Hindawi Publishing Corporation

ISRN Biotechnology
Volume 2013, Article ID 985685, 7 pages
http://dx.doi.org/10.5402/2013/985685

Research Article
Optimization of Cellulase Production from
Bacteria Isolated from Soil

Sonia Sethi, Aparna Datta, B. Lal Gupta, and Saksham Gupta

Department of Biotechnology, Dr. B. Lal Institute of Biotechnology, Malviya Industrial Area, Malviya Nagar, Jaipur 302017, India

Correspondence should be addressed to Sonia Sethi; [email protected]

Received 18 December 2012; Accepted 5 January 2013

Academic Editors: D. Pant and A. Tiessen

Copyright © 2013 Sonia Sethi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cellulase-producing bacteria were isolated from soil and identified as Pseudomonas fluorescens, Bacillus subtilIs, E. coli, and Serratia
marcescens. Optimization of the fermentation medium for maximum cellulase production was carried out. The culture conditions
like pH, temperature, carbon sources, and nitrogen sources were optimized. The optimum conditions found for cellulase production
were 40∘ C at pH 10 with glucose as carbon source and ammonium sulphate as nitrogen source, and coconut cake stimulates the
production of cellulase. Among bacteria, Pseudomonas fluorescens is the best cellulase producer among the four followed by Bacillus
subtilis, E. coli, and Serratia marscens.

1. Introduction However, the application of bacteria in producing cellulase is

not widely used. The cellulolytic property of some bacterial
Cellulose is the most abundant biomass on Earth [1]. It is genera such as Cellulomonas, Cellvibrio, Pseudomonas sp [9].
the primary product of photosynthesis in terrestrial envi- Bacillus, and Micrococcus [7], was also reported. Enzyme
ronments and the most abundant renewable bioresource production is closely controlled in microorganisms and for
produced in the biosphere [2, 3]. Cellulose is commonly improving its productivity these controls can be ameliorated.
degraded by an enzyme called cellulase. This enzyme is pro- Cellulase yields appear to depend upon a complex relation-
duced by several microorganisms, commonly by bacteria and ship involving a variety of factors like inoculums size, pH
fungi [4–7]. value, temperature, presence of inducers, medium additives,
Cellulose is the principal constituent of the cell wall of aeration, growth time, and so forth [7].
most terrestrial plants. The source of cellulose is in plants and Enormous amounts of agricultural, industrial, and
it is found as microfibrils (“2–20 nm” in diameter and “100– municipal cellulosic wastes have been accumulating or used
40,000 nm” long). These form the structurally strong frame- inefficiently due to the high cost of their utilization processes
work in the cell walls. Despite a worldwide and enormous [10]. Cellulose, a polymer of glucose residues connected by
utilization of natural cellulosic sources, there are still abun- beta 1,4 linkages, being the primary structural material of
dant quantities of cellulosic sources and there are still abun- plant cell wall, is the most abundant carbohydrate in nature
dant quantities of cellulose containing raw materials and [11]. Therefore, it has become of considerable economic
waste products that are not exploited or which could be used interest to develop processes for effective treatment and utili-
more efficiently. The problem in this respect is, however, to zation of cellulosic wastes as inexpensive carbon sources.
develop processes that are economically profitable. Complete Cellulase is the enzyme that hydrolyses the beta 1,4 glycosidic
hydrolysis of the enzyme requires synergistic action of 3 bonds in the polymer to release glucose units [12].
types of enzymes, namely, cellobiohydrolase, endoglucanase Cellulose containing wastes may be agricultural, urban,
or carboxymethylcellulase (CMCase), and beta-glucosidases or industrial in origin; sewage sludge might also be consid-
[8]. ered a source of cellulose since its cellulosic content provides
Bacteria which have high growth rate as compared to the carbon needed for methane production in the anaer-
fungi have good potential to be used in cellulase production. obic digestion of sludge. Agricultural wastes include crop
2 ISRN Biotechnology

residue, animal excreta and crop-processing wastes, slashing gelatine hydrolysis. The results were compared with Bergey’s
generated in logging, saw dust formed in timber production, Manual of Determinative Bacteria [19].
and wood products in forestry originated activities. The
previous negative attitude in which wastes were viewed self-
2.3. Enzyme Production Medium. Production medium con-
consciously as valueless and even offensive and for disposal
tained (g/L) glucose 0.5 gm, peptone 0.75 gm, FeSO4 0.01 gm,
only has been replaced in large part by a positive view in
KH2 PO4 0.5 gm, and MgSO4 0.5 gm. Ten millilitres of med-
which wastes are recognized as raw materials of potential
ium were taken in a 100 mL conical flask. The flasks were
value [13].
sterilized in autoclave at 121∘ C for 15 min, and after cooling,
This cellulose-degrading enzyme can be used, for exam-
the flask was inoculated with overnight grown bacterial cul-
ple, in the formation of washing powders, extraction of fruit
ture. The inoculated medium was incubated at 37∘ C in shaker
and vegetable juices, and starch processing [14]. Cellulase is
incubator for 24 h. At the end of the fermentation period, the
produced by a large number of microorganisms. They are
culture medium was centrifuged at 5000 rpm for 15 min to
either cell bound or extracellular. Although a large number
obtain the crude extract, which served as enzyme source.
of microorganisms can degrade cellulose, only a few of them
produce significant quantities of free enzymes capable of
completely hydrolysing crystalline cellulose [15]. 2.4. Enzyme Assay. Cellulase activity was measured follow-
Cellulases are used in the textile industry for cotton soft- ing the method of Miller [18]. Briefly, a reaction mixture
ening and denim finishing; in laundry detergents for colour composed of 0.2 mL of crude enzyme solution plus 1.8 mL
care, cleaning; in the food industry for mashing; in the pulp of 0.5% carboxymethyl cellulose (CMC) in 50 mM sodium
and paper industries for drainage improvement and fibre phosphate buffer (pH 7) was incubated at 37∘ C in a shaking
modification, and they are even used for pharmaceutical water bath for 30 min. The reaction was terminated by adding
applications [16]. In nutshell, the cellulose enzymes will be 3 mL of DNS reagent. The colour was then developed by
commonly used in many industrial applications and the boiling the mixture for 5 min. OD of samples was measured
demands for more stable, highly active and specific enzymes at 575 nm against a blank containing all the reagents minus
will also grow rapidly. So, cellulose enzyme will be the most the crude enzyme.
stirring technology of future. And continuous research for
advances in speckled aspects for cellulose production (such
2.5. Process Optimization for Maximum Cellulase Production
as cost, substrate specificity, and specific activity) is desired
to achieve improved technoeconomic feasibility. The present 2.5.1. pH. Flasks with broth containing the optimum con-
work was carried out to optimize the nutritional and envi- centration of substrate and carbon source are taken and the
ronmental parameters for improving cellulose production by pH of the broth is adjusted to 7.0, 8.0, 9.0, 10.0, and 11.0 in
bacterial strains. different flasks using 1 N HCl and 1 N NaOH and sterilized.
The cultures are inoculated and incubated at particular tem-
perature. At the end of incubation period, the cell-free culture
2. Experimental filtrate is obtained and used as enzyme source.
2.1. Screening and Isolation of Bacteria. Cellulase-producing
bacteria were isolated from soils by the dilution pour plate 2.5.2. Temperature. Production medium at pH 7 was inoc-
or spread plate method using CMC agar media. The plates ulated with overnight grown selected bacterial strain. The
were incubated at 45, 50, and 55∘ C for 24 hours. To visu- broth was incubated at different temperatures from 35, 40,
alize the hydrolysis zone, the plates were flooded with an 45, 50, 55, and 60∘ C for 24 h. At the end of incubation period,
aqueous solution of 0.1% Congo red for 15 min and washed the cell-free culture filtrate is obtained and used as enzyme
with 1 M NaCl [17]. To indicate the cellulose activity of the source.
organisms, diameter of the clear zone around colonies on
CMC agar was measured. Besides, a more quantitative assay 2.5.3. Carbon Sources. The effect of various carbon sources
method was used to determine the cellulose activity of the such as starch, glucose, maltose, lactose, and fructose at the
selected bacterial isolate in liquid medium. The cellulase concentration of 1 to 5% was examined in the production
activity of each culture was measured by determining the medium.
amount of reducing sugars liberated by using a DNS method
[18]. A bacterial isolate with the highest activity was selected
for optimization of cellulose production. 2.5.4. Nitrogen Sources. Various nitrogen sources like yeast
extract, peptone, urea, and ammonium sulphate were exam-
ined for their effect on enzyme production by replacing 0.5%
peptone in the production medium.
2.2. Bacterial Identification. The bacterial isolates were pre-
sumptively identified by means of morphological examina-
tion and some biochemical characterizations. The parameters 2.5.5. Agro-Based Waste Material. To find out the suitability
investigated included colonial morphology, gram reactions, of agro-based waste as substrate for enzyme production,
endospore formation, catalase production, VP reaction, different substrates, that is, groundnut cake, coconut cake, soy
indole production, starch hydrolysis, citrate utilization, and cake, and wheat bran, are taken in the growth medium under
ISRN Biotechnology 3

1 0.8

0.9
0.7
0.8
0.6
0.7

Cellulase activity (U/mL)

Cellulase activity (U/mL)

0.5
0.6

0.5 0.4

0.4
0.3
0.3
0.2
0.2
0.1
0.1

0 0
6 7 8 9 10 11 35 40 45 50 55 60
pH Temperature
E. coli Pseudomonas E. coli Pseudomonas
Bacillus Serratia Bacillus Serratia

Figure 1: Effect of pH on cellulase activity. Figure 2: Effect of temperature on cellulase activity.

submerged condition. The enzyme activity is measured after Immanuel et al. [7] also recorded maximum endoglucanase
24 h for enzyme production. activity in Cellulomonas, Bacillus, and Micrococcus sp. at
40∘ C and neutral pH.
3. Results and Discussion
3.3. Effect of Carbon Source. Various sources of carbon such
Cellulase-producing bacteria were isolated from soil. Based as starch, fructose, maltose, and sucrose were used to
on the morphological and biochemical characteristics, the replace glucose which was the original carbon source in
isolates were identified as Pseudomonas fluorescens, Bacillus growth media. Results obtained showed that glucose brought
subtilus, E. coli, and Serratia marscens. the highest cellulase production compared to other carbon
sources at 24 h incubation. Lactose and fructose also showed
3.1. Effect of pH. All the four isolates were allowed to grow high cellulase production at 24 h of incubation. Hence, glu-
in media of different pH ranging from 6.0 to 11.0. Maximum cose was found to be the best source for cellulase production
enzyme activity was observed in medium of pH 9.0–11.0 in (Figure 3). Glycerol is the best substrate for cellulase pro-
case of E. coli, Pseudomonas fluorescens, Bacillus subtilis, and duction with the efficiency of 28.7% on the added substrate
Serratia marscens (Figure 1). This result was in correlation weight. Ishihara et al. [26] studied the utilization of D-xylose
with the finding of other workers for different Bacillus subtilis as carbon source for the production of cellulase membrane
strains [20–22]. [27] and deduced that xylose is not well metabolized by
any bacterial strains that exhibited high cellulose production
in glucose medium, whereas sucrose, glucose, and mannitol
3.2. Effect of Incubation Temperature. Enzyme activity
were found to be suitable for optimum levels of cellulase
recorded at different temperatures revealed that all the four
production [28].
bacteria yielded maximum cellulase production at 40∘ C
(Figure 2). The temperature was found to influence extra-
cellular enzyme secretion, possibly by changing the physical 3.4. Effect of Different Concentrations of Carbon Sources. Var-
properties of the cell membrane. Optimum temperature for ious concentrations of carbon sources were used to replace 1%
maximum growth of Bacillus subtilis 115 and Bacillus subtilis sugar which was the original concentration in growth media
was 40∘ C [23]. These results are close those of Bakare et al. with 2 to 5%. Results obtained showed that 5% carbon source
(2005) [24] who found that the cellulase enzyme produced by brought the highest cellulase production compared to other
Pseudomonas fluorescence was activated at 30 to 35∘ C showing % carbon sources at 24 h incubation (Figures 4, 5, 6, 7, and 8).
the optimum temperature at 35∘ C. Ray et al. [25] reported
that minimum cellulase yield was observed when fermenta- 3.5. Effect of Nitrogen Source. Production of extracellular
tion was carried out at 45∘ C, while maximum yield was cellulase has been shown to be sensitive to repression by
obtained at 40∘ C by Bacillus subtilis and Bacillus circulans. different carbohydrate and nitrogen sources. The effect of
4 ISRN Biotechnology

1.2 1.2

1
Cellulase activity (U/mL)

1
0.8

Cellulase activity (U/mL)

0.6 0.8

0.4
0.6
0.2

0.4
0
Maltose Fructose Lactose Sucrose Dextrose
Carbon
0.2
E. coli Pseudomonas
Bacillus Serratia
0
Figure 3: Effect of carbon sources. 1 2 3 4 5
Sucrose (%)
1.6 E. coli Pseudomonas
Bacillus Serratia
1.4
Figure 5: Effect of percent sucrose.
1.2
0.8
Cellulase activity (U/mL)

1
0.7

0.8
0.6
Cellulase activity (U/mL)

0.6
0.5

0.4
0.4

0.2
0.3

0
0.2
1 2 3 4 5
Glucose (%)
0.1
E. coli Pseudomonas
Bacillus Serratia
0
Figure 4: Effect of percent glucose. 1 2 3 4 5
Lactose (%)
E. coli Pseudomonas
nitrogen sources was studied in the growth medium, where Bacillus Serratia
peptone was replaced by ammonium sulphate, urea, and Figure 6: Effect of percent lactose.
yeast extract. Among the various nitrogen sources tested,
ammonium sulphate was found to be the best nitrogen source
for cellulase production (Figure 9). Nitrogen is one of the
workers. In the present study, coconut cake was found to be
major cell proteins and stimulation of cellulase activity by
the best inducer of cellulase enzyme production by all the four
ammonium sulphate salt might be due to their direct entry
bacterial isolates (Figure 10).
in protein synthesis [29].

4. Conclusion
3.6. Effect of Agro-Based Waste Material. The effect of agro
based by-products as alternative substrate on bacterial cellu- The aim of the present work was to isolate and identify a
lase production under fermentation was studied by several high cellulase producer from soil. Pseudomonas fulorescens
ISRN Biotechnology 5

0.4 1

0.9
0.35
0.8
0.3

Cellulase activity (U/mL)

0.7
Cellulase activity (U/mL)

0.25 0.6

0.5
0.2
0.4
0.15 0.3

0.1 0.2

0.1
0.05
0
Urea Amm sul. Peptone Yeast
0 extr.
1 2 3 4 5 Nitrogen
Fructose (%) E. coli Pseudomonas
Bacillus Serratia
E. coli Pseudomonas
Bacillus Serratia Figure 9: Effect of nitrogen sources.
Figure 7: Effect of percent fructose.
0.7
0.7
0.6
0.6
0.5
Cellulase activity (U/mL)
Cellulase activity (U/mL)

0.5

0.4
0.4

0.3
0.3

0.2 0.2

0.1 0.1

0 0
1 2 3 4 5 E. coli Bacillus Pseudo Serratia
Maltose (%) Agro-based waste
E. coli Pseudomonas GC Wheat bran
Bacillus Serratia CC Soy cake

Figure 8: Effect of percent maltose. Figure 10: Effect of agro-based waste sources.

among E. coli, Bacillus subtilis, and Serratia marscens pro- have high growth rate as compared to fungi, good potential
duced maximum yield of cellulases. The optimum tempera- to be used in cellulose production. However, the application
ture and pH were determined as 40∘ C and 9–11 pH and best of bacteria in producing cellulase is not widely used.
carbon and nitrogen sources were glucose and ammonium Cellulolytic property of some bacterial genera such as
sulphate. This information has enabled the ideal formulation Cellulomonas, Cellovibrio, Pseudomonas, Sporocytophaga spp.
of media composition for maximum cellulase production by [9], Bacillus, and Micrococcus [7] was also reported. Enzyme
this organism. After optimization, the mass production was production is closely controlled in microorganisms and for
carried in one litre of optimized media at 40∘ C for 48 hrs improving its productivity, these controls can be ameliorated.
at a pH of 10 on a rotary shaker at 110 rpm. Bacteria, which Cellulase yields appear to depend on a complex relationship
6 ISRN Biotechnology

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