International journal of Advanced Scientific and Technical Research
Issue 1, Vol 1 October 2011
ISSN 2249-9954.
OPTIMIZATION FOR CELLULASE PRODUCTION BY Aspergillus niger USING
PADDY STRAW AS SUBSTRATE
S.Siva Sakthi1, *P.Saranraj2 and M.Rajasekar3.
1 - Department of Microbiology, Annamalai University, Chidambaram – 608 002
2 -Research Scholar, Department of Microbiology, Annamalai University
Annamalainagar-608 002, Tamil Nadu, India. Tel no: 9994146964
E-mail: microsaranraj@gmail.com
3 - CAS in Marine Biology, Annamalai University, Parangipettai – 608 502.
Abstract
Microorganisms bring about most of the cellulose degradation occurring in nature. In this
present investigation, Aspergillus niger was isolated from the spoiled coconut and identified
using LPCB staining based on its morphological and cultural features. The paddy straw was
collected and cut into 2 to 3 cm in length and ground with a blender. After that, the grinded
paddy straws were sieved and collected. Cellulase enzyme was produced by the fungus
extracellularly. Optimization of cellulase production was done by using various physical
(Temperature, pH, Salinity and Incubation time) and chemical parameters (Carbon sources and
Nitrogen sources) which could influence the enzyme activity. Cellulase production was
maximum at the temperature 20°C and minimum at 40ºC. The optimal pH for the cellulase
production was observed maximum in 6.0 and minimum in 7.0. Cellulase production was
maximum at 48 hours and minimum at 24 hrs. Cellulase production was maximum with when
fructose was used as a carbon source and minimum with sucrose. Cellulase production was
maximum when Malt extract was used as a nitrogen source and minimum with yeast extract. The
cellulase enzyme extract was partially purified and its protein fraction was stained with its
molecular weight determination by using SDS-PAGE. It revealed two protein bands with the
molecular weights of about 83 kD and 50kD.
Key words: Cellulase, Aspergillus niger, Paddy straw and SDS-PAGE.
*
Corresponding author:
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1. INTRODUCTION
Bioconversion of cellulose-containing raw materials is an important problem of current
biotechnology due to the increasing demand for energy, food and chemicals (Solovyeva et al.,
1997). Cellulases are enzymes which hydrolyse the β-1,4- glycosidic linkage of cellulose and
synthesized by microorganisms during their growth on cellulosic materials (Lee and Koo, 2001).
The complete enzymatic hydrolysis of cellulosic materials needs different types of cellulase;
namely endoglucanase,(1,4-D-glucan-4-glucanohydrolase; EC 3.2.1.4), exocellobiohydrolase(1,
4-D-glucan glucohydrolase) and glucosidase (D-glucoside glucohydrolase) (Yi et al., 1999).
Agriculture wastes contain a high proportion of cellulosic matter which is easily decomposed by
a combination of physical, chemical and biological processes. The bunch consists of 70 moisture
and 30% solid; of which holocellulose accounts for 65.5, lignin 21.2, ash 3.5, hot water-soluble
substances 5.6 and alcohol-benzene soluble 4-1% (Thambirajah et al., 1995). Lignin is an
integral cell wall constituent, which provides plant strength and resistance to microbial
degradation (Shibata et al., 2008).
Cellulases are comprised of independently folding, structurally and functionally discrete
units called domains or modules, making cellulases modular (Henry et al., 1998). A typical free
cellulase is composed of a carbohydrate binding domain (CBD) at the C-terminal joined by a
short poly-linker region to the catalytic domain at the N-terminal. There are only two modes of
action for the hydrolysis of cellulose by cellulases, either inversion or retention of the
configuration of the anomeric carbon. At least two amino acids with carboxyl groups located
within the active site catalyze the reaction by acid-base catalysis.
Fungi are the main cellulase producing microorganisms, though a few bacteria and
actinomycetes have also been reported to yield cellulase activity. Fungal genera like
Trichoderma and Aspergillus are known to be cellulase producers and crude enzymes produced
by these microorganisms are commercially available for agricultural use. The genus Aspergillus
species attack cellulose producing significant amount of cell free cellulase capable of
hydrolyzing cellulose into fermentable soluble sugars such as glucose; an important raw material
in chemical industries (Wainwright, 1992). Aspergillus and Trichoderma species are well known
efficient producers of cellulases (Peij et al., 1998). Several studies have been carried out to
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produce cellulolytic enzymes from biological waste degradation process by many
microorganisms including fungi such as Trichoderma, Penicillium and Aspergillus species etc.,
by Mandels and Reese, (1985).
Cellulases were initially investigated several decades back for the bioconversion of
biomass which gave way to research in the industrial applications of the enzyme in animal
feed, food, textiles and detergents and in the paper industry. With the shortage of fossil
fuels and the arising need to find alternative source for renewable energy and fuels, there
is a renewal of interest in the bioconversion of lignocellulosic biomass using cellulases and
other enzymes. In the other fields, however, the technologies and products using cellulases
have reached the stage where these enzymes have become indispensable.
Cellulase is used to modify the surface properties of cellulosic fibers and fabric in order
to achieve a desired surface effect (Kotchoni et al., 2003). Cellulase has been used to degrade
environmental wastes such as plant wastes (lignocellulosics). Therefore, its production using
readily available sources will help reduce importation costs. The present study was carried out to
evaluate the cellulase activity of the cellulolytic fungi Aspergillus niger isolated from spoiled
coconut using paddy straw as a feed substrate. To understand the biochemistry of cellulose
degrading fungi Aspergillus niger, it is needed to optimize under various physical and chemical
parameters. Cellulase production by different organisms in submerged state fermentation has
received more attention and is found to be cost-prohibitive because of high cost of process
engineering. The present study is focussed on cellulase production by Aspergillus niger using
paddy straw as substrate under optimized condition.
2. MATERIALS AND METHODS
2.1. Isolation and identification of Aspergillus niger
The fungi Aspergillus niger was isolated from the spoiled coconut. A loopful of the
fungal mycelium was taken from the spoiled coconut and inoculated into Sabouraud’s dextrose
agar medium and incubated at room temperature for three successive days. Well grown
Aspergillus niger colonies were maintained on Sabouraud’s dextrose agar slants and stored at
4C. Identification of the Aspergillus niger isolates was carried out by the routine mycological
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methods i.e., by lacto phenol cotton blue staining and plating on Sabouraud’s Dextrose agar
medium.
2.2. Collection of paddy straw
The paddy straw was collected from the Annamalai Nagar, Chidambaram Taluk,
Cuddalore District. The paddy straws were cut into 2 to 3 cm in length and ground with a
blender. After that, the grinded paddy straws were sieved and collected at the range of
0.36-1.00 mm.
2.3. Pretreatment of paddy straw
2.3.1. Alkali pre-treatment
For Treatment-A, the rice straws were pre-treated with different percentage of sodium
hydroxide (NaOH), i.e., 5, 10, 15 and 20% each with different soaking time of 1, 2, 3 and
4 hours, respectively. Whereas in Treatment-B includes the treatment of rice straws with
different percentage of potassium hydroxide, i.e., 5, 10, 15 and 20% concentration each with
soaking time of 1, 2, 3 and 4 hours, respectively. After the alkali treatment, the treated rice
straws were then washed with distilled water as much as possible and the pH was adjusted to 7.8
using 1 M hydrochloric acid.
2.3.2. Heat pre-treatment
After the alkali treatment, the treated rice straws were subjected to the heat treatment for
1 hour at 121°C. Then, the fully treated rice straws were dried in the oven and kept for further
use.
2.4. Production Medium
The composition of production medium used was paddy straw-5g, KCl-2g, KH2PO4-1g,
MgSO4-0.05g, FeSO4-0.02g and Distilled water-1000ml. The pH was adjusted to 6.5 and the
media was sterilized in an autoclave for 15 min at 121°C. The media were inoculated with a loop
full of spore suspension of Aspergillus niger and then incubated in 30ºC in an orbital shaker set
at 100 rpm for 96 hours. The media were centrifuged at 5000 rpm for 15 minutes to obtain crude
enzyme solution.
2.5. Cellulase Assay
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Cellulase activity was determined at 40°C by using carboxymethyl cellulose (Sodium
salt, HiMedia, India) as a substrate. A reactive mixture contains 0.5 ml of 1% (w/v) substrate in
0.1 M citrate buffer (pH 4.8) and 0.5 ml of culture supernatant. The mixture was incubated at
40°C for 30 min. The reducing sugar released was measured using 3,5-dinitrosalicyclic acid
(DNSA) (Miller, 1959). Control was prepared with 10 min boiled enzyme. One unit of
endoglucanase activity was expressed as the amount of enzyme required to release 1 μmol
reducing sugars per ml under the above assay condition by using glucose as a standard curve.
2.6. Optimization of culture conditions
The factors such as temperature, pH, salinity, sources of carbon and nitrogen affecting
production of cellulase were optimized by varying parameters one at a time.
2.6.1. Effect of Temperature
To study the heat stability of the cellulase enzyme production, an experiment conducted
in a 200ml Erlenmeyer flask containing production medium. After sterilization by autoclaving,
the flask were cooled and inoculated with Aspergillus niger culture and maintained at different
temperature (20ºC, 30ºC and 40ºC). The Optical Density (O.D) was taken at 540nm after
24 hours and the OD values were noted.
2.6.2. Effect of pH
To study the pH stability of the cellulase enzyme production, an experiment conducted in
a 200ml Erlenmeyer flask containing production medium, and the pH was adjusted at different
conditions (6, 6.5 and 7) and then the Aspergillus niger culture was inoculated. After 24 hours,
the Optical Density (O.D) was taken at 540nm and the OD values were noted.
2.6.3. Effect of Salinity
To study the Salinity of the cellulase enzyme production, a 200ml Erlenmeyer flask
containing production medium, and the salinity was adjusted at different conditions (0, 20, 40,
50, 60, 80 and 100) and then the Aspergillus niger culture was inoculated. After 24 hours, the
Optical Density (O.D) was taken at 540nm and the OD values were noted.
2.6.4. Effect of Incubation time
To study the incubation time of the cellulase enzyme production , the culture medium
inoculated with Aspergillus niger was incubated at different hours (24, 48, 72, 96, 120 and 144
hours). The Optical Density (O.D) was taken at 540nm and the OD values were noted.
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2.6.5. Effect of Carbon sources
Carbon source (glucose, fructose, lactose, xylose) at different concentrations (1%, 2%,
3%, 4% and 5%) were added in the production medium of cellulase and the Aspergillus niger
culture was inoculated and incubated at 24 hours under room temperature. After 24 hours, the
Optical Density (O.D) was taken at 540nm and the OD values were noted.
2.6.6. Effect of Nitrogen sources
Nitrogen source (peptone, beef extract, yeast extract, malt extract, casein) at different
concentrations (1%, 2%, 3%, 4% and 5%) were added in the production medium of cellulase and
the Aspergillus niger culture was inoculated and incubated at 24 hours under room temperature.
After 24 hours, the Optical Density (O.D) was taken at 540nm and the OD values were noted.
2.7. Determination of molecular weight by SDS-PAGE
Samples were analyzed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS–PAGE, 10% separating and 4.5% stacking) to check the purity and determine the
molecular mass of the purified protein. The electrophoresis apparatus were neatly washed and
assembled the glass plates. The stacking and separating gel were prepared using above
composition. The gel solution was heated up to the appearance of clear solution. Pour the
separating gel from the gap between the glass plates until ¾ is filled and 2 mm layer of distilled
water on top of gel. Let leave the gel up to 30 min for polymerization. The stacking gel was
poured over the separating gel. Immediately insert a clean Teflon comb into the stacking gel
solution and avoid trapping air bubbles. After polymerization comb was removed, the wells are
washed with water. Remove the water by inverting the gel. The gel tanks were filled by running
gel buffer. Then glass plates were fixed in Electrophoresis apparatus. The protein samples were
taken and mixed with three part of SSB. The mix was boiled at 100ºC for 3 min. samples were
loaded on the wells. Electrophoresis apparatus attached to the electric power supply in 20 mA to
the gel. It was run until dye front reaches the bottom gel. The gel plates were removed from the
tank using spatula carefully. Stacking gel was removed completely from separating gel.
3. RESULTS AND DISCUSSION
Cellulase is an important enzyme for hydrolysis of agro wastes and other cellulosic
wastes. The present study was carried out to evaluate the cellulase activity of the cellulolytic
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fungi Aspergillus niger isolated from spoiled coconut using paddy straw as a feed substrate. To
understand the biochemistry of cellulose degrading fungi Aspergillus niger, it is needed to
optimize under various physical and chemical parameters. Cellulase production by different
organisms in submerged state fermentation has received more attention and is found to be costprohibitive because of high cost of process engineering.
The fungi Aspergillus niger was isolated and identified from the spoiled coconut.
Cellulase activity was determined at 40°C by using carboxymethyl cellulose as a substrate and
the results were showed in Figure-1. The growth of the fungi Aspergillus niger was high at 100
mg/ml glucose concentration (0.69 at 540 nm) and very low at 10 mg/ml glucose concentration
(0.09 at 540 nm). The growth of the fungi was increased when the concentration of glucose was
increased.
The production of cellulase enzyme was carried out under different optimization
conditions. The effect of temperature on the cellulase activity of the cellulase was determined at
various temperatures ranging from 20, 30 and 40°C and the results were showed in Figure-2. The
cellulase activity of the cellulase was maximum at 20°C and minimum at 40ºC. The effect of pH
on cellulase activity of the cellulase was examined at various pH ranging from pH 6.0, 6.5 and
7.0 and the results are showed in Figure-3. The optimal pH for the cellulase activity was
observed maximum in 6.0 and minimum in 7.0. The effect of salinity on the cellulase activity of
the cellulase was determined at various salinity conditions ranging from 0, 20, 40, 50, 60, 80
and100 ppt at pH 6.5 for 72 hours. The results are showed in Figure-4. The CMCase activity of
the cellulase was maximum at 50 ppt and minimum at 20ppt. The effect of incubation time on
the cellulase activity of the cellulase was determined at 24,48,72,96,144 hrs. The results are
showed in Figure-5. The CMCase activity of the cellulase was maximum at 48 hrs and minimum
at 24 hrs. The effect of carbon source on the cellulase production of the cellulase was determined
at various sources of glucose, fructose, xylose, lactose, sucrose and the results are showed in
Figure-6. The CMCase activity of the cellulase was maximum in fructose and minimum in
sucrose. The effect of nitrogen source on the cellulase activity of the cellulase was determined at
various sources of Yeast extract, Beef extract, Peptone, Casein, and Malt extract. The results are
showed in Figure-7. The CMCase activity of the cellulase was maximum with Malt extract and
minimum with yeast extract.
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Figure 1 : Absorbance of Glucose at 540 nm
Figure 2: Effect of Temperature on cellulase production by Aspergillus niger
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Figure 3: Effect of pH on cellulase production by Aspergillus niger
Figure 4: Effect of salinity on cellulase production by Aspergillus niger
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Figure 5: Effect of incubation time on cellulase production by Aspergillus niger
Figure 6: Effect of carbon sources on cellulase production by Aspergillus niger
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Figure 7: Effect of nitrogen sources on cellulase production by Aspergillus niger
Figure- 8: Molecular weight determination of protein

Lanes 1 and 2 contains the crude CMCase from Aspergillus niger.
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Lane-3 contains molecular weight markers
Sherif et al., (2008) isolated twelve Aspergillus species from some local soil samples. On
the basis of cellulolytic activity, Aspergillus fumigatus was selected and used for production of
exoglucanase , endoglucanase , CMCase, β-glucosidase and xylanase by adopting SSF condition
using mixed substrate of rice straw amended with wheat bran. Effect of Culture conditions
including; incubation period, initial pH, incubation temperature, moisture level, different
nitrogen sources, different lignocelluloses as carbon source and different ratios of mixed rice
straw and wheat bran were evaluated. The fungus expressed high enzyme production after 4.0
days incubation at moisture level 75%, initial pH 5-6, at 40°C in presence of NaNO3 as an
inorganic nitrogen source. The recorded activities were 14.71, 8.51, 0.93, 0.68 and 42.7 IU g -1
for CMCase, β-glucosidase, exoglucanase, endoglucanase and xylanase, respectively.
The media optimization is an important aspect to be considered in the development of
fermentation technology. To the best of our knowledge, the present findings are perhaps the first
one about the influence of physiochemical properties on cellulase production by a fungi
Aspergillus niger. Among physical parameters, pH of the growth medium plays an important
role by inducing morphological changes in microbes and in enzyme secretion. The pH change
observed during the growth of microbes also affects product stability in the medium (Gupta et
al., 2003).Optimum pH and temperature for maximum production of cellulase were 6.0 and 20˚C
respectively. Maximum cellulase activity in bacteria is reported around neutral pH of the
medium and temperature at 30˚C but fungi vary with respect to pH and temperature to support
maximum production of cellulases (Magnelli and Forchiassin,1999; Pirt,1975; Umekalsom et al.,
1997). In this study, 20˚C temperature was found optimum to support maximum production of
cellulase as observed by the above workers. At higher temperature, the organisms have to spend
a lot of energy for maintenance and at lower temperature, transport of nutrients is hindered. (Pirt,
1975). The incubation period varies with enzyme production (Smitt et al., 1996). Short
incubation period offers potential for inexpensive production of enzyme (Sonjoy et al., 1995). In
the present study the cellulase activity increased steadily and reached maximum at 48 hours of
incubation.
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Cellulase is an inducible enzyme (Ryu and Mandels, 1980; Kubicek, 1992; Kubicek et
al., 1993) and it is affected by the nature of the substrate used in fermentation. Therefore the
choice of an appropriate inducing substrate is of importance. To evaluate the carbohydrates to
cause induction or repression of cellulase was grown on some monosaccharides and
disaccharides. Fructose among the carbon sources examined was found to be the best inducer
and this study substantiates the earlier works: lactose as best inducers of Aspergillus sp. (Bagga
et al., 1989) and fructose is the best inducer of cellulase in Clastiridium thermocellum (Nochure
et al., 1993). Trehalose has been demonstrated as the best inducer of cellulase in Clastiridium sp.
(Thirumale et al., 2001).
The enzyme production is affected significantly by different organic nitrogen sources.
The production of cellulase is sensitive to the nitrogen sources and nitrogen level in the medium
(Desai et al., 1982). The result of the present study showed that the sources have different effect
on the enzyme activity. Among the organic nitrogen sources tested, the enzyme activity was high
with malt extract.
Acharya et al., (2008) focused the factors relevant for improvement of enzymatic
hydrolysis of saw dust by using Aspergillus niger. Different cultural conditions were examined
to assess their effect in optimizing enzyme production. Alkaline pretreated (2 N NaOH) saw dust
at 9.6% concentration gave 0.1813 IU/mL cellulase activity. Optimum pH for cellulase
production was between 4.0 and 4.5. Submerged fermentation at 120 rpm at 28°C gave higher
yields of cellulase compared to static condition. Several other parameters like inoculum size,
time duration, nitrogen source and its concentration were also optimized for the cellulase
production by using saw dust as substrate.
Gautam et al., (2010) compared the production of Cellulase (Filter paper activity,
endoglucanase and β-glucanase) by Aspergillus niger on three different carbon sources. Glucose
containing media gave the highest mycelia weight of 1.294 mg/flask. Maximum Cellulase
enzyme activity (Filter paper activity, endoglucanase and β-glucanase) were obtained from the
culture containing cellulose. The waste cellulosic material can be used as low-cost carbon source
for commercial cellulose production.
Hafiz Iqbal et al., (2010) investigated the potential of a filamentous fungus, Trichoderma
harzianum for hyper-production of third most demanded industrial enzyme carboxymethyl
cellulase using cheap and easily available agro-industrial residue wheat straw as growth
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supporting substrate under still culture solid state fermentation technique. Even though the
fungal strain Aspergillus niger which was isolated from spoiled coconut, it produced more
concentration of cellulase when production medium was prepared with 5 kg paddy straw as a
substrate and utilization of agroindustrual wastes was proved to be one of the method for
recycling wastes and improve the economic condition.
The cellulase enzyme extract was partially purified and its protein fraction was stained
with its molecular weight determination by using SDS-PAGE. The result is shown in Figure- 8.
It revealed two protein bands with the molecular weights of about 83 kD and 50kD. The enzyme
protein was extracted from culture supernatant by the ethanol precipitation method. The crude
enzyme preparation was subjected to SDS-PAGE (containing 0.2% CMC) to determine the
homogeneity and molecular weight of the enzyme. During the electrophoresis of the enzyme,
two bands showing cellulolytic activity were detected. The molecular weights of these proteins
were calculated to be about 83 and 50 kd. These proteins may be isoenzymes or the different
subunits of the same enzyme proteins. A cellulolytic enzyme protein is rich in acidic and
aromatic amino acids. According to other research, two endoglucanase containing fractions were
separated from Aspergillus niger (Lee et al.,2001). These enzymes possess no ability to bind to
or hydrolyse insoluble microcrystalline cellulose, but were active towards soluble carboxymethyl
celullose. The molecular weights of the enzyme protein were of low molecular weights and
hence it has potential for industrial applications.
4. CONCLUSION
Most of the microorganisms can capable to produce various extracellular and intracellular
enzymes using various cheap sources. The effect of enzyme activity is higher than the plant
derived enzymes. Cellulase is an extracellular enzyme, which is produced from various
organisms including bacteria, fungi, and also some Actinomycetes. Research on cellulase has
progressed very rapidly over the last five decades and potential industrial applications of the
enzyme especially in solid waste management have been identified. Major impediments to
exploit the commercial potential of cellulases are the yield, stability and cost of cellulase
production. Although terrestrial strains of microbes have been extensively studied by many
researchers. Aspergillus niger used for the production of cellulase and paddy straw was used as a
substrate.
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