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International Journal of Microbiology

Volume 2012, Article ID 578925, 5 pages
doi:10.1155/2012/578925

Research Article
Isolation of Cellulose-Degrading Bacteria and Determination of
Their Cellulolytic Potential
Pratima Gupta,1 Kalpana Samant,2 and Avinash Sahu2
1 Department
2 Indian

of Biotechnology, National Institute of Technology, Raipur 492 010, India

Institute of Technology Delhi, New Delhi 110016, India

Correspondence should be addressed to Pratima Gupta, prati [email protected]

Received 29 July 2011; Accepted 11 October 2011
Academic Editor: Todd R. Callaway
Copyright 2012 Pratima Gupta 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.
Eight isolates of cellulose-degrading bacteria (CDB) were isolated from four dierent invertebrates (termite, snail, caterpillar, and
bookworm) by enriching the basal culture medium with filter paper as substrate for cellulose degradation. To indicate the cellulase
activity of the organisms, diameter of clear zone around the colony and hydrolytic value on cellulose Congo Red agar media were
measured. CDB 8 and CDB 10 exhibited the maximum zone of clearance around the colony with diameter of 45 and 50 mm and
with the hydrolytic value of 9 and 9.8, respectively. The enzyme assays for two enzymes, filter paper cellulase (FPC), and cellulase
(endoglucanase), were examined by methods recommended by the International Union of Pure and Applied Chemistry (IUPAC).
The extracellular cellulase activities ranged from 0.012 to 0.196 IU/mL for FPC and 0.162 to 0.400 IU/mL for endoglucanase assay.
All the cultures were also further tested for their capacity to degrade filter paper by gravimetric method. The maximum filter paper
degradation percentage was estimated to be 65.7 for CDB 8. Selected bacterial isolates CDB 2, 7, 8, and 10 were co-cultured with
Saccharomyces cerevisiae for simultaneous saccharification and fermentation. Ethanol production was positively tested after five
days of incubation with acidified potassium dichromate.

1. Introduction
Cellulose is a linear polysaccharide of glucose residues with
-1, 4-glycosidic linkages. Abundant availability of cellulose
makes it an attractive raw material for producing many
industrially important commodity products. Sadly, much of
the cellulosic waste is often disposed of by biomass burning,
which is not restricted to developing countries alone, but is
considered a global phenomenon. With the help of cellulolytic system, cellulose can be converted to glucose which
is a multiutility product, in a much cheaper and biologically
favourable process.
Cellulolysis is basically the biological process controlled
and processed by the enzymes of cellulase system. Cellulase
enzyme system comprises three classes of soluble extracellular enzymes: 1, 4--endoglucanase, 1, 4--exoglucanase, and
-glucosidase (-D-glucoside glucohydrolase or cellobiase).
Endoglucanase is responsible for random cleavage of -1,
4-glycosidic bonds along a cellulose chain. Exoglucanase is
necessary for cleavage of the nonreducing end of a cellulose chain and splitting of the elementary fibrils from

the crystalline cellulose, and -1, 4-glucosidase hydrolyses

cellobiose and water-soluble cellodextrin to glucose [1, 2].
Only the synergy of the above three enzymes makes the
complete cellulose hydrolysis to glucose [35] or a thorough
mineralization to H2 O and CO2 possible.
Source for cellulase system extraction is best suitable
from microbial system found in the gut of organisms
thriving on cellulosic biomasses as their major feed. Insects
like termites (Isopteran), bookworm (Lepidoptera), and so
forth, are found to have syntrophic symbiotic microflora
in their guts responsible for cellulosic feed digestion [6, 7].
Many microorganisms have been reported with cellulosic
activities including many bacterial and fungal strains both
aerobic and anaerobic. Chaetomium, Fusarium Myrothecium,
Trichoderma. Penicillium, Aspergillus, and so forth, are some
of the reported fungal species responsible for cellulosic
biomass hydrolysation. Cellulolytic bacterial species include
Trichonympha, Clostridium, Actinomycetes, Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and
Methanobrevibacter ruminantium [8, 9].

2
Cellulase due to its massive applicability has been used in
various industrial processes such as biofuels like bioethanol
[10, 11], triphasic biomethanation [12]; agricultural and
plant waste management [13, 14]; chiral separation and
ligand binding studies [15].
The present work concentrates on the isolation of
cellulose-degrading bacteria from invertebrates such as termites, snails, caterpillars, and bookworms and assessment
of their cellulolytic activity. The coculturing of cellulose-degrading bacteria and yeast was also carried out for simultaneous saccharification and fermentation of cellulose into
ethanol.

2. Materials and Methods

2.1. Sample Collection. Cellulose feeding organisms like
termite, caterpillar, bookworm, and snail were collected for
isolation of cellulose-degrading bacteria from woody habitats. Guts of the collected organism were separately crushed
in 0.9% saline solution under sterile condition.
2.2. Isolation and Screening of Cellulose-Degrading Bacteria.
The macerated gut of the collected organisms was inoculated
in a basal salt media (NaNO3 2.5 g; KH2 PO4 2 g; MgSO4
0.2 g; NaCl 0.2 g; CaCl2 6H2 O 0.1 g in a liter) containing
filter paper (Whatman filter paper no. 1 of area 70.541 cm2 )
for the isolation of cellulolytic bacteria. These cultures were
incubated for 7 days in a shaker incubator at 37 C at 100 rpm.
Bacterial colonies capable of utilizing cellulose as sole source
of carbon were isolated on cellulose agar media composed of
KH2 PO4 0.5 g MgSO4 0.25 g cellulose 2.0 g agar 15 g gelatin
2 g and distilled water lL and at pH 6.87.2.
Confirmation of cellulose-degrading ability of bacterial
isolates was performed by streaking on the cellulose CongoRed agar media with the following composition: KH2 PO4
0.5 g, MgSO4 0.25 g, cellulose 2 g, agar 15 g, Congo-Red
0.2 g, and gelatin 2 g; distilled water 1 L and at pH 6.8
7.2. The use of Congo-Red as an indicator for cellulose
degradation in an agar medium provides the basis for a rapid
and sensitive screening test for cellulolytic bacteria. Colonies
showing discoloration of Congo-Red were taken as positive
cellulose-degrading bacterial colonies [13], and only these
were taken for further study. Cellulose-degrading potential
of the positive isolates was also qualitatively estimated by
calculating hydrolysis capacity (HC), that is, the ratio of
diameter of clearing zone and colony [16].
2.3. Enzyme Production. The selected CDB isolates were
cultured at 37 C at 150 rpm in an enzyme production media
composed of KH2 PO4 0.5 g, MgSO4 0.25 g, and gelatin 2 g,
distilled water 1 L and containing Whatman filter paper No.1
(1 6 cm strip, 0.05 g per 20 mL) and at pH 6.87.2. Broth
culture after three days of incubation period was subjected to
centrifugation at 5000 rpm for 15 min at 4 C. Supernatant
was collected and stored as crude enzyme preparation
at 4 C for further enzyme assays. Pellet recovered after
centrifugation of broth culture was subjected to gravimetric

International Journal of Microbiology

analysis in order to determine the residual cellulose of filter
paper [17].
2.4. Enzyme Assay. Total cellulose activity was determined
by measuring the amount of reducing sugar formed from
filter paper. Endoglucanase (1-4 endoglucanase-EC 3.2.1.4)
activity was assayed by measuring the amount of reducing
sugar from amorphous cellulose. The enzyme activity was
determined according to the methods recommended by
the International Union of Pure and Applied Chemistry
(IUPAC) commission on biotechnology [18]. Endoglucanase
activity was determined by incubating 0.5 mL of supernatant
with 0.5 mL of 2% amorphous cellulose in 0.05 m sodium
citrate buer (pH 4.8) at 50 for 30 min. FPC activity
was determined by incubating 0.5 mL of supernatant with
1.0 mL of 0.05 M sodium citrate buer (pH4.8) containing
Whatman no.1 filter paper strip1.0 6.0 cm (=50 mg).
After incubation for an hour at 50 C, the reaction was
terminated by adding 3 mL of 3, 5-dinitrosalicylic acid
(DNS) reagent to 1 mL of reaction mixture. In these tests,
reducing sugars were estimated spectrophotometrically with
3, 5-dinitrosalicylic acid [19] using glucose as standards. The
enzymatic activity of total FPCase and endoglucanase were
defined in international units (IU). One unit of enzymatic
activity is defined as the amount of enzyme that releases
1 mol reducing sugars (measured as glucose) per mL per
minute.
2.5. Bioethanol Production. A total of four isolates CDB
2, 7, 8, and 10 were grown in mixed culture using basal
salt medium in two dierent sets, one containing filter
paper and the other containing cellulose powder as substrate
for production of cellulolytic enzyme and to initiate saccharification process. Culture was incubated at 37 C with
mixing at 100 rpm for 3 days. After completion of three
days of incubation, the above culture broth was conditioned
for coculturing of Saccharomyces cerevisae by addition of
filter-sterilized salt solution (KH2 PO4 0.4 g, MgSO4 0.02 g,
CaCO3 0.05 g, and NaCl 0.01 g to 1 L culture broth). The
simultaneous saccharification and fermentation was carried
out at 27 C for 5 days in stationary condition. At the end
of incubation, the culture broth was qualitatively tested for
alcohol production using the K2Cr2O7 reagent test [20].

3. Result and Discussion

3.1. Isolation and Screening of Cellulose-Degrading Bacteria.
Cellulose degrading bacteria were enriched and isolated by
inoculating filter paper in liquid medium with macerated
guts from termite, bookworm, snail, and caterpillar separately. All bacterial culture showed growth as the medium
turned cloudy and the filter paper became macerated.
Cellulolytic bacteria were also isolated from gut of insects
by R. J. Dillon and V. M. Dillon. [6], Wenzel et al. [21],
Delalibera et al. [22], and Ramrn et al. [23]. A total of eight
bacterial isolates found to be positive on screening media
(cellulose Congo-Red agar) producing clear zone (as shown
in Figure 1) during aerobic incubation were as follows: CDB

International Journal of Microbiology

Table 1: Maximum clearing zone and hydrolytic capacity (HC) value of CDB on cellulose Congo red agar media. This table shows the
assessment of bacterial isolates from the dierent source organism for cellulose decomposition via measurement of clear zone around the
colony and calculation of hydrolytic value in cellulose Congo Red media. Maximum clearing zone of 50 mm and HC value of 9.8 were
estimated for CDB 10.
Source organism

Termite

Snail
Bookworm
Caterpillar

Isolate number
CDB1
CDB2
CDB8
CDB9
CDB6
CDB10
CDB3
CDB7

Maximum clearing zone (mm)

30
42
45
28
40
50
30
50

Average hc value
5.49
4.29
5.36
4.32
3.45
5.96
3.51
5.35

Maximum HC value
6.77
8.4
9
4.39
6.45
9.8
4.3
8.2

0.45
0.4

Enzyme activity (IU/mL)

0.35
0.3
0.25
0.2
0.15
0.1

CDB 10

CDB 9

CDB 8

CDB 7

CDB 6

CDB 3

CDB 2

0.05
CDB 1

Figure 1: Zone of clearance on cellulose Congo Red agar plates for

isolate CDB 10 after 48 hrs of incubation. The formation of clearing
zone around the colonies confirms the secretion of extracellular
cellulase.

Cellulose-degrading bacterial isolates

1, 2, 8, and 9 from termite, CDB 6 and 10 from snail,

CDB 3 from bookworm, and CDB 7 from caterpillar. The
result showed that clearing zone and HC value ranged to
bebetween 28.0 to 50.0 mm and 4.3 to 9.0 for all isolates
(Table 1). The range of HC value obtained is similar to range
reported by Lu et al. [24] whereas Hatami et al. [25] found
the hydrolytic value between 1.38 to 2.33 and 0.15 to 1.37 of
cellulolytic aerobic bacterial isolates from farming and forest
soil, respectively.
3.2. Cellulolytic Potential of Bacterial Isolates. A total of eight
positive isolates (CDB1, 2, 3, 6, 7, 8, 9, and 10) were
selected for enzyme production and their respective cellulolytic activity was estimated. Enzyme assay for cellulase
activity on filter paper was found to be highest for CDB 10
with 0.194 IU/mL, while for endoglucanase assay maximum
activity was determined to be 0.400 IU/mL by CDB 8. The
activities ranged from 0.012 to 0.196 IU/mL for FPCase and
0.1622 to 0.400 IU/mL for endoglucanase assay. The two
isolates CDB8 and CDB10 exhibited the highest extracellular
cellulase activities compared to other isolates as shown
in activity assay performed for all isolates in Figure 2.
Similar results were reported for Acinetobacter anitratus and

FPCase activity
Endoglucanase activity

Figure 2: Extracellular cellulase activity of two enzymes (FPCase

and endoglucanase) of all CDBs isolates. The activities ranged from
0.012 to 0.196 IU/mL for FPCase and 0.1622 to 0.400 IU/mL for
endoglucanase assay. Values in figure are means of three replicates
with standard deviation.

Branhamella sp. grown in a basic salt medium with glucose

and CMC as sole carbon source separately. Ekperigin [10]
quantitatively determined the cellulase degrading enzyme
of A. anitratus and Branhamella sp. The maximum enzyme
activities of A. anitratus culture supernatant were 0.48
and 0.24 U/mL for CMC and glucose, respectively. For
Branhamella sp., the maximum enzyme activities of the
culture supernatant were 2.56 and 0.34 U/mL for CMC
and glucose, respectively. The filter paper degradation was
observed separately in CDB 2, 3, 6, 7, 8, 9, and 10 as shown
in Figure 3. Gravimetric analysis shows that maximum and
minimum rates of filter paper degradation were 65.7% and
55%, respectively, estimated at third day of incubation. An
average of 57.64% degradation rate was computed. Figure 4

International Journal of Microbiology

FP 7

FP 6

FP 2

FP 9
FP 10

FP 3

FP C

FP 8

Figure 3: Filter paper degradation by isolates CDB 2, 3, 6, 7, 8, 9, and 10 cultured in basal salt medium supplemented with Whatman filter
paper no.1 (1 6 cm strip 2, 0.05 g per 20 mL) at the end of 96 hrs of incubation. Flask FP C is the control for this experimental set up
and does not show any filter paper degradation.

Percent filter paper degradation

80
70
60
50
40
30
20
10
CDB 10

CDB 9

CDB 8

CDB 7

CDB 6

CDB 3

CDB 2

CDB 1

Bacterial isolates

Figure 4: Percent filter paper degradation by various bacterial

isolates obtained from termite, snail, bookworm, and caterpillar
by gravimetric method. Maximum percentage of filter paper
degradation was found to be 65.7% by CDB 8. Values in figure are
means of three replicates with standard deviation.

shows that CDB 8 has highest filter paper degradation rate

of 65.7%. In a result documented by Lu et al. [13], the data
for synergetic cellulose degradation detected in four groups
of mixed cultures were only 23.5%, 26.3%, 19.4%, and
24.5%, respectively. Bichet-Hebe et al. [26] reported the rates
of paper degradation ranged from 31 to 60% after 10 days for
mixed bacterial populations by gravimetric procedure.

3.3. Bioethanol Production. The experiment setup for simultaneous saccharification and fermentation of mixed bacterial
culture (CDB, 2, 7, 8, and 10) with Saccharomyces cerevisiae
resulted in production of ethanol. This result expressed
the high cellulolytic potential of these selected bacterial
isolates for decomposition of cellulose and its fermentation
for production of ethanol. Satheesh Kumar et al. [27]
also used Whatman filter paper and cellulose powder as
substrate in submerged fermentation for production of
cellulolytic enzymes by Bacillus sp. FME (flour mill euent). Coculturing of bacterial strains with yeast sp. and
simultaneous saccharification and fermentation of ethanol
were reported by several workers (Lenziou et al. [28] and
Eklund and Zacchi [29]). Results indicated that significant
synergistic cellulose degradation can be achieved in mixed
culture system of cellulolytic bacteria and noncellulolytic
yeast in which noncellulolytic yeast, Saccharomyces cerevisiae
utilizes the reducing sugar derived from cellulose degradation and converts it to ethanol.
The bacterial isolates showed a potential to convert
cellulose into reducing sugars which could be readily used in
many applications like feed stock for production of valuable
organic compounds; for example in the present study this
has been demonstrated by simultaneous saccharification and
fermentation of cellulose into ethanol.

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International Journal of Microbiology

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