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New Biotechnology �Volume 25, Number 6 � September 2009 RESEARCH PAPER

Cellulase production from Aspergillusniger MS82: effect of temperature and pH

Muhammad Sohail, Roquya Siddiqi, Aqeel Ahmad and Shakeel Ahmed Khan

Department of Microbiology, University of Karachi, Karachi 75270, Pakistan

Abstract

Fungal cellulases are well-studied enzymes and are used in various industrial processes. Much of the

knowledge of enzymatic depolymerization of cellulosic material has come from Trichoderma cellulase

system. Species of Trichoderma can produce substantial amounts of endoglucanase and exoglucanase

but very low levels of b-glucosidase. This deficiency necessitates screening of fungi for cellulytic

potential. A number of indigenously isolated fungi were screened for cellulytic potential. In the present

study, the kinetics of cellulase production from an indigenous strain of Aspergillus niger MS82 is

reported. Product formation parameters of endoglucanase and b-glucosidase (Qp + Yp/s) indicate that A.

niger MS82 is capable of producing moderate to high levels of both endoglucanase and b-glucosidase

when grown on different carbon containing natural substrates, for example, grass, corncob, bagasse

along side purified celluloses. Furthermore, it was observed that the production of endoglucanase

reaches its maximum during exponential phase of growth, while b-glucosidase during the Stationary

phase. Enzyme production by solid-state fermentation was also investigated and found to be promising.

Highest production of cellulase was noted at pH 4.0 at 35 8C under submerged conditions. Growth and

enzyme production was affected by variations in temperature and pH.

IntroductionCellulose is a linear polymer of anhydroglucose units linked

together by b-1,4-glycosidic bonds and found as a major compo-

nent of plant biomass [1]. A variety of fungi and bacteria can

convert this insoluble substrate into soluble compounds by ela-

borating a group of enzymes, cellulases. Endo-b-D-glucanase (EC

3.2.1.4) is one of the major component enzymes of the cellulase

complex and catalyzes the hydrolysis of cellulose by randomly

splitting the sugar residues within the molecule. Exo-b-D-gluca-

nase (EC 3.2.1.19) and b-glucosidase (EC 3.2.1.37) can synergisti-

cally convert cellulose into glucose and hence are used on an

industrial scale [2].

On a commercial scale, conversion of cellulosic biomass

requires the use of cellulases that makes the process costly [3–

5]. Hence, cellulase production from a wide range of microorgan-

Corresponding author: Khan, S.A. (shakeelkhan@uok.edu.pk)

1871-6784/$ - see front matter � 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2009.02.002

isms has been studied extensively [6]; therefore, the screening and

characterization of novel isolates is essential to make the enzyme

production process feasible [7].

Many organisms produce cellulases to perform cellulolysis

necessary for growth and product formation under appropriate

conditions. Commercial cellulase preparations from Trichoderma

reesei are popular as it contains high activities of both exo-gluca-

nase and endo-glucanase but a low levels of b-glucosidases [8];

therefore, attention has recently been diverted to other micro-

organisms including the members of genus Aspergillus. Aspergillus

niger has widely been exploited as it possesses all the three essential

components of cellulase system [9]. Studies on fermentation para-

meters to obtain maximum yields of cellulase enzymes seem

necessary. Temperature and pH are regarded as the most important

features beside the use of suitable inducers in this regard. In an

earlier study a pH-dependent expression of cellulases has been

reported in Trichoderma reesei [10].

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RESEARCH PAPER New Biotechnology � Volume 25, Number 6 � September 2009

FIGURE 1

Growth and enzyme production kinetics of Aspergillus niger MS82. Key: Dry

mass ~, endoglucanase (+), b-glucosidase (�).

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Jahangeer et al., has previously reported the cellulolytic poten-

tial of some indigenously isolated fungi [11]. In the present study,

the kinetics of cellulase production from an indigenous strain of A.

niger MS82 was investigated. Growth kinetics of A. niger MS82 and

production kinetics of endoglucanase and b-glucosidase were

studied under submerged as well as solid-state fermentation by

using crude lignocellulosic materials from plants in addition to

commercially available cellulose.

Materials and methodsGrowth and enzyme production kinetics by A. niger MS82A large number of fungi were isolated from indigenous sources and

screened for cellulytic potential. One of the best cellulase producing

strains (A. niger MS82) was selected. A. niger MS82 was grown in basal

medium [12], which contains (g/L) KH2PO4, 2.0; (NH4)2SO4, 1.4;

Urea, 0.3; MgSO4.7H2O, 0.3; CaCl2, 0.1; Peptone, 1.0; Tween 80, 2.0;

FeSO4.7H2O, 0.005; MnSO4.H2O, 0.0016; ZnSO4.7H2O, 0.0014 and

CoCl2.6H2O, 2.9; supplemented with 1% (w/v) of various carbon

sources (e.g. low viscosity carboxymethyl cellulose, CMC; salicin or

glucose), and incubated in an orbital shaker at 180 rpm for six days.

Growth and enzyme production kinetics was studied at 258, 308, 358and 40 8C, in the basal medium at pH 4.0, 5.0, 6.0 and 7.0, followed

by separation of cells and residual insoluble carbon source by

filtration through a thick layer of glass wool. Filtrate was subjected

to centrifugation at 6000 rpm for 20 min and the clear cell-free

culture supernatant thus obtained was used for further studies.

Growth was measured by taking dry mass. Enzyme production

kinetics was only studied in medium containing insoluble carbon

sources. Cell-free culture supernatants were assayed to study

enzyme production pattern over different phases of growth. All

experiments were performed in triplicate.

Enzyme assaysEndoglucanase and b-glucosidase activity was determined by

incubating enzyme preparation (500 ml) in the presence of

500 ml of substrate 1% (w/v) low viscosity carboxymethyl cellulose

for endoglucanase; 1% (w/v) salicin for b-glucosidase in 50 mM

sodium citrate buffer (pH 4.8). The reaction was carried out at

50 8C for 30 min, as the assay was linear for 30 min. The amount of

reducing sugar was determined by dinitrosalisylic acid (DNS)

method [13]. One International Unit (IU) of enzyme was defined

as the amount of enzyme that releases one mmol of reducing sugar

per minute under standard assay conditions.

Solid-state fermentation (SSF)Solid-state fermentation (SSF) was carried out according to the

modified method of Kang et al. [14]. Briefly, dried and ground

lignocellulosic substrates (grass, corn cob and bagasse) 2 g each

were placed in a 250 ml Erlenmeyer flask, Petri dishes (8 inches

diameter), and test tubes (18 mm � 150 mm), carbon-deficient

basal medium was added to attain a moisture level �65% and

autoclaved at 121 8C for 30 min. Spore suspension (5 � 105 spores/

g substrate) was inoculated, mixed and incubated at 35 8C. Sam-

ples were periodically drawn by agitating the contents in 50 ml of

sodium citrate buffer (50 mM, pH 4.8) followed by filtration

through four layers of muslin cloth and centrifugation at 5000 g

for 20 min. The supernatant was used as crude preparation to study

the enzyme production kinetics.

438 www.elsevier.com/locate/nbt

ResultsGrowth and enzyme production kinetics in shake flaskGrowth and enzyme production kinetics revealed that in the

presence of CMC (Figure 1) and salicin (data not shown) the

endoglucanase production started earlier (i.e. during lag phase)

than b-glucosidase and its titer continued to increase until early

stationary phase and then remained constant. While b-glucosidase

production was started at the beginning of log phase and con-

tinued to increase until the end of the experiment (i.e. 225 h). A

similar pattern of enzyme production, with variations in titer, was

noted at different incubation temperatures and pH. No endoglu-

canase or b-glucosidase production was noted in the presence of

glucose as sole carbon source.

The highest titer of endoglucanase was obtained when A.niger MS82 was grown at 30 8C as well as 35 8C with an initial

pH 4.0 of the medium. Enzyme production decreased

drastically at pH 5 and remained approximately constant up to

7. The endoglucanase titer was slightly decreased when the

incubation temperature was 25 8C; however, the level of

enzyme continued to decrease with the increase in the pH of

the medium. An increase in incubation temperature to 40 8Cdrastically affected the endoglucanase production at all pH

(Figure 2a).

Unlike endoglucanase, the highest levels of b-glucosidase were

obtained at 25 8C at pH 4.0, which decreased drastically with an

increase in pH of the medium to 5 and continued to decrease

slightly with an increase in pH to 7. There was a slight reduction in

the rate of production when the incubation temperature was 30 8Cand 35 8C. The b-glucosidase production was drastically affected at

40 8C and continued to decrease with an increase in pH and

become negligible at pH 7.0 (Figure 2b).

The growth kinetic studies on A. niger MS82 indicate that there

was a maximum rate of growth at 30 8C in a medium having initial

pH of 4.0 with a generation time (g) of 1.7 h. The generation time

continued to gradually increase with an increase in the pH of the

growth medium up to 7.0 (g 9 2.6 h). Nonetheless, the variation in

the pH value did not affect the growth rate at the lowest and the

New Biotechnology �Volume 25, Number 6 � September 2009 RESEARCH PAPER

FIGURE 2

Effect of growth temperature and pH on volumetric production of

endoglucanase (a) and b-glucosidase (b). Key: 25 8C &, 30 8C *, 35 8C ~

and at 40 8C !.

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highest incubation temperatures, that is, 258 and 40 8C. The gen-

eration time, g, was slightly increased to 2.4 h at 35 8C and it was

further increased to 3.1 h at 40 8C (Table 1).

In all the experiments, the pattern of change in pH during the

growth was also studied (data not shown). It was noted that there

was a sharp decrease in pH to 4.0 within first 20 h of incubation,

especially when the initial pH of the medium was adjusted to 6.0 or

7.0; the pH remained constant thereafter. However, when the pH

of the medium was 4.0 or 5.0, it decreased slowly and became

steady when it reached to �3.5 when the cells were in late log

TABLE 1

Effect of temperature and pH on generation time (g; in hours)

Temperature (8C) pH of the medium

4.0 5.0

25 2.3 � 0.05 2.3 �30 1.68 � 0.02 1.71

35 2.42 � 0.04 2.53

40 3.0 � 0.001 3.05

phase. It was also observed that endoglucanase and b-glucosidase

production was not initiated until the pH of the medium was

lowered to �4.0.

Solid State Fermentation (SSF)Significant differences in the levels of endoglucanase and b-glu-

cosidase were noted when SSF was carried out in different vessels

containing grass and corncob as substrates (Table 2). However,

there was not much difference in the levels of these enzymes when

bagasse was used as substrate. The highest titers of endoglucanase

and b-glucosidase were obtained from grass when SSF was carried

out in Petri plates and in test tubes; however, the enzyme produc-

tion was decreased in flasks. Contrary to grass, corncob gave

maximum yield of these enzymes when flasks were used for SSF.

Higher levels of endoglucanase were obtained in Petri plates con-

taining corncob, whereas, tubes gave higher yield of b-glucosidase.

A comparison among the levels of enzymes between the sub-

merged conditions and SSF using carboxymethyl cellulose (CMC),

cellulose acetate, sigma-cell, avicel and filter paper along side the

crude carbon substrate revealed the highest levels of enzymes in

grass containing medium that was comparable to the level of

enzyme production in phosphoric acid swollen cellulose (PASC)

containing medium under similar conditions. These results

further indicate that the use of crude carbon substrates by the

strain A. niger MS82 may be used for the production of enzymes in

a cost effective manner (Table 3).

DiscussionA large number of microorganisms are capable of producing

cellulases; however, fungi are considered most active against the

most abundantly available natural polymer. Different strains

belonging to Trichoderma sp. have been most extensively studied

in this regard. A number of studies revealed that Aspergillus sp.

produce relatively large quantities of endoglucanase and b-gluco-

sidase, but low levels of exoglucanase, together with high levels of

protein make it an ideal organism for industrial applications

[7,15].

Studies conducted by Iyayi [16] and Kang et al. [14] revealed the

production of higher levels of cellulases by indigenous strains. In

the present study, enzyme production by the indigenous strain of

A. niger MS82 also revealed that it was capable of growing not only

on various commercial preparations of cellulose but also on agri-

cultural residues as well, with high titers of enzymes. The finding

of the present study is also in line with a study carried out by Kim

et al. [17] that indicates the considerable levels of cellulases and

xylanases by A. niger KK2 strain under submerged conditions. In a

later study, Kang et al. [14] reported that the KK2 strain of A. niger

6.0 7.0

0.05 2.31 � 0.07 2.33 � 0.01

� 0.02 2.05 � 0.02 2.4 � 0.06

� 0.05 2.78 � 0.1 2.85 � 0.05

� 0.02 3.05 � 0.04 3.1 � 0.02

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RESEARCH PAPER New Biotechnology � Volume 25, Number 6 � September 2009

TABLE 2

Endoglucanase and b-glucosidase production (IU/ml) using SSF of lignocellulosic substrates in different fermentation vessels

Lignocellulosic substrate Enzyme Fermentation vessel

Flask Petri dish Test tubes

Grass Endoglucanase 0.11 � 0.006 0.43 � 0.02 0.35 � 0.08b-glucosidase 0.17 � 0.01 0.46 � 0.03 0.27 � 0.1

Corncob Endoglucanase 0.35 � 0.02 0.22 � 0.01 0.08 � 0.007

b-glucosidase 0.24 � 0.03 0.05 � 0.006 0.11 � 0.005

Bagasse Endoglucanase 0.31 � 0.01 0.35 � 0.05 0.35 � 0.07

b-glucosidase 0.31 � 0.04 0.28 � 0.008 0.27 � 0.05

TABLE 3

Product formation parameters on different cellulosic substrates at 35 8C and pH 4.0

Substrate Enzyme Qp (IU/L h)a Yp/s (IU/g cellulosic material)b

Carboxymethyl cellulose Endoglucanase 2.648 � 0.05 50.32 � 1.3b-glucosidase 1.735 � 0.01 25.16 � 1.1

Phosphoric acid swollen cellulose Endoglucanase 2.84 � 0.02 48.1 � 1.2

b-glucosidase 4.65 � 0.11 77.7 � 4.3

Filter paper Endoglucanase 1.95 � 0.08 24.05 � 1.0

b-glucosidase 2.76 � 0.03 31.82 � 0.8

Sigma-cell Endoglucanase 2.91 � 0.1 40.7 � 2.8b-glucosidase 3.34 � 0.09 38.48 � 1.9

Cellulose acetate Endoglucanase 1.56 � 0.05 19.2 � 1.1

b-glucosidase 2.371 � 0.06 18.5 � 0.9

Grass Endoglucanase 4.11 � 0.1 59.2 � 3.5b-glucosidase 6.896 � 0.15 100 � 6.6

a Qp, volumetric rate of enzyme production is defined as total IU of enzyme produced in one hour when organism was grown in 1 L medium.bYp/s, specific rate of enzyme production, total IU of enzyme produced per g of cellulosic material.

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can produce cellulases under solid-state fermentation conditions.

Other studies on fermentation of agro-industrial by-products, by

A. niger, A. flavus and Penicillium sp., led to a conclusion that the

strains of A. niger are better in degrading cellulose in the crude

natural substrates [16]. The present strain of A. niger MS82 was also

efficiently able to utilize lignocellulosic biomass (grass) with high

volumetric levels (Qp) of endoglucanase and b-glucosidase, up to

4.11 and 6.896 IU, respectively, in contrast to 1.7 FPU/ml of

cellulase in Czapek-Dox medum containing 1% cellulose, by

another strain of A. niger [18].

Juhasz et al. [19] reported about the higher levels of endoglu-

canase than b-glucosidase from Trichoderma reesei Rut C30 strain

when grown on lignocellulosic materials after seven days. The

other studies with Trichoderma viride and Penicillium janthinellu-

mare revealed the higher levels of b-glucosidase and xylanase when

bagasse was used compared with purified cellulose; however, lower

yield of FPAse was noted in the same medium [20]. Our strain, A.

niger MS82 also produces higher levels of cellulases in grass con-

taining media compared with other natural lignocellulosic mate-

rials.

Victor et al. [21] obtained 0.0743, 0.0573 and 0.0502 IU/ml of

cellulase within 12 h when sawdust, bagasse and corncob,

respectively, were used as substrates from a strain of Aspergillus

flavus. Spiridonov and Wilson [22] revealed the higher yield of

cellulases from Thermomonospora fusca when grown on solka-

floc, the enzyme yield was however decreased on ground grass; a

440 www.elsevier.com/locate/nbt

further 12–30 times decrease in enzyme yield was noted on

glucose and xylan containing medium. A. niger MS82 has also

shown a similar pattern of both, endoglucanase and b-glucosi-

dase production.

Present study indicates that the yields of b-glucosidase and

endoglucanase from A. niger MS82 were increased with the

increase in the amount of growth that was in agreement with

another study conducted by Sachslehner [23], where the levels

of mannanase, cellulase and xylanases increased with an

increase in the concentration of mycelia by Sclerotium rolfsii

(0.5–2.0 mg/ml).

Xiong et al. [24] reported that there was a correlation between

the initial pH of the medium and cellulase titers by Trichoderma

reesei Rut C-30. It was reported that Trichoderma reesei Rut C-30

produced higher yields of cellulases when the pH of the medium

containing lactose was adjusted to 4.0–4.5. This study also suggests

that the acidic pH (4.0) of the medium induces the production of

cellulases, as mentioned earlier [10,24] either by glycosylation of

cellulases [25] or by some other means. Slow growth of A. niger

MS82 and longer lag phase was observed at higher temperatures

(358 and 40 8C), which was followed by a shorter log phase.

Contrary to this finding, Castro et al. [26] reported a longer log

phase and higher levels of xylanase by Aspergillus FP-470 at 45 8C.

However, the enzyme production ability of A. niger MS82 reaches

its peak level during log phase, for endoglucanase, and stationary

phase of fungal growth for b-glucosidase. This observation is

New Biotechnology �Volume 25, Number 6 � September 2009 RESEARCH PAPER

similar to an earlier study carried out by Tamburini [27]. These

findings suggest that A. niger MS82 strain is capable of producing

higher titers of cellulases and may be used for possible biotechno-

logical applications.

AcknowledgementAuthors gratefully acknowledge the support of Higher Education

Commission, Pakistan to one of the authors under 200 Merit

Scholarship Scheme for Ph.D. studies.

References

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[1] Beguin, P. and Aubert, J.P. (1994) The biological degradation of cellulose. FEMS

Microbiol. Lett. 13, 25–58

[2] Bhat, M.K. (2000) Cellulases and related enzymes in biotechnology. Biotechnol.

Adv. 18, 355–383

[3] Wayman, M. et al. (1992) Bioconversion of waste paper to ethanol. Proc. Biochem.

27, 239–245

[4] Szijarto, N. et al. (2004) Cellulase fermentation on a novel substrate (waste

cardboard) and subsequent utilization of home-produced cellulase and

commercial amylase in a rabbit feeding trial. Indust. Crops Prod. 20, 49–57

[5] Zhang, Y.H.P. et al. (2006) Outlook for cellulase improvement: screening and

selection strategies. Biotechnol. Adv. 24, 452–481

[6] Lynd, L.R. et al. (2005) Consolidated bioprocessing of cellulosic biomass: an

update. Curr. Opin. Biotechnol. 16, 577–583

[7] Duarte, J.C. and Costa-Ferreira, M. (1994) Aspergilli and lignocellulosics:

enzymology and biotechnological applications. FEMS Microbiol. Rev. 13, 377–386

[8] Rosgaard, L. et al. (2006) Efficiency of new fungal cellulase systems in boosting

enzymatic degradation of Barley Straw Lignocellulose. Biotechnol. Prog. 22, 493–498

[9] Shaukat, F. et al. (2002) Kinetics of b-glucosidase production by Escherichia coli

recombinants harboring heterologous bgl genes. Biotechnol. Lett. 24, 1803–1806

[10] Sprey, B. and Lambert, C. (1983) Titration curves of cellulases from Trichoderma

reesei: demonstration of a cellulase-xylanase-b-glucosidase-containing complex.

FEMS Microbiol. Lett. 18, 217–222

[11] Jahangeer, S. et al. (2005) Screening and characterization of fungal cellulases

isolated from the native environmental source. Pak. J. Bot. 37, 739–748

[12] Mandels, M. and Weber, J. (1969) The production of cellulases and their

applications. Adv. Chem. Ser. 95, 391–414

[13] Miller, R.G. and Phillips, R.A. (1969) Separation of cells by velocity

sedimentation. J. Cell. Physiol. 73, 191–201

[14] Kang, S.W. et al. (2004) Prodcution of cellulases and hemicellulases by Aspergillus

niger KK2 from lignocellulosic biomass. Bioresour. Technol. 91, 153–156

[15] De Vries, R.P. and Visser, J. (2001) Aspergillus enzymes involved in degradation of

plant cell wall polysaccharides. Microbiol. Mol. Biol Rev. 65, 497–522

[16] Iyayi, A.E. (2004) Changes in the cellulose, sugar and crude protein contents of

agro-industrial by-products fermented with Aspergillus niger. Aspergillus flavus and

Penicillium sp. Afr. J. Biotech. 3, 186–188

[17] Kim, S.W. et al. (1997) Cellulase and xylanase production by Aspergillus niger KKS

in various bioreactors. Bioresour. Technol. 59, 63–67

[18] Narasimh, G. et al. (2006) Nutrient effects on production of cellulytic enzymes by

Aspergillus niger. Afr. J. Biotechnol. 5, 472–476

[19] Juhasz, T. et al. (2005) Characterization of cellulases and hemicellulases produced

by Trichoderma reesei on various carbon sources. Process Biochem. 40, 3519–3525

[20] Adsul, M.G. et al. (2004) Polysaccharides from bagasse: applications in cellulose

and xylanase production. Carbohydr. Polymers 57, 67–72

[21] Victor, O.T. et al. (2003) Celluluase production by Aspergillus flavus Linn isolate

NSPR 101 fermented in saw dust, bagasse and corn cob. Afr. J. Biotechnol. 2, 150–

152

[22] Spiridonov, N.A. and Wilson, D.B. (1998) Regulation of biosynthesis of

individual cellulase in Thermomonsopora fusca. J. Bacteriol. 180, 3529–3532

[23] Sachslehner, A. et al. (1998) Induction of mannanase, xylanase and

endoglucanase activities in Sclerotium rolfsii. Appl. Environ. Microbiol. 64, 594–600

[24] Xiong, H. et al. (2004) Influence of pH on the production of xylanase by

Trichoderma reesei Rut C-30. Process Biochem. 39, 729–733

[25] Stals, I. et al. (2004) Factors influencing glycosylation of Trichoderma reesei

cellulases. I: Postsecretorial changes of the O- and N-glycosylation pattern of

Cel7A. Glycobiology 14, 713–724

[26] Castro, L.P.M. et al. (1997) Thermostable xylanases produced at 37 8C and 45 8C

by a thermotolerant Aspergillus strain. FEMS Microbiol. Lett. 146, 97–102

[27] Tamburini, E. Perito, B. and Mastromi, G. (2004) Growth phase-dependent

expression of an endoglucanase encoding gene (egl s) in Streptomyces rochei A2.

FEMS Microbiol. Lett. 237, 267–272

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