking rate of 150-200 rpm for B. alcalophilus S39 and
3
B. amyloliquefaciens C2 . No s ignifica nt ly d iffere nt was no ticed in e nzyme a ct ivit y p r o d u ce d at ra te of 150
an d 200 rp m.
Similar data was found by Bin Amwarali Khan and Hus aini (2006) who noticed a remarkable increase of
cellulase activity in fermentation medium under shaking condition compared to static condition. It was observed
more th a n 2 fo ld h ig h e r cellu la s e enzyme a c t iv ity in s haking co n d it ion (2.97 IU/ ml) c o mp are to n o n s h a kin g
co ndition (1.38 IU/ml) for Bacillus amyloliquefa ciens UMA S 1002 strain. They also reported that the highest
cellulase enzyme production by Bacillus amyloliquefaciens UMAS 1002 strain were 2.97 and 2.89 IU /ml at
ag it a tion spee d o f 100 and 200 rp m res p ect iv ely.
Inoculum Size:
Fig . (6) s h o ws that the in o c u lu m size of 3.0 % achieved the highest cellulase enzyme production being
2.40 & 2.39 U/ml o f CM Ca s e ; 1.20 & 1.18 U/ml o f F Pa s e and 3.61 & 3.53 U/ ml o f â-glucosidases by B.
3
alcalo ph ilu s S39 and B. amyloliqu efaciens C2 , respectively.
These res ults were almost similar with findings collected by Ray et al. (2007) e lucid a t ed th e enzyme
production increased gradually up to 3% inoculum size, but decreased thereafter. The enzyme pro d u ctio n b y
both strains Bacillus subtilis and Bacillus circulans in 3% ino culum size was n o t s ig n ifican tly different (P <
0.05) from that in 2% inoculum size.
In the present study, it could be concluded that carbon and nitrogen s o urces , p H value, temperature,
inoculum size and aeration play a most crucial role in cellulase production by B . alcalophilus and B.
amyloliquefaciens.
Fig. 5: Effect of shaking ra te on cellulas e p rodu ction of by Bacillus strains.

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2435
Fig. 6: Effect of inoc ulum size o n t he prod uction o f ce llulase p rod uction of b y Bacillus strains.
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