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Production of cellulases from Aspergillus niger NS-2 in solid state fermentation

on agricultural and kitchen waste residues

Namita Bansal a, Rupinder Tewari b, Raman Soni c, Sanjeev Kumar Soni a,⇑

a Department of Microbiology, Panjab University, Chandigarh 160014, Indiab Department of Biotechnology, Panjab University, Chandigarh 160014, Indiac Department of Biotechnology, D.A.V. College, Chandigarh 160011, India

a r t i c l e i n f o

Article history:

Received 4 August 2011

Accepted 8 March 2012

Available online 12 April 2012

Keywords:

Aspergillus niger

Agro waste

Kitchen waste

Cellulases

Solid state fermentation

a b s t r a c t

Various agricultural and kitchen waste residues were assessed for their ability to support the production

of a complete cellulase system by Aspergillus niger NS-2 in solid state fermentation. Untreated as well as

acid and base-pretreated substrates including corn cobs, carrot peelings, composite, grass, leaves, orange

peelings, pineapple peelings, potato peelings, rice husk, sugarcane bagasse, saw dust, wheat bran, wheat

straw, simply moistened with water, were found to be well suited for the organism’s growth, producing

good amounts of cellulases after 96 h without the supplementation of additional nutritional sources.

Yields of cellulases were higher in alkali treated substrates as compared to acid treated and untreated

substrates except in wheat bran. Of all the substrates tested, wheat bran appeared to be the best suited

substrate producing appreciable yields of CMCase, FPase and b-glucosidase at the levels of 310, 17 and

33 U/g dry substrate respectively. An evaluation of various environmental parameters demonstrated that

appreciable levels of cellulases could be produced over a wide range of temperatures (20–50 C) and pH

levels (3.0–8.0) with a 1:1.5 to 1:1.75 substrate to moisture ratio.

2012 Elsevier Ltd. All rights reserved.

1. Introduction

During the last few decades, interest in the use of lignocellulosic

residues for biofuel production has increased due to their relative

abundance, renewable nature and availability as almost zero cost

substrates. Cellulose, the main component of lignocellulosic bio-

mass, has attracted worldwide attention in its capacity to produce

greener and cleaner fuels by producing fermentable sugars that can

then be converted to second-generation bioethanol. The biocon-

version of cellulose to fermentable sugars requires the synergistic

action of complete cellulase system comprising of endoglucanases

(EC 3.2.1.4) which act randomly on soluble and insoluble cellulose

chains, exoglucanases (cellobiohydrolases; EC 3.2.1.91) which

liberate cellobiose from the reducing and non-reducing ends of

cellulose chains, and b-glucosidases (EC 3.2.1.21) which liberate

glucose from cellobiose (Milala et al., 2005; Bansal et al., 2011;

Deswal et al., 2011). The costs of cellulase account for more than

40% of the total processing cost (Ahamed and Vermette, 2008; Des-

wal et al., 2011). The availability of low-cost cellulases could be

one solution to meet the increasing demand of biofuels. Hence,

the use of low-cost technologies as well as cheaper substrates

can help to reduce cellulase prices. Moreover, the ability of some

microorganisms to make use of lignocellulosic substrates as their

growth medium to produce cellulases can make the bioconversions

more economically viable.

Cellulases are produced by several microorganisms including

bacteria, actinomycetes and fungi, but the latter are of great inter-

est because they excrete their enzymes extracellularly (Bollok and

Reczey, 2005). Trichoderma reesei is the most efficient producer of

endo- and exo-glucanases (Miettinen-Oinnonen and Suominen,

2002), but does not excrete a sufficient amount of b-glucosidase

(Bollok and Reczey, 2005) for which Aspergillus strains are known

to be good producers ( Juhasz et al., 2003). The major obstacle to

using cellulosic residues for biofuel production is the recalcitrant

nature, low yields and high cost of cellulases. The recalcitrant nat-

ure can be overcome by physical, chemical and thermal pretreat-

ments while the enzyme yields can be enhanced by exploring the

diverse environments for efficient natural microbial variants or tai-

loring the existing strains. On the other hand, the enzyme produc-

tion costs can be reduced by adopting suitable fermentation

processes that employ cheap and waste cellulosic residues as the

inducers. Taking into consideration all the above mentioned prob-

lems, scientific dedication targets the economyof the cellulase pro-

duction. Both solid and liquid fermentation systems have been

used for enzyme production, but the former has greater advantages

as it requires less capital, lower energy, a simple fermentation

medium, has superior productivity, does not require a rigorous

control of fermentation parameters and produces less wastewater.

0956-053X/$ - see front matter 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.wasman.2012.03.006

⇑ Corresponding author. Tel.: +91 172 2534149; fax: +91 172 2541770.

E-mail address: [email protected] (S.K. Soni).

Waste Management 32 (2012) 1341–1346

Contents lists available at SciVerse ScienceDirect

Waste Management

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / w a s m a n

http://dx.doi.org/10.1016/j.wasman.2012.03.006mailto:[email protected]://dx.doi.org/10.1016/j.wasman.2012.03.006http://www.sciencedirect.com/science/journal/0956053Xhttp://www.elsevier.com/locate/wasmanhttp://www.elsevier.com/locate/wasmanhttp://www.sciencedirect.com/science/journal/0956053Xhttp://dx.doi.org/10.1016/j.wasman.2012.03.006mailto:[email protected]://dx.doi.org/10.1016/j.wasman.2012.03.006


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Moreover, an easy control of bacterial contamination and lower

costs of downstream processing make it more attractive (Pandey,

1994; Krishna, 2005; Sherief et al., 2010). Solid state fermentation

(SSF) when carried out with different agricultural and kitchen

waste residues adds value by decreasing the cost of enzyme pro-

duction, reducing the quantity of solid waste and boosting the

environmentally friendly management of agricultural and domes-

tic wastes including corn cobs, carrot peelings, composite, grass,

leaves, orange peelings, pineapple peelings, potato peelings, rice

husk, sugarcane baggage, saw dust, wheat bran, wheat straw,

which are increasing as a result of the rising population (da Silva

et al., 2005; Bansal et al., 2011). The Aspergillus species are known

to use a broad range of lignocellulosic substrates (Lockington et al.,

2002; Kang et al., 2004; Wang et al., 2006; Gao et al., 2008) for the

production of cellulases. In the present work, focus was placed on

the production of complete cellulase complex from locally isolated

Aspergillus niger under SSF on different agricultural and kitchen

waste residues.

2. Materials and methods

2.1. Microorganism

A. niger NS-2 used in the present study was isolated locally from

decaying agricultural residues (Bansal et al., 2011) and maintained

on a potato dextrose agar.

2.2. Substrates and pretreatment

Various agro- and kitchen waste residues (corn cobs, carrot

peelings, composite, grass, leaves, orange peelings, pineapple peel-

ings, potato peelings, rice husk, sugarcane baggage, saw dust,

wheat bran, wheat straw) were collected locally. These were dried

by keeping them overnight in a hot air oven (70 C), before being

finely crushed and stored in air tight containers until further use.

These substrates were pretreated separately with 1.0% (v/v)H2SO4 and 1% (w/v) NaOH. Twenty grams of each substrate was

dispensed in 500 ml Erlenmeyer flasks, already containing 100 ml

of acid or alkali and left at room temperature for 2 h. These were

then washed thoroughly with distilled water to remove traces of

acid and base followed by drying as mentioned above.

2.3. Cellulase production under solid state fermentation

Solid state fermentation was carried out in separate sets of

250 ml Erlenmeyer flasks, each having 5.0 g of untreated or treated

(acid and alkali) dry substrates moistened with distilled water (pH

6.5) to obtain a final substrate to moisture ratio of 1: 1.5. These

were autoclaved at 121 C for 20 min, cooled and inoculated with

5 discs (7 mm) cut from the periphery of actively growing coloniesof 72 h-old culture of A. niger NS-2 on PDA plates followed by incu-

bation at 30 C for 96 h under static conditions. The enzymes were

extracted by adding 50 ml of tap water to the solid state cultures

and shaking the contents on a rotary shaker at 150 rpm for 30–

45 min at room temperature (Bansal et al., 2011). The contents of

the flasks were then filtered through a metallic sieve and the solid

residue was pressed to release leftover liquid, centrifuged

(10,000 g ; 4 C) for 10 min and the clear supernatant was analyzed

for cellulase enzyme complex.

2.3.1. Enzyme assays

The components of the cellulase systemwere measured at 50 C

in terms of carboxymethyl cellulase (CMCase), filter paper activity

(FPase) and b-glucosidase according to the methods of Mandels etal. (1976). One unit (U) of the CMCase, FPase,b-glucosidase was ex-

pressed as being equivalent to the enzyme that releases 1 lmole of glucose from CMC, Whatman filter paper and salicin respectively in

0.1 M acetate buffer, pH 4.0, in 1 min under assay conditions using

dinitrosalicylic acid reagent (Miller, 1959). The enzyme productiv-

ities have been expressed in terms of U/g dry substrate (gds) used

in the production medium.

2.4. Time course of cellulase production on wheat bran

Wheat bran was chosen as a substrate for further studies be-

cause no pretreatment was required for inducing the maximum

production of enzyme components. The time course of enzyme

production was studied by preparing different sets of 250 ml Erlen-

meyer flasks, each containing 5 g wheat bran moistened with 1.5

parts of distilled water. These were sterilized and inoculated, as

mentioned in Section 2.3, and incubated at 30 C for 10 days. The

flasks, in duplicate, were withdrawn at regular intervals of 24 h

to study the production profiles of the cellulase system.

2.5. Optimization of environmental factors for cellulase production

on wheat bran

The effect of various parameters such as incubation tempera-

ture (20–50 C), substrate to moisture ratio (1:0.25–1:3.0), initial

pH of the water used as a moistening agent (3.0–8.0), inoculum

size (number of disc varied from 1 to 10) was investigated on the

production profile of the cellulase system using wheat bran in

SSF by varying one variable at a time.

3. Results and discussion

The use of abundantly available and cost-effective agricultural

and kitchen waste residues that were once considered to be of

no value are presently being recognized as raw materials of poten-

tial value (Karmakar and Ray, 2010) to achieve higher cellulase

yields using SSF, thereby reducing the overall cost of enzymeproduction.

3.1. Evaluation of different agro- and kitchen waste residues for

enzyme production in solid state fermentation

The production of cellulases with SSF is gaining interest as a cost

effective technology with an almost tenfold predicted reduction in

costs and much higher yields as compared to submerged fermenta-

tion (Tengerdy, 1996; Singhania et al., 2006). The nature of solid

substrate is the most important factor in SSF for cellulase produc-

tion as it not only supplies nutrients to the culture, but also serves

as an anchorage for the microbial cells. Therefore, the particle size,

chemical composition, cost and availability of the substrate are of

critical importanceduring the selection of substrates. An ideal solidsubstrate should provide all the necessary nutrients to the growing

microorganism for optimal function. However, some of the nutri-

ents may be available in sub-optimal concentrations, or not even

present in the substrate. In such cases, it would be necessary to sup-

plement them externally. It has also been common practice to pre-

treat some substrates before use in SSF processes, making them

more easily accessible for microbial growth (Pandey et al., 2001).

The cellulase yields obtained on various raw and acid/alkali treated

substrates are revealed in Tables 1–3. As compared to pre-treated

substrates most of the untreated raw substrates had lower cellulase

yields probably due to higher lignin content and firm binding, mak-

ing them less accessible to the organism. Higher productivities of

cellulases were noted in alkali-treated substrates than acid treated

which can be related to the release of lignin component in the caseof alkali treatment causing solubilization and modifications in the

1342 N. Bansal et al. / Waste Management 32 (2012) 1341–1346


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crystalline state of the cellulose, while in case of acid treatment, the

hemicelluose component becomes solubilized producing mono-

mers, furfural, hydroxymethylfurfurals and other volatile products

which, along with lignin, produce a negative environment for an

organism to grow, thereby lowering the amount of cellulases

(Hendriks and Zeeman, 2009). Several agricultural crop residues

in the form of flours, brans, straws, hulls, residues of the fruit pro-

cessing industries, waste of the oil processing mills have been suc-

cessfully used in solid state fermentation by many workers,

however there is hardly any report on the use of kitchen waste res-

idues in the process. Wheat bran has been the prime among many

solid state fermentation processes developed for the production of

bulk chemicals and value-added fine products (Pandey et al.,

2001). Various reports on cellulase production on oil palm waste

(Alam et al., 2005), groundnut waste (Vyas and Vyas, 2005), cassava

waste (Pothiraj et al., 2006), pineapple waste (Omojasola et al.,

2008), pretreated lignocellulosics (Sridevi et al., 2009), waste paper

( Juwaiedet al., 2010), sugarcanebagasse( Ja’afaruand Fagade, 2010)

agro waste (Karmakar and Ray, 2010), coir waste and saw dust

(Samuel et al., 2010), molasses (Shabeb et al., 2010) are available,

butthe yields are quite lowwhen compared with those from A. niger

NS-2.

Untreated wheat bran induced the maximum production of all

the components of cellulases and yielded 310.6, 16.8, 33.0 U/gds

of CMCase, FPase, b-glucosidase, respectively. Wheat bran is the

outer 15% of the wheat seed and is composed predominantly of

non-starchycarbohydrates(58%), starch (19%) andcrudeprotein

(18%), with the non-starch polysaccharides being primarily 70%

Table 1

Cellulase production by Aspergillus niger NS-2 on substrates without pre treatment.

Substrates (untreated) CMCase (U/gds) FPase (U/gds) b-glucosidase (U/gds)

Corn cobs 10.0 ± 0.4 3.1 ± 0.06 1.8 ± 0.072

Carrot peelings 4.9 ± 0.003 0.3 ± 0.001 5.0 ± 0.048

Composite kitchen waste 48.6 ± 1.4 10.3 ± 0.1 19.5 ± 0.39

Grass 17.6 ± 0.01 5.1 ± 0.5 10 ± 0.14

Leaves 12.1 ± 0.36 8.0 ± 0.1 6.8 ± 0.064

Orange peelings 1.0 ± 0.003 1.9 ± 0.001 3.0 ± 0.018Pineapple peelings 2.6 ± 0.02 1.0 ± 0.01 2.8 ± 0.013

Potato peelings 31.3 ± 0.3 5.9 ± 0.17 18.3 ± 0.43

Rice husk 14.1 ± 0.4 3.1 ± 0.006 10.6 ± 0.31

Sugarcane baggase 5.0 ± 0.1 1.5 ± 0.005 3.0 ± 0.03

Saw dust 1.8 ± 0.2 2.0 ± 0.021 1.0 ± 0.005

Wheat bran 310.6 ± 3.0 16.8 ± 0.15 33.0 ± 1.2

Wheat straw 11.2 ± 0.01 2.8 ± 0.01 5.5 ± 0.06

Table 2

Cellulase production by Aspergillus niger NS-2 on H2SO4 pre-treated substrates.

Substrates (H2SO4 treated) CMCase (U/gds) FPase (U/gds) b-glucosidase (U/gds)

Corn cobs 89.3 ± 1.4 20.2 ± 0.31 6.3 ± 0.11Carrot peelings 100.0 ± 3.2 13.0 ± 0.1 11.0 ± 0.19

Composite kitchen waste 111 ± 4.3 11.0 ± 0.5 15.0 ± 0.43

Grass 60.1 ± 1.2 11.0 ± 0.43 6.9 ± 0.24

Leaves 80.0 ± 1 12.9 ± 0.03 4.2 ± 0.51

Orange peelings 68.6 ± 1.9 15.0 ± 0.38 1.8 ± 0.001

Pineapple peelings 70.0 ± 2.1 12.0 ± 0.13 1.5 ± 0.1

Potato peelings 110.6 ± 3.4 18.0 ± 0.42 3.5 ± 0.06

Rice husk 60.0 ± 0.9 16.0 ± 0.6 1.0 ± 0.031

Sugarcane baggase 72.8 ± 2.6 20 ± 0.12 5.8 ± 0.11

Saw dust 55.4 ± 1.5 18.1 ± 0.65 3.0 ± 0.21

Wheat bran 240.0 ± 4.5 10.2 ± 0.31 24.1 ± 0.14

Wheat straw 100.9 ± 3.2 19.4 ± 0.11 4.3 ± 0.04

Table 3

Cellulase production by Aspergillus niger NS-2 on NaOH pretreated substrates.

Substrate (NaOH pretreated) CMCase (U/gds) FPase (U/gds) b-glucosidase (U/gds)

Corn cobs 108.8 ± 3.5 40.5 ± 0.98 18.1 ± 0.21

Carrot peelings 121.76 ± 2.1 21.0 ± 0.67 13.6 ± 0.44

Composite kitchen waste 145.7 ± 3.8 16.0 ± 0.13 19.0 ± 0.11

Grass 81.6 ± 0.11 20.0 ± 0.005 13.1 ± 0.1

Leaves 100.8 ± 1.2 17.8 ± 0.12 6.3 ± 0.12

Orange peelings 90.1 ± 0.99 21.0 ± 0.01 4.0 ± 0.09

Pineapple waste 100.0 ± 1.6 18.1 ± 0.04 3.4 ± 0.01

Potato peelings 144.0 ± 3.7 24.0 ± 0.1 17.8 ± 0.02

Rice husk 75.6 ± 3.1 14.0 ± 0.004 14.1 ± 0.16

Sugarcane baggase 90.7 ± 2.1 15.3 ± 0.12 16.3 ± 0.1

Saw dust 73.8 ± 1.1 6.9 ± 0.18 15.7 ± 0.32

Wheat bran 261.2 ± 5.3 18.0 ± 0.1 11.0 ± 0.12

Wheat straw 127.5 ± 2.7 30.6 ± 0.12 5.1 ± 0.09

N. Bansal et al./ Waste Management 32 (2012) 1341–1346 1343


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arabinoxylans, 24% cellulose and 6% b-(1–3),(1–4)-glucans (Sun

et al., 2008). The optimal growth of the fungus in untreated wheat

bran and the production of significant levels of cellulases was prob-

ably due to the presence of adequate amounts of various nutrients,

higher porosity and efficient aeration (Roussos et al., 1991; Babu

and Satyanarayana, 1995; Baysal et al., 2003). Appreciable yields

were also obtained on a pretreated composite mixture as well as

on potato peels with alkali treatment, thereby promoting better

growthand enzyme yields (Tables2 and 3). An untreated composite

mixture containing a variety of nutrients may probably have an

inhibitory effect of some components, thus leading to a lesser pro-

duction of enzymes as compared to wheat bran. The use of wheat

bran and sugarcane bagasse for cellulase production has been re-

ported earlier (Kang et al., 2004; Haq et al., 2006). In a study by

Sun et al. (2008) it was reported that the regulation of wheat bran

composition is needed for the improved induction of cellulases in

P. decumbans. The media formulations in most of the studies in-

volves the exogenous addition of various carbon, nitrogen and min-

eral sources. The novelty of the present study is the appreciable

yields of complete cellulase system on various waste residues with-

out the supplementation of any nutrient.

3.2. Time course of cellulase production on wheat bran

The evaluation of the time course is of prime importance for cel-

lulase biosynthesis by fungi (Kuhad and Singh, 1993). In the pres-

ent study, maximum enzyme production occurred after 96 h with

the yields of CMCase (296.0 ± 12.7 U/gds), FPase (17.08 ± 4.7 U/

gds), b-glucosidase (32.0 ± 0.1 U/gds) as depicted in Fig. 1. On fur-

ther incubation, the enzyme yields declined gradually at the end of

216 h probably due to the release of proteases and the drop in the

pH of the medium. In a similar study for cellulase production by T.

reesei, cellulase yields remained fairly constant over the 72–120 h

incubation period with maximum levels observed after 96 h

(Singhania et al., 2006). Cellulase production by Trichoderma sp.

on apple pomace revealed a progressive increase in the enzyme

activity with the incubation time from 0 to 120 h with the maxi-

mum reaching (2.3 U/gds) at 120 h (Sun et al., 2010).

3.3. Optimization of environmental factors for cellulase production on

wheat bran

Environmental factors are known to affect microbial growth

and enzyme production. In the present study, cellulase production

by A. niger NS-2 on wheat bran was optimized with respect to tem-

perature, pH, moisture content and inoculums size.

3.3.1. Temperature

Incubation temperature is an important factor affecting enzyme

production in SSF. The temperature normally achieved in SSF

ranges from 25 to 30 C (Singhania et al., 2006). Highest yields of

CMCase (297.00 ± 10.46 U/gds), FPase (15.9 ± 1.74 U/gds), b-gluco-

sidase (33.2 ± 1.8 U/gds) were obtained at 30 C after 96 h as de-

picted in Fig. 2. The maximum yields were obtained at 30 C, but

appreciable yields of enzymes were obtained at 40 and 50 C,

exhibiting productivities of 255.36 ± 4.46 U/gds (CMCase),

10.60 ± 1.91 U/gds (FPase), 30.0 ± 1.1 U/gds (b-glucosidase) and

224 ± 7.6 U/gds (CMCase), 7.2 ± 1.47 U/gds (FPase), 22.4 ± 0.53 U/

gds (b-glucosidase) respectively. A. niger NS-2 exhibited a wider

range of temperatures for its growth and cellulase production in

the present study. The optimum temperature in a range of 25–

30 C has been reported for cellulase production in various fungi

(Gautam et al., 2011) suggesting that cellulase production varies

with the strain. The optimum temperature of 30 C was reported

for cellulase production by A. niger YL128 ( Ja’afaru and Fagade,

2010). Narasimha et al. (2006) used saw dust for production of cel-

lulase by A. niger with 0.775 U/ml cellulase activity obtained at

28 C. Temperature from 28 to 34 C did not affect significantly

the production of endoglucanase (10.3 U/ml) by A. niger ( Jecu,

2000). Trichoderma harzianum enzyme activity increased with a

rise in temperature up to 35 C with maximum activity of

422 ± 3.21lM/ml/min (Iqbal et al., 2010). The optimum tempera-ture for cellulase production by Aspergillus fumigatus was reported

to be 32 C (Gilna and Khaleel, 2011). In a study by Haq et al.

(2006) a temperature range starting from 25 to 40 C was investi-

gated and among these, 30 C was optimized for the best growth of

A. niger and Trichoderma viride. It is a well-known fact that higher

temperatures (above 30 C) alter the cell membrane composition

and stimulate protein catabolism, causing cell death. In a study

by da Silva et al. (2005) Thermoascus aurantiacus grew and pro-

duced enzymes at 50 C.

3.3.2. Moisture content

Water is essential for the microbial metabolism and its deple-

tion affects the diffusion of solutes, gases and osmotic changes

brought about by excessive metabolites in the vicinity of cells

(Todd, 1972; de Loecker et al., 1993). During solid-state fermenta-

tion, both high and low moisture contents affect productivity. A

higher moisture level decreases porosity, changes wheat bran par-

ticle structure, promotes the development of stickiness and lowers

oxygen transfer, whereas lower moisture content causes a reduc-

tion in the solubility of nutrients of the solid substrate, a lower

degree of swelling and higher water tension (Anto et al., 2006).

Fig. 1. Time course of CMCase, FPase and b-glucosidase production by the solidstate culture of A niger NS-2.

Fig. 2. Effect of incubation temperature on CMCase, FPase and b-glucosidaseproduction by solid state cultures of A niger NS-2 on wheat bran.



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The substrate-moisture ratio of 1:1.5 (60.0% water level) was best

suited for CMCase and FPase revealing the yields of 298.0 ± 7.29

and 21.0 ± 1.7 U/gds as depicted in Fig. 3. b-glucosidase yields of

50.2 ± 1.76 U/gds was observed at a substrate to water ratio of

1:1.75. The reduction in enzyme production in all other ratios

may be attributed to lesser growth, a reduction in substrate poros-

ity, changes in the structure of substrate particles and a reduction

of gas volume(Babu and Satyanarayana, 1995). In one of the earlier

reports, A. niger produced the highest FPase (2.9 U/g) under a mois-

ture content level of 50.0% (Chandra et al., 2007) while Lee et al.,

2011 reported the maximum FPase yield of 2.3 U/g in A. niger un-

der 70% moisture content, which also proved to be optimal initial

moisture level for the highest cellulase production by Trichoderma

sp. GIM 3.0010 (Sun et al., 2010). The optimal water fractions in the

solid substrate appeared to be 40.0–60.0% (by mass) in the case of

Trichoderma koningii for producing FPase yield of 4.3 U/g and

CMCase yield of 10.5 U/g (Liu and Yang, 2007). In a similar study

by Soni et al. (2010) on Aspergillus sp. S4B2F, 1:1.5 was the best sui-

ted substrate to moisture ratio for producing the highest cellulase

yield.

3.3.3. Initial pH of the moistening agent To study the effect of pH on cellulase production, the pH of

moistening agent was adjusted between 3.0 and 8.0. The produc-

tion profile of all the three components as shown in Fig. 4 depicts

the highest CMCase 394.6 ± 1.23 U/gds, FPase 28.00 ± 1.8 U/gds

and b-glucosidase 46.0 ± 1.81 U/gds at pH 7.0, respectively. At pH

3.0 and pH 8.0, the CMCase yields were 235.5 ± 8.5 and

381.9 ± 6.45 U/gds, respectively. On the other hand, FPase produc-

tivities at pH 3.0 and pH 8.0 were 16.8 ± 0.2 and 22.49 ± 1.3 U/gds,

while b-glucosidase yields under these conditions were 22.0 ± 1.79

and 31.3 ± 0.87 U/gds, respectively. The enzyme activity increased

gradually up to the optimum level, followed by slight decrease in

activity. The results obtained in this study are in agreement with

Gautam et al. (2011) who reported the highest production of cellu-

lases by A. niger and Trichoderma sp. at pH 6.5.

3.3.4. Inoculum size

The medium inoculated with 5 mycelial discs (7 mm dia) pro-

duced the highest cellulase yields of 340.0 ± 4.5 U/gds (CMCase),

20.9 ± 0.29 U/gds (FPase), 34.9 ± 3.0 U/gds (b-glucosidase) which

gradually decreased with an increased no of mycelia discs, as de-

picted in Fig. 5. Lower cellulase biosynthesis at lower inoculum is

probably due to less conidial cells which are insufficient to use

the fermentation medium in a better way, while the decreased

yield at higher inoculums size is probably due to anaerobic condi-

tions in the medium, owing to initial high concentration of conidial

cells or nutritional imbalance due to tremendous growth (Haq

et al., 1993). In a study by Acharya et al. (2008) employing 5, 10,

15 and 20 mycelial discs (7 mm dia) of A. niger as inoculum, the

highest cellulase productivity was obtained with 10 discs. The ef-

fect of different inoculum sizes studied for the production of the

carboxymethyl cellulase enzyme from T. harzianum on pretreated

wheat straw in SSF revealed maximum cellulase productivity at

10% inoculum size, while further increases in an inoculum level

showed a decline in enzyme activity (Iqbal et al., 2010).

4. Conclusions

Successful attempts have been made to make use of various

agro- and kitchen waste residues as substrates for the production

of complete cellulase complex by A. niger NS-2 under solid state

fermentation, with a view to developing a low cost production sys-

tem. The study is novel because without any supplementation of

exogenous nutrients in the solid medium consisting of various

waste residues, simply moistened with distilled water, fairly good

amounts of each of CMCase, FPase and b-glucosidase were ob-

tained. Untreated wheat bran induced the maximum production

of enzyme components followed by alkali treated composite kitch-

en waste and potato peelings. An appreciable production of cellu-lases on different kitchen waste residues highlighted the potential

Fig. 3. Effect of moisture content on the solid medium on CMCase, FPase and b-glucosidase production by solid state cultures of A niger NS-2 on wheat bran.

Fig. 4. Effect of pH of the moistening agent on CMCase, FPase and b-glucosidase

production by solid state cultures of A niger NS-2 on wheat bran.

Fig. 5. Effect of inoculum size on CMCase, FPase and b-glucosidase production bysolid state cultures of A niger NS-2 on wheat bran.

N. Bansal et al./ Waste Management 32 (2012) 1341–1346 1345


6/6

of these wastes as possible raw materials for enzyme production,

thereby reducing the cost of cellulases. Further optimization and

scale up studies need to be carried out in order to exploit these

inexpensively produced cellulases in second-generation biofuel

production.

Acknowledgments

This work was supported by University Grants Commission

(UGC), New Delhi, under the special assistance programme (SAP)

and Department of Science & Technology (DST), Government of In-

dia under PURSE grant.

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