IOSR Journal of Environmental Science, Toxicology and Food Technology (IOSR-JESTFT)

e-ISSN: 2319-2402,p- ISSN: 2319-2399.Volume 10, Issue 5 Ver. II (May. 2016), PP 68-73
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Optimization of Cellulase Enzyme from Vegetable Waste by

Using Trichoderma atroviride in Solid State Fermentation
Sakshi Bairagi
(Department of Environmental Sciences, M.D. University, Rohtak (Haryana), India)

Abstract: The present study deals with production of cellulase enzymes from filamentous soil fungus
Trichoderma atroviride by using vegetable waste as substrate through the process of solid state fermentation.
The enzyme production was further improved by optimizing a number of physical parameters such as incubation
time, temperature, pH, inoculums size and nutritional factors (carbon source, nitrogen source and detergents).
By optimization of different parameters, the maximum activities of cellulase synthesized by Trichoderma
atroviride were observed after 5 days incubation at H 6 and 30C temperature with sucrose, yeast extract and
Tween -80 as carbon, nitrogen and detergents supplements respectively. The high activity of cellulase produced
by the fungus suggests its potential for commercial scale production for various industrial applications.
Keywords: Cellulase, optimization, solid state fermentation, Trichoderma atroviride, vegetable waste.

I.

Introduction

The need for utilizing renewable resources to meet the future demand for fuel has increased the
attention on cellulose. Cellulose is the structural component of the primary cell wall of plant biomass. It is a
polymer of -1, 4 linked glucose units. It is considered as one of the most abundant renewable carbon source on
earth and the dominating waste material from agriculture [1]. It represents about 1.5 x 1012 tons of the annual
biomass production through photosynthesis and is considered to be an almost inexhaustible source of raw
material. Its crystalline structure and insoluble nature represents a big challenge for enzymatic hydrolysis.
Cellulose is generally degraded by multi-complex enzyme called cellulases [2]. Cellulase (E.C 3.2.1.4) refers to
a class of enzyme that catalyzes the hydrolysis of 1, 4 -D glycosidic linkages in cellulose component [3].
Cellulase enzymes play an important role in natural biodegradation processes in which plant materials
are effectively degraded by cellulolytic fungi, bacteria, actinomycetes and protozoa. In industry, these enzymes
have its importance due to major role in the production of fermentable sugars and ethanol, organic acids and
chemicals. The major other applications are biostoning of jeans and biopolishing of cotton and other cellulosic
fabrics, paper recycling and as animal feed additives for improving the nutritional quality and digestibility [4]. It
is also used for deinking of paper industries and to enhance pulp drainage in textile industries. It is used for
bioremediation, waste water treatment and also for single cell protein [5]. It has importance in food sciences like
food processing, drying of beans in coffee. Main function of cellulase enzyme in food industry is extraction,
clarification and stabilization of fruit juices and vegetables.
Bioconversion of cellulosic materials mainly depends on the nature of cellulose component, sources of
cellulase enzyme and optimal conditions for production of enzymes. Cellulase enzymes are synthesized by a
number of microorganisms. Fungi and bacteria are the main natural agents of cellulose degradation [6].
However, fungi are well known agents of decomposition of organic matter due to their elongated hyphea that
creates mechanical pressure on the cellulose structure, causing them to production of large amounts of cellulase
enzymes [7]. Cellulase production from fungi is highly useful for the enzyme production as compared to other
microorganisms. Fungi such as Trichoderma sp., Aspergillus sp. and Penicillium sp. are the most commonly
cellulase producers. However, the most extensively studied cellulases are produced by efficient lignocellulose
degrading fungi, particularly Trichoderma sp. [8].
Improvement of microbial strains for the over-production of industrial products can reduce the process
cost and may also possess some specialized desirable characteristics. Several lignocellulosic materials are
efficient substrates for white-rot fungi, which produce industrially important cellulolytic enzymes. Among
processes used for enzyme production, solid state fermentation (SSF) using agro-wastes are an attractive and
cost effective option because it presents higher productivity involving a simpler operation [9]. Solid state
fermentation (SSF) is gaining interest as a cost effective technology for the production of higher yields of
cellulase as compared to liquid culture. SSF has many advantages over other fermentation processes due to
simple process, energy saving, less water consumption and less production of waste products. Production of
cellulases by the fungal isolates requires optimal conditions for their growth which leads to the release of
extracellular enzymes. The growth conditions as well as extracellular enzyme production conditions is likely to
vary among isolates. Certain fermentation parameters such as temperature, incubation period, carbon source, pH
etc. were found to be critically affecting the cellulase yield [10].
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Optimization of Cellulase Enzyme From Vegetable Waste By Using Trichoderma Atroviride in Solid..
Therefore, the present study aims to enhance the production of extracellular cellulases by filamentous fungi
Trichoderma atroviride under solid state fermentation using vegetable waste as substrate and optimizing cultural
parameters favoring the maximal exploration of fungal capacity for overproducing of cellulase.

II.

Materials And Methods

2.1 Microorganism and Growth Conditions

Trichoderma atroviride, originally isolated from soil was used for this study. The culture was
maintained on potato dextrose agar (PDA) slants at 4C and subcultured every 6 months.
2.2 Cellulase production from fungal strain in solid state fermentation
Solid state fermentation was carried out in 250 ml Erlenmeyer flasks containing mandel and weber
media supplemented with 5 g of vegetable waste as substrate. Each flask was autoclaved at 121C for 20 min.
After cooling, each flask was inoculated with 2 ml of the spore suspension of Trichoderma atroviride prepared
in Tween-80 from 5 days grown old slants, and the inoculated flasks were incubated in an incubator. Crude
enzyme was extracted from fermented substrate by adding of 0.05 M citrate buffer and the contents were mixed
for 1 hr at 180 rpm in an orbital shaker and filtered through a muslin cloth by squeezing. The extract was
centrifuged at 10,000 rpm for 10 min and the supernatant was used for determination of enzyme activity. The
enzyme activity was determined by carboxymethyl cellulase assay and filter paper assay.
2.3 Carboxymethyl Cellulase (CMCase) Assay
0.5 ml of crude enzyme supernatant was incubated with 0.5 ml of 1% (w/v) CMC (carboxymethyl
cellulose) in 0.05 M citrate buffer solution of pH 4.8 for 30 minutes at 50C. The resulted reducing sugars were
determined according to Millers method with glucose as a standard [11].
2.4 Filter Paper (FPase) Assay
For FPase activity 0.5 ml of crude enzyme supernatant was incubated with 1 ml of 0.1M citrate buffer
of pH 4.8 containing 50mg Whatman no. 1 filter paper. After incubation of 1 hour at 50C, obtained reducing
sugars were estimated by Millers method.
2.5 Optimization of culture conditions for cellulase production
To optimize the cellulase production, effects of fermentation conditions such as the incubation period,
pH, temperature, carbon source, nitrogen source and detergents were studied.
2.5.1. Effect of incubation period
Effect of incubation period on enzyme production was checked by incubating the fermentation medium
for different incubation time (3 to 8 days) and enzyme assay was carried out at interval of 24 hours by DNS
method.
2.5.2. Effect of temperature
In order to determine the effective temperature for cellulase production by the Trichoderma atroviride,
fermentation was carried out at 5C intervals in the range of 25, 30, 35 and 40C.
2.5.3 Effect of pH
To determine optimal pH, fungus cultures were cultivated in a 250 mL flask containing optimized
medium with different pH ranges from 3.0 to 8.0. The pH of the medium was adjusted by using 1 N HCl or 1 N
NaOH. The flasks were kept at 30C for 5 days of cultivation.
2.5.4 Effect of carbon sources
Effects of various carbon compounds namely, sucrose, dextrose, starch and lactose were used for
studying. The broth was distributed into different flasks and 1.0% of each carbon sources were then added
before inoculation of the strain and after culture inoculation, the flasks were incubated for 5 days at 30C.
2.5.5 Effect of nitrogen sources
The fermentation medium was supplemented with organic and inorganic compounds (yeast extract,
peptone, beef extract and sodium nitrate) and was incubated for 5 days at 30C.
2.5.6 Effect of different detergents
Four different surfactants for increasing of cellulase production such as Tween 20, Tween 80, SDS
(sodiumdodecyl sulphate) and PEG-6000 (Polyethylene Glycol) were used.
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Optimization of Cellulase Enzyme From Vegetable Waste By Using Trichoderma Atroviride in Solid..
2.5.7 Statistical Analysis
Data presented on the average of three replicates (SE) obtained from their independent experiments.

III.

Result And Discussion

3.1 Optimization of parameters for cellulase enzyme production

Medium parameters such as temperature, pH, incubation time, and carbon, nitrogen and detergent
source play very important role in production of enzyme and greatly influence the enzyme activity. CMCase and
FPase test were used to check the enzyme activity at variable parameters.
3.1.1Effect of incubation time
Time of fermentation has an important impact on the product formation. Enzyme production is related
to time of incubation [12]. Time course for production of cellulase enzyme was investigated and fermentation
for a period of 3 to 8 days were carried out. Enzyme activity was investigated after interval of 24 hours. The
results showed that the maximum CMCase activity (77.39 U/g) and FPase activity (42.08 U/g) were observed at
incubation time of 120 hours or 5 day was for Trichoderma atroviride. Decrease in cellulase activity was
recorded with further incubation (Fig. 1). Beyond 5th day, a steep loss in enzyme production was observed. This
trend of decreased enzyme activity may be due to the depletion of macro and micronutrients in fermentation
medium with the lapse in time, which stressed fungal physiology resulting in the inactivation of secretary
machinery of enzymes [13]. This result of incubation time for Trichoderma atroviride was found comparable
with the reports of Karthikeyan et al. [14] who reported 120 hours to be optimum incubation time for the
production of cellulase.

Enzyme Activity (U/g)

CMCase

FPase

100
80
60
40
20
0
3

Incubation Period (Days)

Fig. 1: Effect of incubation period on cellulase production.

3.1.2 Effect of temperature
Temperature is the important parameter that effects the cellulase production. Even slight changes in
temperature can affect enzyme production. Presently, the optimal temperature for maximum cellulase
production was studied by incubation of the inoculated media at different temperature ranging from 25 - 40C
and optimum temperature for CMCase and FPase activity was found to be 30 with FPase activity of 43.11 U/g,
CMCase activity of 85.31 U/g. Enzyme activity was decreased at 35C for (Fig. 2). These results of present
study are comparable with the results of Maurya et al. and Shafique et al. [15, 16] who reported 30C optimum
temperature for cellulase production.

Enzyme Activity (U/g)

CMCase

FPase

100
80
60
40
20
0
25

30

35

40

Incubation Temperature (C)

Fig. 2: Effect of incubation temperature on cellulase production.

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Optimization of Cellulase Enzyme From Vegetable Waste By Using Trichoderma Atroviride in Solid..
3.3.3 Effect of pH
Among physical parameters, pH of the growth medium plays an important role by inducing
morphological changes in microbes and in enzyme secretion [17]. The pH change observed during the growth of
microbes also affects product stability in the medium. Trichoderma atroviride was allowed to grow in media of
different pH ranging from 3.0 to 8.0. Maximum enzyme production was observed in medium of pH 6 (Fig 3).
Trichoderma atroviride showed maximum CMCase (88.91 U/g) and FPase (50.96 U/g) production at pH 6
during fifth day of incubation at 30C. Reduction in enzymes activities after 6.0 pH may be ascribed to the fact
that change in pH may change the three dimensional structure of the enzymes. These results are in agreement
with the observations of Baig et al. [18] wherein Trichoderma lignorum favoured a pH of 6.0 as optimum for
maximum CMCase production from using banana waste. Our results are also similar with Jaradat et al. [19] who
reported that the cellulase enzyme from the active isolate J2 was active over a pH range of 47 with maximum
activity at pH 6.

Enzyme Activity (U/g)

CMCase

FPase

100
80
60
40
20
0
3

pH

Fig. 3: Effect of pH on cellulase production.

3.3.4 Effect of carbon sources
Carbon sources play a crucial role in the cell metabolism and synthesis of cellulase. Trichoderma
atroviride was grown on various carbon sources at 1% concentration to determine their potential to support
cellulase enzyme activities. Various sources of carbon such as sucrose, dextrose, starch and lactose were used in
growth media. Results obtained showed that Trichoderma atroviride in presence of sucrose brought about the
maximum CMCase (120.81 U/g) and FPase (86.15 U/g) production compared to other carbon sources (Fig 4).
Gautam et al. [20] also observed that sucrose (1.0%) was found to be the main carbon source for cellulase
production by using Trichoderma sp..

Enzyme Activity (U/g)

CMCase

FPase

140
120
100
80
60
40
20
0
Control Sucrose Dextrose Starch

Lactose

Carbon Source (1 %)

Fig. 4: Effect of carbon source on cellulase production.

3.3.5 Effect of nitrogen sources
The nitrogen source is important for industrial fermentation medium designing to meet maximum
enzyme production. The effect of nitrogen sources on enzyme production of Trichoderma atroviride by
incorporating various nitrogen sources at 1% concentration into Mandel and Weber media was studied and the
results are shown in Fig. 5. The maximum activities of CMCase and FPase by Trichoderma atroviride using
yeast extract as best nitrogen source were 176.29 and 93.46 U/g, respectively. Whereas minimum CMCase
(91.06 U/g) and FPase (53.05 U/g) is recorded in control. Yeast extract was found to be the best organic source
in enhancing cellulase production probably because this complex nitrogen source contains more elements that
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Optimization of Cellulase Enzyme From Vegetable Waste By Using Trichoderma Atroviride in Solid..
are necessary for the metabolism of fungi. The results of present study regarding best nitrogen source were
found to be similar with the results of the previous studies where yeast extract was the best nitrogen source for
the production of cellulase by Trichoderma.

Enzyme Activity (U/g)

CMCase

FPase

180
160
140
120
100
80
60
40
20
0
Control Yeast Peptone Beef Sodium
Extract
Extract Nitrate
Nitrogen Source (1%)

Fig. 5: Effect of nitrogen sources on cellulase production.

3.3.6 Effect of detergents
Surfactants alter cell permeability of microorganisms which lead to increased protein secretion or
surface effects on cell-bound enzymes. The detergents had various effects on different enzymes and most
commonly used detergents were Tween 20, Tween 80, Polyethylene glycol (PEG-6000) and sodium dodecyl
sulfate (SDS) used in this study. In control experiment, no detergent was added. Maximum CMCase (178.92
U/g) and FPase (98.89 U/g) activities by Trichoderma atroviride was observed with Tween 80 as shown in Fig.
6. It was observed that medium containing Tween 80 had the highest positive influence on production of
cellulase compared to other surfactants. The addition of Tween 80 increased the cellulase production by several
folds. Similar observation was earlier reported by Liu et al. [21] for cellulase and xylanase production in SSF
method by Trichoderma viride. The result of this study therefore, revealed that Tween 80 at a concentration of
1% was the best surfactant for optimum production of the cellulase.

Enzyme Activity (U/g)

CMCase

FPase

180
160
140
120
100
80
60
40
20
0

Detergents (1%)

Fig. 6: Effect of detergents on cellulase production.

In the present study temperature of 30C, pH 6, incubation time of 5 days, sucrose as carbon source and
yeast extract as nitrogen source and Tween -80 as detergent source were found to be optimum for Trichoderma
atroviride as maximum cellulase enzyme production was observed with these parameters.

IV.

Conclusions

The cost-effective technologies are needed for economical production of cellulases using vegetable
waste as substrate. Cellulase yields appear to depend on a complex relationship involving a variety of factors
like incubation time, pH value, temperature, presence of carbon, nitrogen and detergent sources. Major
parameters affecting the fermentation process for enzyme production were studied and optimal levels were
identified. Presently our studies investigated that the fungi belonging to Trichoderma atroviride effectively
produced cellulase under laboratory conditions and can be used for various industrial applications.

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Optimization of Cellulase Enzyme From Vegetable Waste By Using Trichoderma Atroviride in Solid..
Acknowledgement
The author gratefully acknowledges support by getting Senior Research Fellowship from the
University Grant Commission (UGC) and lab facilities from Deptt. of Environmental Sciences, M.D.University,
Rohtak.

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