International Journal of Environment and Bioenergy, 2012, 3(3): 145-153

International Journal of Environment and Bioenergy

ISSN: 2165-8951
Florida, USA
Journal homepage: www.ModernScientificPress.com/Journals/IJEE.aspx
Article

Isolation and Characterization of Cellulase Produced by

Cellulolytic Bacteria from Peat Soil of Ogan Komering Ilir,
South Sumatera
Puspita Lisdiyanti 1, *, Eko Suyanto 1, Niken Financia Gusmawati 2, Wiwik Rahayu 3
1

Research Center for Biotechnology, Indonesian Institute of Science, Cibinong 16911, Indonesia
Research Center for Marine Technology, Ministry of Marine Affairs and Fisheries, Pasir Putih
Ancol, Indonesia
3
Civil Engineering, Faculty of Engineering, University of Indonesia, Depok, Indonesia
2

* Author to whom correspondence should be addressed; E-Mail: puspita.lisdiyanti@lipi.go.id,

p_lisdiyanti@yahoo.com; Tel.: +62-21-8754587: Fax: +62-21-8754588.
Article history: Received 12 June 2012, Received in revised form 7 August 2012, Accepted 8 August
2012, Published 20 August 2012.

Abstract: Peat soil composes of organic compounds derived from plant debris with
organic carbon content higher than 25%. The organic fraction of peat soil generally
contains lignin, cellulose, hemicellulose, proteins, waxes, tannins, resins, and suberin. In
this study, the cellulolytic bacteria were isolated from peat soils from Ogan Komering Ilir,
South Sumatera, Indonesia, tested for their cellulase activities, and the potential isolates
were identified into genus or species level. The degradation ability of cellulose was
screened using 1% CMC (carboxy methyl cellulose) as a substrate and the cellulase
activities of crude enzyme were determinated using DNS (3,5-dinitro salicyclic acid)
method. As results, we selected four bacterial isolates (S3B40, S3B32, S3B37, and AB16)
that have high cellulase activities. Among 4 isolates, Paenibacillus elgii S3B40 has the
highest cellulase activity in all tested. Optimum activity of isolate S3B40 at pH 8 and
temperature 60 oC was 14.5 U/mL, S3B32 at pH 8 and temperature 80 oC was 0.506 U/mL,
AB16 at pH 5 and temperature 50 oC was 0.361 U/mL, and S3B37 at pH 5 and temperature
30 oC was 1.167 U/mL, respectively.
Keywords: peat soil; cellulase; bacteria; isolation; characterization.

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1. Introduction
Marshes and other types of wetlands are globally important as reservoirs of soil organic carbon
(SOC) and locally important as hydro-regulators and biodiversity hotspots. These wetlands cover
approximately 3% of the land-surface, store up to 30% of the earths terrestrial carbon and play a vital
role in carbon cycling (Gorham, 1995). Their biomass production exceeds decomposition due to low
pH and the anoxic conditions often found in the soil caused by high ground water or flooding
(Augustin et al., 1996). Microorganisms play a major role in the turnover of energy and the matter in
the soil, there have been only a few attempts to study the microorganisms especially bacteria in the
peat soils (Dedysh et al., 2006; Kraigher et al., 2006). Peat soils are formed largely from inhibited
decomposition of various plant materials in the waterlogged environment of marshes, bogs and
swamps. The plant materials were identified such as cellulose and lignin derivatives in the large-scale
and fine-grained humic materials in peat (Viraraghavan and Rana, 1991). Also, peat soils are highly
acidic, having a pH of between 3 and 4.5 and they are also very low in ash, normally less than 3% wt.
According to the world resource of peat, Indonesia has 17 106 ha, dry weight 68 109 tonne, and
total energy 135.32 1010 GJ of peat soils.
Cellulase provides a key opportunity for achieving tremendous benefits of biomass utilization
(Wen et al., 2005). Cellulose is extracellular enzyme, which is a polymer of -1,4-linked glucose units,
a major polysaccharides constituent of plant cell walls. Therefore, it has become of considerable
economic interest to develop processes for the effective treatment and utilization of cellulosic wastes
as inexpensive carbon sources. Cellulases are inducible enzymes which are synthesized by
microorganisms during their growth on cellulosic materials (Lee and Koo, 2001). The breakdown of
lignocellulose polysaccharides requires a combination of enzymes, which split off glucosidic lingkages
between -D-xylopyranosyl and glucopyranosil residues. A complete cellulase systems consists of
three classes of enzymes: exo-1,4--glucan cellobiohydrolase (EC 3.2.1.91), which cleave cellobiosyl
units from the ends of cellulose chains; endo-1,4--glucanases (EC 3.2.1.4), which cleave internal
glucosidic

bonds;

-D-glucosidase

(EC

3.2.1.21),

which

cleave

glucose

units

from

cellulooligosaccharides (Bhat and Bhat, 1997). For cellulase system, a number of components and
functional characteristics lead to efficient hydrolysis of cellulase (Cohen et al., 2005).
A number of fungi and bacteria had reported capable of utilizing cellulose as a carbon source.
Cellulases are produced by fungi and bacteria such as from the genus Aspergillus, Rhizopus and
Trichoderma (Murashima et al., 2002; Saito et al., 2003; Fahrurrozi et al., 2010), and the genus
Bacillus (Lee et al., 2008). Cellulases from both bacterial and fungal sources appear to function very
similarly when acting on crystalline cellulosic substrates because of their common structural
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147

similarities: each consists of a distinct catalytic domain joined to a cellulose-binding domain via an
interdomain-linker peptide (Henrissat, 1994).
The aim of this study is to search the bacteria hydrolyzing of cellulose substrate from peat soil
from Ogan Komering Ilir, South Sumatra, Indonesia which has the possibility to be implemented to
consolidate of peat soil.

2. Research Methods
2.1. Isolation and Screening of Cellulase-prooducing Bacteria
One g of peat soil was suspended with serial dilution of aquadest steril and the 100 L of
suspension was cultivated at room temperature (29 2 oC) for 3 days in a CMC solid medium
containing (g/L), 10-CMC, 0.2-MgSO4.7H2O, 0.75-KNO3, 0.5-K2HPO4, 0.02-FeSO4.7H2O, 0.04CaCl2, 2-yeast extract, 1-D-glucose, and 1.5% (w/v) agar. This CMC medium also was used for
screening and production of cellulase of the isolates. Colonies of bacteria were picked up and purified
using fourway streak method to obtain pure bacterial isolates. Colonies of the isolates were spotted on
the above medium, incubated at room temperature for 2 days, the plate was flooded with 0.1% Congo
red for 15 to 20 min, washed with 1 M NaCl for 15-20 min, and observed for clear zone around the
colony (Hendricks et al., 1995). The clear zone formed by isolates is used as indicator for cellulse
activity.
2.2. Production and Measurement of Crude Cellulase
The isolates were grown at room temperature for 3 days with rotary shaking in 50 mL
erlenmeyer containing 25 mL of CMC liquid medium. The cell free supernatant containing the crude
extracellular enzyme was collected after centrifugation of the culture in 7,000 g at 4 oC for 20 min. The
crude enzyme was kept in 4 oC refrigerator for further analyses. Cellulase activity was determined by
using DNS (3,5-dinitrosalicyclic acid) method, through the determination of the amount of reducing
sugars. The mixture was incubated for 30 min at 37 oC and the reaction was stopped by the addition of
DNS solution. The treated samples were boiled for 15 min, cooled in room temperatur for 10 min and
the optical density was measured at 540 nm. A unit (U) of cellulase activity was defined as the
amount of enzyme that produced reducing sugars corresponding to 1 mol glucose equivalents from
carboxymethylcellulose per minute under the assay condition.
2.3. Effect of pH on Enzyme Activity
The optimum pH of crude enzyme for hydrolysis of CMC was determined by incubating the
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mixture of crude enzyme and 1% (w/v) CMC for 30 min in the presence of appropriate buffers; 0.2 M
phosphate citrate buffer (pH 4-5), 0.2 M phosphate buffer (pH 6-8), and 0.2 M Tris-HCl buffer (pH 89). The reaction mixtures in various pH buffers were incubated for 30 min at 37 oC and the cellulase
activity was assayed by DNS.
2.4. Effect of Temperature on Enzyme Activity
The optimum temperature of the crude enzyme at optimum pH was determined by incubating
the mixture of crude enzyme and 1% (w/v) CMC for 30 min at different temperatures ranging from 30
to 80 oC. The reaction was stopped and assayed by the addition of DNS solution.
2.5. Molecular Identification of the Isolates
Total DNA of the bacteria was extracted using an InstaGene Matrix Kit (BioRad). One-day old
bacterial colonies were added to microcentrifuge tube with 1.0 mL of sterile water in order to get
suspension of bacteria, and then centrifuged at 10,000-12,000 rpm for 1 min. The supernatants were
decanted and the pellets were resuspended with 50 L InstaGene matrix. The bacterial suspension was
incubated at 56 C for 15-30 min on a head block and then vortexed with high speed for 10 sec and
returned to the heat block, incubated at 100 C for 8 min and vortexed again with high speed for 10
sec, and centrifuged at 10,000-12,000 rpm for 2-3 min. The supernatants containing the DNA of
bacteria were stored at -20C. The 16S rRNA gene was amplified by using the primers 9F (5'AGRGTTTGATCMTGGCTCAG-3') and 1492R (1492R: 5'-TACGGYTACCTTGTTAYGACTT-3')
(Position-base sequence numbering based on Escherichia coli numbering system, accession number
V00348). The PCR reaction conditions were 96 C, 5 min; 96 C, 30 seconds, 55 C, 30 seconds, 72
C, 1 min, for 30 cycles; and 72 C, 7 min. Sequence of 16S rRNA gene was analyzed and identified
their

similarity

ti

data

base

in

GeneBank/DDBJ/EMBL

based

on

BLASTN

(www.ncbi.nlm.nih.gov/blast/).

3. Results and Discussion

3.1. Isolation and Cellulase Activity of Selected Isolates
Twenty eight bacterial isolates were isolated from peat soils collected from Ogan Komering
Ilir, South Sumatra, Indonesia by using CMC solid medium. All the isolates had capability to degrade
CMC and showed the cellulose activity when screened by Congo red. Figure 1 showed the clear zone
for cellulase activity by cellulolytic bacteria isolated from peat soils. The clear zone formation
differred for each bacteria, it means that each bacteria is able to produce in different cellulase complex.
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149

Figure 1. Cellulase activity in CMC Congo red agar plates.

Congo red (sodium diphenyl-bis-alpha-naftilamin sulfonate) solution is a type of synthetic dyes

consisting of unsaturated organic substances with chromophores as the color bearer and color
auxochrome as a binder. Chromophore groups will occupy the space between cellulose fibrils and has
a non-covalent affinity to cellulose fibrils containing -1,4-D-glugopiranosil.
All of cellulase-producing bacteria were inoculated to the CMC broth medium containing 1%
(b/v) CMC as inducer component and 0.2% (b/v) yeast extract and 0.1% (b/v) D-glucose as initiation
of growth phase. After the concentration of glucose is low, the bacteria will use cellulose as carbon
source to produce cellulase. We selected four bacteria for degrading cellulose based on CMC Congo
red test. Table 1 showed that cellulase activities of 4 selected isolates AB16, S3B37, S3B40, and
S3B32 are 0.193, 0.014, 3.082, and 0.021 (U/mL), respectively. Apparently, IS value is not show
correlation with cellulase activity. Production of extracellulase depends greatly on the composition of
medium (Deswal et al., 2011) and substrate concentration (Reczey et al., 1996).

Table 1. Cellulase activity for four bacteria

No.

Isolates

IS

Sugar reducer (mg/mL)

Cellulase activiy (U/mL)

1
2
3
4

AB16
S3B37
S3B40
S3B32

2.18
8
4
8

0.1045
0.0075
1.6642
0.0112

0.193
0.014
3.082
0.021

3.2. Effects of pH and Temperature

Ingledew (1990) reported that pH 6-7 is the optimum pH for the growth of the genus of
Bacillus. Lee et al. (2008) also reported that cellulase activity of Bacillus amyoliquefaciens DL-3 was
optimal at pH 7. In this result, isolate AB16 and S3B37 had the optimum pH 5 where the highest
celluase activities were 0.331 and 0.567 U/mL, respectively. While, isolate S3B40 and S3B32 had
optimum pH 8 where the highest cellulase activities were 15.755 and 0.062 U/mL, respectively (Fig.2).
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In overall, the cellulase activity of isolate S3B40 was the highest in pH 5, 7, 8, and 9.

Figure 2. Effect of pH on cellulase activity for isolates AB16, S3B37, S3B40 and S3B32.

In addition to pH, temperature plays an important role in enzymatic reactions. Krisna (2005)
and Ko et al. (2010) reported that temperature is one of the most important physical variable affecting
in solid-state fermentation. When the temperature increases up to optimum temperature, enzyme
reaction rate increase due to the increasing of kinetic energy. Increasing in kinetic energy will
accelerate the movement of vibration, translation, and rotation of both enzyme and substrate. This will
enlarge enzyme and substrate to react. As results, isolate S3B40 has the highest cellulase activity at 60
o

C (14.55 U/mL), isolate AB16 has at 50 oC (0.361 U/mL), isolate S3B37 has at 30 oC (1.167 U/mL),

and isolate S3B32 has at 80 oC (0.506 U/mL) (Figure 3). Several data have not shown because the
enzyme activity is lower than another. Same in the effect of various pH, the cellulase activity of isolate
S3B40 was the highest in all tested parameters. Lee et al. (2008) reported that cellulase activity from
B. amyoliquefaciens DL-3 has the optimum temperature 50 oC at pH 8.

Figure 3. Effect of temperature on optimum pH to cellulase activity for isolates AB16, S3B37, S3B40
and S3B32.
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151

3.3. Molecular Identification of the Selected Isolates

Based on 16S rRNA gene analysis, the three isolates belong to the genus Bacillus (Table 2).
Isolate AB16 was identified as Bacillus pumilus, S3B37 as Bacillus cereus, and S3B32 as Bacillus sp..
While, isolate S3B40 was identified as Paenibacillus elgii. Paenibacillus species are capable to
hydrolyze plant materials and frequently isolated and identified from soil and plant related sources
(Rivas et al., 2006). Several members of the genus Paenibacillus secrete diverse assortments of
extracellular polysaccharide hydrolyzing enzymes and their xylanolytic and cellulolyticsystems are
gradually being identified (Pastor et al., 2001; Pason et al., 2006; Wang et al., 2008; Akaracharanya et
al., 2009). Paenibacillus campinasensis BL11, isolated from a high temperature, alkaline environment,
expresses several kinds of polysaccharide hydrolases, including carboxymethyl cellulases (CMCase),
avicelase, xylanase, amylase, b -glucanase, and pectinase (Ko et al., 2007).
Table 2. Molecular identification of cellulase-producing bacteria based on 16S rRNA gene
Isolate

Identified as

Closed related to strain with

Acc Number

BLAST Homology Value

AB16
S3B37
S3B40
S3B32

Bacillus pumilus
Bacillus cereus
Paenibacillus elgii
Bacillus sp

AY112667
JN700160.1
HQ236086.1
GU113075.1

99%
97%
98%
94%

4. Conclusions
Cellulase-producing bacteria form peat soil, Ogan Komering Ilir were succesfully isolated.
Four selected isolates were identified as AB16 (Bacillus pumilus), S3B37 (Bacillus cereus), S3B40
(Paenibacillus elgii), and S3B32 (Bacillus sp). Cellulases from the selected isolates are thermozyme
and influenced by pH and temperature conditions. The highest cellulase activity was shown by isolate
S3B40.

Acknowledgment
We thank to Siti Muslikah for her effort to take the samples of peat soil from Ogan Komering
Ilir, South Sumatra.

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