Brazilian Journal of Microbiology (2011) 42: 1119-1127

ISSN 1517-8382

PRODUCTION OF CELLULASE BY ASPERGILLUS NIGER UNDER SUBMERGED AND SOLID STATE

FERMENTATION USING COIR WASTE AS A SUBSTRATE

Soma Mrudula*, Rangasamy Murugammal

Department of Microbiology, M.G.R. College, Dr. M.G.R. Nagar, Hosur, T.N - 635 109, India.

Submitted: February 28, 2010; Returned to authors for corrections: November 17, 2010; Approved: March 14, 2011.

ABSTRACT

Aspergillus niger was used for cellulase production in submerged (SmF) and solid state fermentation (SSF).
The maximum production of cellulase was obtained after 72 h of incubation in SSF and 96 h in Smf. The
CMCase and FPase activities recorded in SSF were 8.89 and 3.56 U per g of dry mycelial bran (DBM),
respectively. Where as in Smf the CMase & FPase activities were found to be 3.29 and 2.3 U per ml culture
broth, respectively. The productivity of extracellular cellulase in SSF was 14.6 fold higher than in SmF. The
physical and nutritional parameters of fermentation like pH, temperature, substrate, carbon and nitrogen
sources were optimized. The optimal conditions for maximum biosynthesis of cellulase by A. niger were
shown to be at pH 6, temperature 30 ºC. The additives like lactose, peptone and coir waste as substrate
increased the productivity both in SmF and SSF. The moisture ratio of 1:2 (w/v) was observed for optimum
production of cellulase in SSF.

Key words: Aspergillus niger, coir waste, cellulase, submerged fermentation, solid-state fermentation.

INTRODUCTION cleave cellobiose and other soluble oligosaccharides to

glucose (10). These enzymes find potential applications in the
Complete cellulose hydrolysis to glucose demands the production of food, animal feed, textile, fuel, chemical,
action of exoglucanases, endoglucanases and -glucosidases. pharmaceutical industries and in waste management (9, 40).
Exoglucanases (1,4- -D-glucancellobiohydrolase, EC The production of cellulase has been reported from a wide
3.2.1.91) are usually active on crystalline cellulose and cleave variety of bacteria (22) and fungi (4, 34). However,
disaccharide units either from non-reducing or reducing end. filamentous fungi are preferred for commercial enzyme
Endoglucanases (1,4- -D-glucan-4-glucanohydrolase, EC production, because the level of the enzymes produced by these
3.2.1.4) are more active against the amorphous regions of cultures is higher than those obtained from yeast and bacteria
cellulose and they can also hydrolyze substituted celluloses, (8). Almost all fungi of genus Aspergillus synthesize cellulase,
such as carboxymethyl cellulose (CMC) and hydroxyethyl therefore this genus has the potential to dominate the enzyme
cellulose (HEC) internally. -Glucosidases (EC 3.2.1.21) industry. Aspergillus and Trichoderma spp. are well known

*Corresponding Author. Mailing address: Department of Microbiology, M.G.R. College, Dr. M.G.R. Nagar, Hosur, T.N - 635 109, India.; Tel.: + 91 (04344)
261004 Fax: +91 (04344) 260573.; E-mail: [email protected]

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Mrudula, S. et al. Production of cellulase by A. niger

efficient production of cellulases (33). subcultured and maintained on Czapek-Dox-agar slants and
Industrially important enzymes have traditionally been stored at 4 °C in a refrigerator, until needed.
obtained from submerged fermentation (SmF) because of the
ease of handling and greater control of environmental factors Pre-treatment of substrates
such as temperature and pH. However, solid state fermentation The procured cellulosic substrates such as rice husk, rice
(SSF) technique can improve the yield and reduces the cost of bran, coir waste, wheat bran and saw dust were ground to fine
enzyme production (16, 21). Filamentous fungi are the most powder and the substrates were individually treated with 1%
commonly used microorganisms in SSF because they are able (w/v) NaOH solution in the ratio of 1:10 (substrate: solution)
to grow on solid materials with low water contents (30). There for 1h and was brought to neutral pH by washing thoroughly
are several reports describing use of agro industrial residues for with distilled water and dried at room temperature. The treated
the production of cellulose such as wheat straw, wheat bran and substrates were autoclaved at 121°C for 1 h (18).
rice straw as substrates (11, 35, 2, 19). The other advantages of
SSF include superior productivity, simple technique; low Preparation of inoculum
capital investment, low energy requirement and less water The inoculum was prepared by growing the organism in
output (42, 38), better product recovery and lack of foam build 250 ml Erlenmeyer flask with 100 ml of Czapek-Dox broth
up and reported to be most appropriate process for developing containing g/l of sucrose, 30; sodium nitrate, 3; K2HPO4, 1;
countries (39). MgSO4, 0.5; KCl, 0.5; FeSO4, trace; agar, 15. The medium
The cellulase production by filamentous fungi in SmF and was inoculated from the Czapek-Dox agar slants and
SSF has been studied extensively (15, 1, 38). However, there is incubated at 30 °C for 3 days in a shaker (200 rpm) before it
no report on comparison of the cellulase production by SmF was used for the fermentation process.
and SSF conditions. From this point of view, the organism was
isolated and demonstrated for their improved efficiency in SmF Submerged fermentation (SmF)
and SSF for the production of cellulase using agro-industrial Submerged fermentation was carried out in 250 ml
waste as raw material. The influence of various parameters was Erlenmeyer flasks containing 100 ml of fermentation medium.
evaluated under two different fermentation conditions. In the The composition of the medium contained the following g/l of
present study, we describe the comparison of cellulase distilled water. L-Glutamic acid, 0.3; NH4NO2, 1.4; K2HPO4,
production by Aspergillus niger in SmF and SSF systems. 2.0; CaCl2, 2.0; MgSO4, 0.3; protease peptone, 7.5; FeSO4,
5.0; MnSO4, 1.6; ZnSO4, 1.4; tween 80, 20 % (v/v) ; coir
MATERIALS AND METHODS waste, 30. The medium was sterilized by autoclaving at 121°C
for 15 min. Each flask was inoculated with 1ml of the above

Microorganism said inoculum. The cultures were incubated on a rotary shaker

The fungus was isolated from coir retting ground area of (120 rpm) at 30°C for 72 h.

Karimangalam, Dharmapuri Dist, Tamil Nadu, India. The

isolate was grown on CMC agar medium and screened Solid state fermentation (SSF)
according to Wood and Bhat (41). The culture was identified Solid state fermentation was carried out in 250 ml
based on colony morphology and microscopic examination Erlenmeyer flasks that contained 10 g of coir waste and 15 ml
(Devenathan et al. 2007). The isolated fungal colony was of distilled water (moistening agent). The flasks were sterilized

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at 121°C for 15 min and cooled to room temperature. About Effect of supplements (carbon & nitrogen sources)
1ml of inoculum was added, mixed well and incubated at 30°C Smf: Various carbon and nitrogen sources at
in a humidified incubator for 96 h. The flasks were periodically concentration of 5% w/v were supplemented as individual
mixed by gentle shaking. components to the fermentation medium containing coir waste
as substrate. The medium was inoculated and incubated at 30
Enzyme extraction ºC for 3 days in an orbital shaker incubator (120 rpm). The
At the end of the fermentation the culture broth from broth was centrifuged and the enzyme assay was carried out.
submerged fermentation was centrifuged at 6000 rpm for 15 Care was taken to see that monosaccharides and disaccharides
min and the supernatant was used as a source of extracellular were prepared as 10 X solution and sterilized separately by
enzyme. In solid state fermentation (SSF) the enzyme was autoclaving at 10 lbs for 10 min. and required concentration
extracted from the coir waste by mixing homogenously the was added to the medium before inoculation.
entire waste with (1:10 w/v) distilled water and agitated on a SSF: Various carbon and nitrogen sources at
rotary shaker (120 rpm) at 30 °C with a contact time of 1h. concentration of 4% w/v were supplemented as individual
Dampened cheese cloth was used to filter the extract and components to the flasks that contain 10 g of coir waste and
pooled extracts were centrifuged at 6000 rpm for 15min and moistened with 15 ml of distilled water (moistening agent).
the clear supernatant was used as a source of extracellular The flasks were sterilized and cooled to room temperature.
enzyme. About 1 ml of inoculum was added, mixed well and incubated
at 30ºC in a humidifying incubator for 4 days. At the end of
Enzyme assay fermentation the contents from each flask were extracted with
Cellulase [(filter paperase (FPase) and carboxymethyl distilled water and the supernatant was used for enzyme assay.
cellulase (CMCase)] activities were assayed according to the The effect of moisture content in SSF was studied by
method described by Ghose (17).One unit of enzyme activity varying the ratio of coir waste to distilled water (2.5 to 12.5).
(CMCase and FPase) is defined as the amount of enzyme The optimum conditions for enhanced cellulase
which releases 1 µmole of reducing sugars per min with production were verified by performing the experiments using
glucose as standard, under the assay condition described above. the selected parameters at their respective optimum conditions.
The values of enzymatic activity were expressed as U/ml for
SmF and U/g of dry mycelial bran (DMB) for SSF. Comparative evaluation of SmF and SSF systems for
enzyme production
Optimization of process parameters in SmF and SSF The enzyme yield produced in SmF was compared with
Evaluation of optimized culture conditions in SSF and the yield obtained in SSF using coir waste as substrate
SmF using Aspergillus niger: The protocol adopted for the according to Solis Pereira et al (36).
standardization of fermentation parameters was to evaluate the
effect of an individual parameter. The parameters optimized RESULTS AND DISCUSSION
were: substrates (rice husk, rice bran, coir waste, wheat bran,
saw dust), temperature (20 to 40 °C), pH (4.5 to 8), Isolation and screening of cellulolytic organism
fermentation period [(24 to 192 h in SmF) and (24 to 120 h in The soil sample collected from coir retting zone area was
SSF)]. serially diluted in saline and was spread on to the

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carboxymethyl cellulose (CMC) agar plates. The plates were systems. In a study conducted by Ojumu et al (29) on
incubated at 30ºC for 3-5 days. Cellulase producing fungal Aspergillus flavus reported that the saw dust, corncobs and
colonies were selected after flooding the plates with congo red bagasse were found to be the best substrates for the production
(0.1% w/v), followed by destaining with NaCl (0.1 M). The of cellulase. Oberoi et al (28) reported kinnow pulp as the best
colony that showed largest halo forming zone was selected for substrate for the production of cellulase in SSF. Sugarcane
the present study. bagasse, tea production waste, coconut coir pith, rice husk,
wheat bran, rice bran etc., have been employed for production
Identification of fungal isolates of cellulase using a variety of microorganisms such as
Colony morphology: The selected strain was subcultured Trichoderma, Aspergillus, Penicillium, Botrytis, Neurospora
on CMC agar medium. Initially the colonies were white and etc., (31).
changed to black, as culture matured. When immature the
colonies were covered with white fluffy aerial mycelia, while Temperature
mature colonies showed salt and pepper effect which was Incubation temperature plays an important role in the
covered with black spores and reverse of the colony was buff metabolic activities of a microorganism. In the present study
colored. the optimum temperature for maximum enzyme production
was recorded at 30ºC under SmF and SSF (Table 1). About 83
Microscopic examination of the isolates % of cellulase production was observed at 35ºC. Whereas more
The matured colonies were subjected to lacto phenol than 50 % of cellulase was produced when grown at 20, 25 and
cotton blue staining and microscopic examination was made. 40ºC in SmF. 55 and 68% of cellulase production was recorded
From the microscopic observation it was clear that the colony when the organism was grown at 40 and 20ºC, respectively in
showed the hyaline septate hyphae. The conidial head was SSF. The results of SmF process in the present study confirm
large and appeared black to brownish black. The conidiophores the findings of Devanathan et al (12) and Kathiresan and
were hyaline or brownish near the vesicle. Each vesicle Manivannan (23) for Aspergillus sp. Asquieri and Park (6)
appeared globose in shape and cover with brownish sterigmata found that the optimum temperature for production of CMCase
on the entire surface in two series. Based on the colony from thermostable Aspergillus sp. was 37ºC, whereas the
morphology and microscopic observation the strain was maximum cellulase production was observed at 40ºC for
confirmed as Aspergillus niger (12). Aspergillus terreus QTC 828 (3). In general the temperature
maintained in SSF system is in the range of 25 to 35ºC and
Optimization of fermentation parameters in SmF and SSF depends on the growth kinetics of the microorganism employed
Substrates: Among the 5 substrates screened, coir waste rather than on the enzyme produced (25). Ali et al. (3) reported
gave the maximum cellulase production when fermented with maximum yield of cellulase from Aspergillus niger Z10 strain
Aspergillus niger under SmF and SSF (Table 1). Considerable and A. terreus at 40ºC, respectively in SSF.
amount of enzyme production was observed on wheat bran and
rice bran. Comparatively less enzyme production was observed p
with the rest of the substrates in both SmF and SSF. Since coir Among physical parameters, pH of the growth medium
waste showed maximum production of cellulase, it was plays an important role by inducing morphological changes in
selected for further optimization studies for SmF and SSF microbes and in enzyme secretion. The pH change observed

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Mrudula, S. et al. Production of cellulase by A. niger

during the growth of microbes also affects product stability in maximum at 96 h of incubation when grown in SmF. These
the medium (20). Optimum pH for maximum production of results are in agreement with the reports made by Devanathan
cellulase was 6.0 when grown in SmF and SSF (Table 1). et al (12) and Acharya et al (1). However, further increase in
Similar observation was made for cellulase production by A. the incubation time reduced the enzyme production. It might be
terreus QTC 828 in SmF by Ali et al (3) and Trichoderma due to the depletion of macro and micronutrients in the
reesei in SSF by Doppelbauer et al (13), whereas pH 7 was fermentation medium with the lapse in time, which stressed the
reported by Krishna (24) for the production of bacterial fungal physiology resulting in the inactivation of secreting
cellulases by using banana wastes in SSF. machinery of the enzymes (27). Incubation time necessary for
optimal production was observed at 72 h in SSF. Short
Fermentation period incubation period for enzyme production offers the potential for
Table 1 shows the effect of fermentation period on the inexpensive production of enzyme (37). Muniswaran and
production of the cellulase in SmF and SSF. In the present Charyulu (26) have also reported similar trend in cellulase
study cellulase activity increased steadily and reached production using Trichoderma viride.

Table 1. Effect of physical parameters on cellulase production in SmF and SSF by A. niger

SmF (U/ml) SSF (U/g DMB)

Parameter CMCase FPase CMCase FPase
Substrates
Rice husk 0.31 0.25 0.49 0.47
Rice bran 0.51 0.22 1.1 0.5
Coir waste 0.8 0.51 3.42 1.77
Wheat bran 0.72 0.43 1.77 0.8
Saw dust 0.18 0.12 1.2 0.52
Temperature (OC)
20 1.5 0.8 3 1.51
25 1.5 1.3 3.6 2
30 3.4 1.7 4.44 2.51
35 3.1 1.3 3.57 2.45
37 1.9 1.2 3.51 1.46
40 1.7 1.1 2.5 1.3
pH
4.5 0.6 0.3 8.2 3.3
5.0 0.81 0.61 9.2 4.1
5.5 1.5 0.82 9.6 4.3
6.0 2 0.91 9.8 4.6
6.5 1.81 0.82 9 2.9
7.0 1.71 0.81 7.3 2.5
7.5 1.3 1.26 6.8 2.2
8.0 0.9 0.5 6.6 2.1
Fermentation period (h)
24 2.46 1.37 4.5 2.4
48 2.51 1.46 5.3 2.5
72 3 1.53 9 3.6
96 3.46 0.88 7.1 2.3
120 2.2 1 6.9 2.1
144 1.5 0.51 − −
168 0.52 0.49 − −
192 0.5 0.47 − −

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Mrudula, S. et al. Production of cellulase by A. niger

Moisture content cellulase production with an increase in inoculum

Moisture content is a critical factor in SSF processes concentration from 5 to 15 % and found to be maximum at
because this variable has influence on growth, biosynthesis and 15%. Higher or lower inoculum size resulted in a significant
secretion of enzyme. In the present study the maximum decrease in enzyme production (Fig.2). Similar findings have
enzyme production was obtained at coir waste to distilled water been reported for the production of bacterial cellulases by solid
ratio of 1:2. (Fig.1). Any further increase and decrease in the state bioprocessing of banana wastes (24). In contrast 10% of
ratio resulted in decreased enzyme activity. Similar inoculum was reported to be optimum for the production of
observation was made by Anto et al (5) for production of - cellulase by Aspergillus niger under SSF (15).
amylase by Bacillus cereus, whereas Babu and Satyanaryana
(7) reported maximum -amylase enzyme production by
thermophilic Bacillus coagulans at a high level of substrate
moisture ratio of 1: 2.5. According to Lonsane et al (25), lower
moisture content causes reduction in solubility of nutrients of
the substrate, low degree of swelling and high water tension.
On the other hand, higher moisture levels can cause a reduction
in enzyme yield due to steric hindrance of the growth of the
producer strain by reduction in porosity (interparticle spaces)
of the solid matrix, thus interfering oxygen transfer.

Figure 2. Effect of inoculum size on cellulase production by

A. niger in solid state fermentation with coir waste as substrate

Carbon sources
Cellulase production was found to be dependent upon the
nature of the carbon source used in the culture media. To
evaluate the carbohydrates to cause induction or repression of
cellulase, the organism was grown on monosaccharide and
disaccharides. In general, enhanced production of enzyme was

Figure 1. Effect of moistening agent on cellulase production observed with all the tested sugars when compared to the

by A. niger in solid state fermentation with coir waste as control. Among the carbon sources examined, lactose was

substrate found to be the best inducer in SmF and SSF (Table 2). This
study substantiates the work of Kathiresan and Manivannan
(23) and Devanathan (12) who demonstrated lactose as best
Inoculum size inducer of Aspergillus sp. Nochure et al (27) identified
In the present study there is a significant increase in fructose as the best inducer of cellulase in Clostridium

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Mrudula, S. et al. Production of cellulase by A. niger

thermocellum. In another study dextrin was found to enhance the nitrogen sources enhanced cellulase production when compared to
production of cellulase by Trichoderma sp. in SSF (32). Increase control. Among them peptone supported maximum enzyme
in enzyme production with additional carbon sources have been production followed by beef extract, groundnut oilcake, yeast
demonstrated by both the SmF and SSF systems as a result of extract and casein. These results are in agreement with the reports
good growth (36). of Kathiresan and Manivannan (23) and Devanathan et al. (12) for
production of cellulase by Penicillium fellutanum and Aspergillus
Nitrogen sources niger, respectively in SmF. Enari et al. (14) reported that good
The effect of supplementation of nitrogen sources in SmF and cellulase production can be obtained with peptone as the organic
SSF system on the enzyme production is shown in Table 2. All the nitrogen source in SSF.

Table 2. Effect of carbon and nitrogen sources on cellulase production in SmF and SSF by A. niger

SmF (U/ml) SSF (U/g DMB)

Supplement
CMCase FPase CMCase FPase
Carbon sources
(5 % w/v in SmF and 4 %
w/w in SSF)
Control 0.7 0.4 3.7 2
Glucose 2.52 0.54 12.1 6.5
Xylose 2.2 1.42 15.7 6.6
Lactose 3 1.71 18 10.9
Maltose 2.51 1.5 17.5 6.3
Sucrose 2.54 1.51 13.7 6.2
Nitrogen sources
(5 % w/v in SmF and 4 %
w/w in SSF)
Control 0.56 0.4 3.8 2
Yeast extract 1.1 0.6 8.2 4.2
Beef extract 1.53 0.9 11 4.5
Peptone 2.1 1.36 13.8 6.2
Groundnut oil 1.61 1.1 8.1 4.3
Casein 0.53 0.47 4.2 3.8
Sodium nitrate − − 4 3.6

Enzyme yields dividing the yield obtained from SSF in U/g DMB with the
Under the optimum conditions, the strain produced 2.04 yield from SmF in U/ml culture broth. With this method,
units of cellulase per ml of culture broth (Fig. 3) in SmF and cellulase production by A. niger in SSF and SmF using coir
29.11units of cellulase per gram of dry mycelial bran in SSF. waste as substrate, were compared in terms of their
extracellular enzyme production in U/g DMB and U/ml,
Comparative evaluation of SmF and SSF system for respectively (Fig. 3). When comparison made between SmF
enzyme titres and SSF, production of total cellulase by SSF was 14.6 fold
The method adopted for comparison of submerged and higher than that of SmF.
solid state fermentation is the same as reported by Solis-Pereira In conclusion the result of the present study clearly
et al. (36) for pectinase production from Aspergillus niger by indicates the potential of Aspergillus niger can be successfully

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Mrudula, S. et al. Production of cellulase by A. niger

cultivated under SSF conditions for the production of cellulase Bacillus cereus MTCC 1305 using solid state fermentation. Food Tech.
Biotechnol., 44 (2), 241-245.
using coir waste as substrate. The coir waste is abundantly
6. Asquieri, E.R.; Park, Y.K. (1992). Production of extracellular cellulases
available in India as it is one of the major producers of
from the thermostable Aspergillus sp. Rev. Microbiol., 23, 183-188.
coconut. Having considered the means of reducing disposal 7. Babu, K.; Sathyanarayana, T. (1995). -Amylase production by
problem, therefore it can be effectively utilized by a potential thermophilic Bacillus coagulans in solid state fermentation. Process
Biochem., 30 (4), 305-309.
strain like Aspergillus niger for production of cellulase which
8. Bakri, Y.P.; Jacques, P.; Thonart. (2003). Xylanase production by
is commercially important.
Penicillum canescens 10-10c in solid state fermentation. Appl. Biochem.
Biotechnol., 108 (1-3), 737-748.
9. Beguin, P. (1990). Molecular biology of cellulose degradation. Ann. Rev.
Microbiol., 44 (2), 19-248.
10. Bhat, M.K.; Bhat, S. (1997). Cellulase degrading enzymes and their
potential industrial applications. Biotechnol. Adv., 15 (3-4), 583-620.
11. Biswas, S.R.; Jana, S.C.; Mishra, A.K.; Nanda, G. (1990). Production,
purification and characterization of xylanase from a hyperxylanotic
mutant of Aspergillus ochraceus. Biotechnol. Bioeng., 35, 244-251.
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mangrove debris. Trends. Appl. Sci. Res., 2, 23-27.
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Steinmuller, H. (1987). The use of lignocellulosic wastes for production
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485-494.
14. Enari, T.M.; Markenan, P.; Fiechter, A. (1977). Production of
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