African Journal of Biotechnology Vol. 2 (6), pp.

150152, June 2003

Available online at http://www.academicjournals.org/AJB
ISSN 16845315 2003 Academic Journals

Cellulase Production by Aspergillus flavus Linn Isolate

NSPR 101 fermented in sawdust, bagasse and corncob
OJUMU, Tunde Victor1*, SOLOMON, Bamidele Ogbe2,e, BETIKU, Eriola2,, LAYOKUN, Stephen
Kolawole 2, and AMIGUN, Bamikole3
1
Engineering Materials Development Institute, P.M.B 611, Akure Nigeria.
2
Department of Chemical Engineering, Obafemi Awolowo University, Ile-Ife, Nigeria.
3
Department of Food Science and Technology, Federal Polytechnic Ado-Ekiti, Ekiti State, Nigeria.
Accepted 23 May 2003

Bagasse, corncob and sawdust were used as lignocellulosic substrates for the production of cellulase
enzyme using Aspergillus flavus after ballmilling and pretreatment with caustic soda. From the
fermentation studies, sawdust gave the best result with an enzyme activity value of 0.0743IU/ml while
bagasse and corncob gave 0.0573IU/ml and 0.0502IU/ml respectively. The three lignocellulosics gave
their maximum enzyme activities at about the twelfth hour of cultivation, suggesting that the 12th hour
is the optimum time when the enzyme may be harvested.

Key words: Aspergillus flavus, cellulase activity, lignocellulosics.

INTRODUCTION

Agricultural wastes and in fact all lignocellulosics can be years. It is now a subject of intensive research as a
converted into products that are of commercial interest contribution to the development of a large-scale
such as ethanol, glucose, and single cell protein conversion process beneficial to mankind (Kumakura,
(Solomon et al., 1999). Cellulase enzyme has been 1997). Such process as suggested by Fan et al. (1987)
reported (Fan et al., 1987; Wu and Lee, 1997; Solomon and Kumakura (1997) would help alleviate shortages of
et al., 1999; Kansoh et al., 1999) for the bioconversion of food and animal feeds, solve modern waste disposal
lignocellulosics to these useful products. Solomon et al. problem, and diminish mans dependence on fossil fuels
(1990) achieved hydrolysis of sawdust using cellulase by providing a convenient and renewable source of
with activity of 0.0561IU/ml. energy in the form of glucose.
Lignocellulosics are abundant sources of carbohydrate, Some features of natural cellulosic materials are known
continually replenished by photosynthetic reduction of to inhibit their degradation/bioconversion (Solomon et al.,
carbon dioxide by sunlight energy (Fan et al., 1987). 1990, 1999). These are degree of crystallinity and
Thus they are the most promising feedstock for the lignification and the capillary structure of cellulose. The
production of energy, food and chemical (Wu and Lee, crystallinity and lignification limit the accessibility and
1997; Solomon et al., 1999). The bioconversion of susceptibility of cellulose to cellulolytic enzymes and
cellulosic materials has been receiving attention in recent other hydrolytic agents (Fan et al., 1987). However, many
physical, chemical and microbial pre-treatment methods
for enhancing bioconversion of cellulosic materials have
been reported (Kansoh et al., 1999; Depaula et al., 1999;
Solomon et al., 1999; Kumakura, 1997; Wu and Lee,
*Correspondence Author; E-mail: [email protected] 1997). Pre-treatment of cellulose opens up the structure

and removes secondary interaction between glucose

Present Address: German Research Centre for Biotechnology chains (Tang et al., 1996; Fan et al., 1987). Solomon et
(GBF), Biochemical Engineering Division, Mascheroder Weg 1, al. (1999) produced cellulase of 0.056425 IU/ml from the
D-38124 Braunschweig, E-mail: [email protected],
growth of Aspergillus flavus on bagasse pre-treated with
[email protected]
using ballmilling and caustic soda.
e
E-mail: [email protected] Since the production of cellulase enzyme is a major
factor in the hydrolysis of cellulosic materials, it is
 Ojumu et al. 151

important to make the process economically viable. After sterilisation, the vessel was cooled to room temperature
Although much work has been done on the production of and the impeller shaft was coupled and filters were plugged into
their receptacles. The inoculum was introduced aseptically and
cellulase from lignocellulosics (Solomon et al., 1999; fermentation proceeded at agitation of rate of 200 rpm and aeration
Depaula et al., 1999; Kansoh et al., 1999), emphasis has maintained at 1.0 vvm. The fermentation was maintained at 35oC
been placed much on bagasse. This work focused at for 52 h, sample of the medium was withdrawn every 4 h and the
improving its yield by using various sources of supernatant was analysed for reducing sugar and enzyme activity
lignocellulosic namely; sawdust, corncob and bagasse after centrifugation.
inclusive. Thus assessing the effect of the various
lignocellulosic source on the yield of cellulase by the Determination of reducing sugars and cellulase activity
growth of A. flavus.
The total amount of reducing sugars (expressed as equivalent
glucose) in 1.0 ml supernatant was determined by the modified
MATERIALS AND METHODS dinitrosalicyclic acid (DNS) method of Miller (1959) as applied by
Solomon et al. (1999). Cellulase activity was determined as a Filter
Lignocellulosic sources and pre-treatment Paper Activity (FPA) by the method of Ghose (1987) also applied
by Solomon et al. (1999).
The substrates used for this work are bagasse, sawdust and
corncob; they are cheap and readily available sources of
lignocellulosics. The bagasse was collected from Nigeria Sugar
Company, Bacita, Kwara State, Nigeria. The corncob was obtained 0.09
from Obafemi Awolowo University Research Farm and the sawdust Bagasse
from a sawmill in Ile-Ife, Nigeria. The substrates were sundried for 0.08 Sawdust
two days so as to reduce the moisture content and make them Corncob
more susceptible to ballmilling. 0.07
The substrates were ballmilled at about 60 rpm for 48 h after
Cellulase activity (IU/ml)

which the ballmilled substrates were individually screen analysed in

the Endecott test sieve shaker and each sample was made to pass 0.06
through a 0.5 mm screen. The samples were then soaked in 1%
(w/v) sodium hydroxide solution at a ratio of 1:10 0.05
(substrate:solution) (Gharpuray et al., 1983; Solomon et al., 1999)
for 2 h at room temperature after which it was washed free of the 0.04
chemicals and autoclaved at 121oC (15 psig steam) for 1 h. The
treated substrate was then filtered and washed successively with
distilled water until the wash water was neutral. 0.03

0.02
Inoculum Preparation
0.01
A pure culture of A. flavus Linn Isolate NSPR 101 was provided by
the Department of Microbiology, Obafemi Awolowo University, Ile-
0
Ife, Nigeria. This was used throughout this study. The organism
was maintained as direct stock culture from which inocula were 0 10 20 30 40 50 60
prepared. It was grown on malt extract agar slant at 30oC for 5 Time (h)
days and stored at 4oC with regular subculturing. 200 ml of the
optimised medium (Solomon et al., 1999) of each sample with A.
Figure 1. Substrate effect on A. flavus cellulase activity grown in
flavus from a 4 day culture was used as inoculum prepared in a 250
optimised medium at 200 rpm agitation and 1.00 vvm aeration.
ml. The inoculum was shaken continuously on an environment-
controlled incubator shaker (New Brunswick Scientific Co., USA) at
200 rpm and 35oC for 24 h before it was used for the fermentation
process.
RESULTS AND DISCUSSION
Fermentation Experiment Figure 1 shows the plot of enzyme activities in the
Different fermentation runs were carried out on the batch fermentor
various substrates (measured as filter paper activity) at
unit of the double-unit Microferm Fermentor. Two litre of the the time interval of 4 h during the fermentation studies of
optimised medium containing: 30 g/l pre-treated substrate, 0.3 g/l of A. flavus. The figure shows the effect of the various
L-glutamic acid, 1.4 g/l NH4NO3, 0.2% of Tween 40, 2.0 g/l of lignocellulosics on the production of cellulase enzyme
KH2PO4, 0.3 g/l of CaCl2, 0.3 g/l of MgSO4, 0.75 g/l protease produced. The results presented here show the extent of
peptone, 5.0 mg/l of FeSO4.7H2O, 1.6 mg/l of MnSO4.H2O, 1.4 mg/l production (yield) of cellulase enzyme for a period of 52 h
of ZnSO4.7H2O and 2.0 mg/l of CoCl2 were mixed in a 7 litre
fermentation vessel made of borosilicate glass and sterilised with its measured as enzyme activity and also the production
accessories at 121oC for 15 min. rate of the enzyme measured as the ratio of the yield to
152 Afr. J. Biotechnol.

time. Cellulase activity increased to the maximum at Hatakka AI (1983) Pretreatment of wheat straw by white-rot fungi for
enzymatic saccharification of cellulose. Eur. J. Appl. Microbiol.
about 12 h of production for all the lignocellulosic Biotechnol. 18: 350-357.
materials used. The stability of the cellulase spanned Howell JA (1978). Enzymatic deactivation during cellulose hydrolysis.
from 12 to 40 h except in corncob where there was a Biotechnol. Bioeng. 20: 847-863
sluggish decrease from 28 to 40 h before the eventual Kansoh AL, Essam SA, Zeinat AN (1999). Biodegradation and
utilization of bagasse with Trichoderma reesei. Polym. Degrad. Stab.
decrease in the activity. Solomon et al. (1999) have 62: 273-278.
previously reported that the enzyme could be harvested Kumakura M (1997). Preparation of immobilized cellulase beads and
at about 12th h, when the activity is highest. their application to hydrolysis of cellulosic materials. Process
A. flavus grown on sawdust gave the highest cellulase Biochem. 32: .555-559
Miller GL (1959). Use of dinitrosalicyclic acid reagent for determination
activity of 0.0743 IU/ml, while bagasse and corncob gave
of reducing sugar. Biotechnol. Bioeng. Symp. 5: 193-219
0.0573 and 0.0502 IU/ml, respectively. The depression Solomon BO, Layokun SK, Nwesigwe PK, Olutiola PO (1990).
in cellulase activity between 20th and 30th h, common to Hydrolysis of sawdust by cellulase enzyme derived from Aspergillus
all three substrates, may be due to cumulative effect of flavus Linn Isolate NSPR 101 beyond the initial fast rate period.
JNSChE. 9: 1-2.
cellobiose, a dimer of glucose which is known to inhibit Solomon BO, Amigun B, Betiku E, Ojumu TV, Layokun SK (1999).
both endoglucanase and b-glucosidase (Howell, 1978). Optimization of Cellulase Production by Aspergillus flavus Linn
Hatakka (1983) also suggested that delignification Isolate NSPR 101 Grown on Bagasse. JNSChE. 16: 61-68
produces aromatic water-soluble products which can Tang LG, Hon DNS, Pan SH, Zhu YQ, Wang Z, Wang ZZ (1996).
Evaluation of microcrystalline cellulose changes in ultrastructural
repress the cellulolytic action of the enzyme. characteristics during preliminary acid hydrolysis. J. Appl. Polym.
The value obtained when the lignocellulosic was Sci. 59: 483-488.
bagasse compares favourably with 0.056425 IU/ml Wu Z, Lee YY (1997). Inhibition of the enzymatic hydrolysis of cellulose
reported by Solomon et al. (1999) using the same by ethanol. Biotechnol. Lett. 19: 977-979.
operating conditions. The estimated production rates are
0.00478, 0.00619 and 0.00418 IU/(ml-h) for bagasse,
sawdust and corncob, respectively. The highest cellulase
productivity with sawdust may be due to its very high
percentage of cellulose which is the major component of
cell walls of wood.
In conclusion, A. flavus is capable of producing
cellulase from sawdust and corncob in addition to
bagasse that was previously used. Cellulase enzyme
produced from sawdust corncob and bagasse could be
harvested at 12 hours of production, the time at which the
activity is highest. Sawdust is most suitable for cellulase
production compared to bagasse and corncob as it gave
the highest yield of the enzyme.

ACKNOWLEDGEMENT

Prof. B.O. Solomon wishes to thank the International

Foundation for Science (IFS) for the financial support
used for the procurement of all the chemicals needed for
this work.

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