Isolation, Optimization and Production of Cellulase by Aspergillus niger from Agricultural Waste

Abstract

Cellulases are the group of hydrolytic enzymes such as endoglucanase (CMCase), exoglucanase, β-glucosidase (BGL) and FPase which are responsible for release of sugars in the bioconversion of the cellulosic biomass into a variety of value-added products. The cellulase producing fungi were isolated from various agriculture fields. Total 21 isolates were obtained on Czapek's Dox agar medium. Aspergillus niger was selected as most efficient enzyme producer by screening technique. Optimization of some nutritional and environmental factors like nitrogen source, temperature, pH and fermentation time were studied under submerged culture condition for cellulolytic enzyme production. Different agriculture waste material was used as carbon source. Maximum cellulolytic activity was observed in 4.2 pH media at 28°C after 96 hours in submerge condition. Wheat straw showed maximum activity of CMCase, exoglucanase, β β β β β-glucosidase and FPase were 8.38 IU/ml, 5.21 IU/ml, 0.30 IU/ml and 8.08 IU/ml, respectively followed by baggase.

Full text

* To whom all correspondence should be addressed.]
E-mail: hitjasani1610@gmail.com
JOURNAL OF PURE AND APPLIED MICROBIOLOGY, June 2016. Vol. 10(2), p. 1159-1166
Isolation, Optimization and Production of Cellulase
by Aspergillus niger from Agricultural Waste
Hitesh Jasani1*, Nimita Umretiya1, Darshan Dharajiya1,
Manthan Kapuria2, Shilpa Shah2 and Jagdish Patel3
1Department of Plant Molecular Biology and Biotechnology, C. P. College of Agriculture,
Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar - 385506, India.
2Shree P. M. Patel Institute of Post Graduate Studies & Research in Science, Anand - 388001, India.
3Department of Biochemistry, PDPIAS, Charotar University
of Science and Technology, Changa - 388421, India.
(Received: 07 February 2016; accepted: 03 April 2016)
Cellulases are the group of hydrolytic enzymes such as endoglucanase (CMCase),
exoglucanase, ββ
ββ
β-glucosidase (BGL) and FPase which are responsible for release of sugars
in the bioconversion of the cellulosic biomass into a variety of value-added products. The
cellulase producing fungi were isolated from various agriculture fields. Total 21 isolates
were obtained on Czapek’s Dox agar medium. Aspergillus niger was selected as most
efficient enzyme producer by screening technique. Optimization of some nutritional and
environmental factors like nitrogen source, temperature, pH and fermentation time were
studied under submerged culture condition for cellulolytic enzyme production. Different
agriculture waste material was used as carbon source. Maximum cellulolytic activity was
observed in 4.2 pH media at 28°C after 96 hours in submerge condition. Wheat straw
showed maximum activity of CMCase, exoglucanase, ββ
ββ
β-glucosidase and FPase were 8.38
IU/ml, 5.21 IU/ml, 0.30 IU/ml and 8.08 IU/ml, respectively followed by baggase.
Keywords: Aspergillus niger, Cellulase, Lignocellulose, Wheat straw, Rice burn, Banana waste.
The importance of cellulose as a
renewable source of energy has made cellulose
hydrolysis the subject of intense research and
industrial importance1. It is the primary product of
photosynthesis in terrestrial environments and the
most abundant organic substrate (100 billion dry
tons/year) on earth for the production of glucose,
for fuel and as chemical feed stock2-3. Cellulase
enzymes, which can hydrolyze cellulose forming
glucose and other commodity chemicals, can be
divided into three categories: endoglucanase (EC
3.2.1.4); exoglucanase (EC 3.2.1.91) and β-
glucosidase (EC 3.2.1.21)4-5. Scientists have strong
interests in cellulases because of their various
applications include starch processing, animal food
production, alcohol fermentation, malting and
brewing, extraction of fruit and vegetable juices,
fuel, pulp and paper industry, waste management,
protoplast production, genetic engineering and
pollution treatment medical/pharmaceutical
industry and textile industry6-9.
The cost of production and low yields of
cellulase enzymes are the major problems for
industrial application. Therefore, investigations on
the ability of the lignocellulose hydrolyzing
microbial strains to utilize inexpensive substrate
have been done10. The lignocellulosic biomass is
mainly composed of cellulose, hemicellulose, and
lignin that are strongly intermeshed and chemically
bonded by noncovalent interactions and by
covalent crosslinkages11. Because of the use of

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1160 JASANI et al.: STUDY OF CELLULASE BY A. niger FROM AGRICULTURAL WASTE
machinery in agriculture, the livestock population
with farmer decreases and resulted in the addition
of these agro-wastes in the biosphere leading to
environmental pollution in the form of carbon
dioxide, methane etc12-13.
Every part of plant such as leaves, straws,
stems, stalks, corncobs, bran, baggase, etc., comes
under plant biomass and it was decomposed by
microorganisms such as bacteria, actinomycetes
and fungi14. Bacteria, actinomycetes and fungi
have been found to produce cell-bound enzymes
and multi-protein complexes expressing cellulases
and hemicellulases called cellulosomes. The
cellulosome was first discovered in 1983 from
Clostridium thermocellum which is anaerobic and
thermophilic spore-forming bacteria15. The
production of cellulase generally depends on
variety of growth parameters which includes pH
value, temperature, time, pre-treatments
lignocellulosic material and medium nutrients16.
This work focuses on different factors
relevant to improvement of enzymatic hydrolysis
of lignocellulosic materials such as wheat straw,
rice straw, baggase and banana agro-waste. To
understand the biochemistry of cellulose degrading
fungi, it is needed to optimize various conditions.
MATERIALS AND METHODS
Isolation and maintenance of cellulase producing
fungi The soil and agro-waste samples were
collected from various farm including banana, rice,
wheat and sugarcane near Anand, Gujrat, INDIA.
Crushed agro-waste samples were used for
preparing suspension. The samples were sprinkled
over the Czapek’s Dox agar plates and incubated
for 7 days at 28±0.5°C. The growth of fungal
colonies was observed and individual colony were
isolated and restreaked on the same agar. The
isolate was identified based on of their colony
characteristics, microscopic examination and
morphological observation. Isolated fungi were
maintained on modified Czapek Dox agar slant &
PDA slant and stored at 4°C.
Screening for cellulose producing fungi
Plate screening using cellulose as carbon source
After 7 days of growth on modified
Czapek’s Dox agar medium, plates were sprayed
with iodine solution. It was allowed to stand at
room temperature for 5 minutes.
Plate screening using carboxymethylcellulose as
carbon source
Spores from one week old PDA plates
were suspended in sterile D/W. A small well created
in the middle of the carboxymethylcellulose agar
medium and spores of each isolates were inoculated
into the well. Plates were incubated at 28±0.5 ºC for
three days followed by 18 hours at 50ºC. Plates
were stained with 0.1% Congo red dye for 0.5-1
hour and distained with 1 M NaCl solution for 15-
20 min17.
Production of cellulase enzyme by submerge
fermentation process
Media composition described by Mandels
& Weber18 was used under fermentation process
for enzyme production. The composition of media
was: (NH4)2SO4-1.4 gm, KH2PO4-2.0 gm, CaCl2-0.3
gm, Urea*#-0.3 gm, MgSO4.7H2O*-0.3 gm,
FeSO4.7H2O*-5.0 mg, MnSO4.7H2O*-1.6 mg,
ZnSO4.7H2O*-1.4 mg, COCl2-2.0 mg, Proteose
peptone-1.0 gm, Tween 80-1.0 ml, Cellulose-10.0
gm, Distilled water-1000 ml, pH-5.0±0.2.
(*Components were added after autoclaving, #Urea
was sterilized by filtration).
For preparation of inoculum, those
isolates showed a maximum zone of hydrolysis in
screening were used. 1.0 ml of a spore suspension
(~108 spores/ml) was inoculated into production
medium and kept at 28±0.5 °C in orbital shaker
incubator at 110-120 rpm.
Cellulase enzyme activity assay
Culture was harvested at 24 hours interval
and homogenized at 8,000 rpm at 4°C for 10 min.
The supernatant was used as the crude extracellular
enzyme source for enzyme assay. Isolates which
showed maximum cellulase production was used
for the further study.
Enzyme activity was assayed according
to the DNSA (3,5-dinitrosalicylic acid) methods
recommended by the International Union of Pure
and Applied Chemistry (IUPAC) commission of
Biotechnology19. One unit of enzyme activity is
defined as 1 µmol glucose equivalents released
per minute (µmol/ml/min).
DNSA method for Endoglucanase assay
Endoglucanase activity was determined
by incubating 0.5 ml of supernatant with 0.5 ml of

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1161JASANI et al.: STUDY OF CELLULASE BY A. niger FROM AGRICULTURAL WASTE
1% carboxymethylcellulose (CMC) in 0.05M citrate
buffer (pH 4.8) at 50°C for 30 minute. Appropriate
control without of enzyme was simultaneously run.
The reaction was terminated by addition of 3 ml
dinitrosalicylic acid reagent. The tubes were placed
in boiling waterbath for 10 minutes & cooled at
room temperature. Absorbance was read at 540
nm.
FPase assay
Filter paper activity was determined by
adding 0.5 ml of culture supernatant with 1 ml of
0.05 M citrate buffer (pH 4.8) containing a 1cm×6cm
strip (50 mg) of Whatman No.1 filter paper, and
incubated for 1 hour at 50°C20. Reaming steps was
followed as per endoglucanase assay.
Exoglucanase assay
Exoglucanase activity was determined by
adding 1.0 ml of culture supernatant with 1.0 ml of
0.1 M citrate buffer (pH 4.8) containing 50 mg of
absorbent cotton incubated for 24 hour at 50°C.
Reaming steps was followed as per endoglucanase
assay.
ββ
ββ
β-glucosidase assay
β-glucosidase activity was determined by
incubating 1 ml of culture supernatant with 10 mg
salicin in 1 ml 0.05 M citrate buffer (pH 4.8) at 50°C
for 30 minute21. Reaming steps was followed as per
endoglucanase assay.
Protein estimation
Protein estimation was carried out using
folin lowery’s method using Bovine serum albumin
as standard22.
Optimization of pH, temperature and incubation
period To select the optimum pH, temperature,
incubation period for fermentative production of
the enzyme the selected fungal strain were
cultivated with varying pH ranges 4.2, 5.2, 6.2, 7.2
and 8.2, temperatures of 20°C, 28°C, 37°C, 55°C
and 75°C, incubation period range of 24 to 144
hours, by keeping all other parameters constant.
Cellulose used as carbon source in fermentation
media. The flaks were inoculated with 1.0 ml of
spores and incubated at 28±0.5 °C in shaker
incubator at 110-120 rpm. Enzyme assays were
carried out at regular intervals.
Optimization of nitrogen source
Different types of nitrogen sources
(Peptone, (NH4)2SO4 and Urea) were used for
optimization. Peptone was used from 0.05, 0.075,
0.1, 0.125 and 0.15 gm%. (NH4)2SO4 was used from
0.1, 0.12, 0.14, 0.16 and 0.18 gm% and Urea was
used in range from 0.01 to 0.05 gm% in cellulose
containing fermentation medium. The flaks were
inoculated with 1.0 ml of spore and incubated it at
28±0.5 °C in shaker incubator at 110-120 rpm.
Enzyme assays were carried out at regular intervals.
Optimization of carbon source
Beside cellulose, different types of
agriculture material were used as carbon sources
which included wheat straw powder*, rice straw
powder*, baggase powder* and banana agro waste
powder* without and with pre-treatment (1N
NaOH) (*Particle size: 180 & B.S.S. Mesh No. 85).
The flaks were inoculated with 1 % carbon source
and 1.0 ml of spores into modified production
medium and incubated at 28±0.5 °C in shaker
incubator at 110-120 rpm. Enzyme assays were
carried out at regular intervals.
RESULTS AND DISCUSSION
Isolation and screening for cellulase producing
fungi Total 21 isolates were obtained on
Czapek’s Dox agar medium, from which 10 fungi
that showed clear zone on addition of Iodine
solution (Figure 1 & 2). Further these fungi were
grown on the CMC agar plates. After 5 days of
incubation, the appearance of the clear zone around
Table 1. Comparison of cellulose and wheat straw for the production of cellulase enzyme
Endoglucanase Exoglucanase β-glucosidase FPase
Unit Specific Unit Specific Unit Specific Unit Specific
Carbon activity activity activity activity activity activity activity activity
source IU/ml U/mg IU/ml U/mg IU/ml U/mg IU/ml U/mg
Cellulose 0.24 1.12 0.03 0.12 0.21 1.10 0.21 0.98
Wheat 8.38 10.58 5.21 6.57 0.30 1.18 8.08 10.20

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1162 JASANI et al.: STUDY OF CELLULASE BY A. niger FROM AGRICULTURAL WASTE
Fig. 1. Cellulase positive 10 isolated fungi on
Czapek’s Dox agar medium Fig. 2. Clear zone on Czapek’s Dox agar medium
Fig. 3. Clear zone on CMC agar medium Fig. 4. Effect of pH on Aspergillus niger cellulase
activity using cellulose substrate
Fig. 6. Effect of time on Aspergillus niger cellulase
activity using cellulose substrate
Fig. 5. Effect of temperature on Aspergillus niger
cellulase activity using cellulose substrate

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1163JASANI et al.: STUDY OF CELLULASE BY A. niger FROM AGRICULTURAL WASTE
the surrounding the small well when added Congo
Red solution was strong evidence that the fungi
produced cellulase in order to degrade cellulose.
The clearing zone diameter was measured and
maximum zone of clearance producing fungus was
selected for further studies (Figure 3).
Cellulase enzyme activity assay
The protein concentration in crude
samples was determined with bovine serum
albumin (BSA) as standard. The enzyme unit (IU/
ml) of crude enzyme was determined by using
DNSA method and their specific activity U/mg was
calculated. In which isolate No. 3 showed higher
endoglucanase (0.2389 IU/ml), exoglucanase
(0.0255 IU/ml), β-glucosidase (0.2059 IU/ml) and
FPase activity (0.2100 IU/ml) among 10 isolates.
On the basis of enzyme activity, specific activity,
microscopic examination and morphological
observation isolate no. 3 was Aspergillus niger.
Fig. 7. Effect of (NH4)2SO4 on Aspergillus niger cellulase
activity using cellulose substrate
Fig. 9. Effect of urea on Aspergillus niger cellulase
activity using cellulose substrate Fig. 10. Effect of different lignocellulose material
on Aspergillus niger cellulase activity with
cellulose substrate as standard
Optimization of pH, temperature and incubation
period The maximum cellulase activity was
reported at pH 4.2-5.2. The enzyme activities of
CMCase, exoglucanase, β-glucosidase, FPase were
0.51 IU/ml, 0.19 IU/ml, 0.23 IU/ml and 0.29 IU/ml,
respectively (Figure 4). Maximum cellulase activity
was observed at 28°C, the activity of CMCase,
exoglucanase, β-glucosidase, FPase was 0.51 IU/
ml, 0.19 IU/ml, 0.23 IU/ml and 0.29 IU/ml,
respectively (Figure 5) and Endoglucanase,
exoglucanase and FPase activity was found
maximum 0.24 IU/ml, 0.03 IU/ml, 0.21 IU/ml,
respectively at 96 hour while β-glucosidase activity
was observed maximum (0.21 IU/ml) at 96 hour
(Figure 6).
Similar finding was observed when rice
bran and orange peel used as substrates and
highest cellulase activity reported at 4.0 pH with
Fig. 8. Effect of peptone on Aspergillus niger
cellulase activity using cellulose substrate

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1164 JASANI et al.: STUDY OF CELLULASE BY A. niger FROM AGRICULTURAL WASTE
by Aspergillus niger23. A highest level of β-
glucosidase was obtained at pH 4.0, which
decreased drastically with an increase in pH of the
medium to 5 by Aspergillus niger 24. When media
was cultured with pineapple peel, orange peel and
CMC using Aspergillus niger at pH 4 gave
maximum cellulase activities of 0.270, 0.200 and 0.173
mg/ml, respectively25. Optimum temperature for
maximum enzyme production was recorded at 30ºC
under SmF and SSF for Aspergillus spp.26.
Maximum endoglucanase activity was reported at
28°C when Aspergillus niger using wheat
straw:wheat bran as carbon source27. Rice bran
produced maximum cellulase activity at 45 °C with
value of 20.35µmol/min23. Maximum activity of
cellulases and hemicellulases by Aspergillus niger
KK2 from lignocellulosic biomass was reported
after 96 hours10. Secretion of maximum activity of
cellulase enzymes was reported at after 7 days by
Aspergillus niger13. Highest production of
CMCase by Aspergillus niger using plant wastes
as substrate was reported after 144 hours12.
Optimization of nitrogen source
The maximum CMCase activity 0.25 IU/
ml, exoglucanase 0.03 IU/ml, â-glucosidase 0.20 IU/
ml, FPase 0.20 IU/ml were observed after 96 hours,
When Ammonium sulphate applied at 0.14 gm%
(Figure 7).
Peptone at 0.125 gm% was showed
maximum CMCase activity 0.20 IU/ml,
exoglucanase 0.03 IU/ml, β-glucosidase 0.17 IU/
ml, FPase 0.17 IU/ml after 96 hours (Figure 8).
Urea at 0.04 gm% was observed maximum
CMCase activity 0.25 IU/ml, exoglucanase 0.03 IU/
ml, β-glucosidase 0.20 IU/ml, FPase 0.23 IU/ml after
96 hours (Figure 9).
According to previous studies at 0.03%
urea, peptone and NaNO3 used as nitrogen source,
the activity of cellulase were obtained 0.824, 0.421
and 0.401 IU/ml, respectively28. Cellulase activities
were obtained 0.1196, 0.1528 and 0.1528 IU/ml using
peptone (0.125%), (NH4)2SO4 (0.15%) and urea
(0.03), respectively using saw dust as substrate29.
Optimization of different carbon source
Different agricultural wastes such as
wheat straw, rice straw, baggase and banana agro-
waste were tested for the production of enzyme.
Of all the substrates tested, wheat straw was found
to the best substrates for the production of cellulase
followed by baggase. The other substrates gave
comparatively less enzyme production. The highest
CMCase (8.39 IU/ml), exoglucanase (5.21 IU/ml),
β-glucosidase (0.30 IU/ml), FPase (8.08 IU/ml)
activities were reported in lignocellulosic material
wheat straw (Figure 10) as well as specific activity
were also higher compare to cellulose as standard
carbon source (Table 1).
A result was agreement with previously
finding that Aspergillus fumigateus grown on
wheat straw gave maximum β-glucosidase (0.1320
IU/ml) and CMCase (0.225 IU/ml) only after 24 hrs30.
0.72 IU/ml CMCase and 0.43 U/ml FPase under
submerged condition when Aspergillus niger grow
on wheat bran28. The maximum CMCase activity of
0.499 IU/ml was achieved with 4% wheat bran
concentration14. Maximum titers of FPase, CMCase
and BGL obtained on ricebran + Wheatbran
combination were 2.632, 2.478 and 2.984 IU/ml in
submerged condition15. 14.88 IU/L CMCase
activity was reported in wheat bran by A.
awamori31.
Present study was aimed at isolation of
promising cellulase producing fungus and its
identification, optimization of cultural conditions
for production of cellulolytic enzymes. Fungal
culture was initially identified as species of the
genera of Aspergillus niger based on cultural,
morphological and microscopic characteristics. The
cellulolytic activity of the culture was studied by
standard CMC and Congo Red plate assay method.
Cellulase production with Aspergillus spp. in liquid
state fermentation was highest at temperature 28°C,
pH-4.2, incubation time (4 days), 0.04 % urea as
nitrogen source and in presence of carbon
substrates (Wheat straw). This study revealed that
the successful use of lignocellulosic material as
carbon source is dependent on the development
of economically feasible process for cellulases
production.
ACKNOWLEDGEMENTS
All the authors would like to thank Shree
P. M. Patel Institute of Post Graduate Studies &
Research in Science, Anand, Gujarat, India, for
providing us funds and facilities to successfully
complete our research.

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1165JASANI et al.: STUDY OF CELLULASE BY A. niger FROM AGRICULTURAL WASTE
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