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Egyptian Journal of Aquatic Research (2015) 41, 257–264

H O S T E D BY
National Institute of Oceanography and Fisheries

Egyptian Journal of Aquatic Research


http://ees.elsevier.com/ejar
www.sciencedirect.com

FULL LENGTH ARTICLE

Aquatic Bacillus cereus JD0404 isolated from the


muddy sediments of mangrove swamps in Thailand
and characterization of its cellulolytic activity
Aiya Chantarasiri *

Faculty of Science, Energy and Environment, King Mongkut’s University of Technology North Bangkok,
Rayong Campus, Bankhai, Rayong 21120, Thailand

Received 11 August 2015; revised 31 August 2015; accepted 31 August 2015


Available online 26 September 2015

KEYWORDS Abstract This study aimed to conduct the isolation, screening and identification of bacteria with a
Bacillus cereus; high level of cellulolytic activity from the muddy sediments of mangrove swamps in Thailand. One
Cellulolytic activity; hundred and ninety aquatic bacterial isolates were isolated from different muddy sediments and
Mangrove swamp; eighty one isolates were determined to be cellulolytic bacteria. The cellulolytic bacterium identified
Muddy sediment as Bacillus cereus JD0404 showed maximum hydrolysis activity on carboxymethylcellulose agar
plates. Its cellulolytic performance for CMCase activity, Avicelase activity and b-glucosidase
activity was 1.778 ± 0.003 U/mL, 0.079 ± 0.001 U/mL and 0.048 ± 0.002 U/mL, respectively.
The optimum temperature and pH for the enzyme activity were determined to be 50 °C and 7.0
respectively. The cellulolytic activity was greatly enhanced by Mn2+ and considerably inhibited
by EDTA and toluene. Preliminary bioconversion application showed that the B. cereus JD0404
could be used for the hydrolysis of cellulose-based biomass. This study demonstrated a feasible
bacterium for environmentally friendly industries and biotechnology.
Ó 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Sandilyan and Kathiresan, 2014), including coastal protection


against natural disasters, storage of organic material, habitats
Mangrove swamps or mangrove forests are the unique coastal for estuarine organisms and mitigation of the global warming
wetland ecosystems which are found along tropical and sub- phenomenon. Mangrove ecosystems can store large amounts
tropical coastlines dominated by halophilic plants in the gen- of organic carbon and are rich in organic carbon in sediments
era Rhizophora and Avicennia (Mitsch and Gosselink, 2015). which mainly originated from litter falls and the underground
The mangrove swamps and neighbouring coastal environ- roots of mangrove plants (Yong et al., 2011). The mangrove
ments provide many ecological benefits (Ghosh et al., 2010; microbial communities play a vital role in the organic carbon
cycle. Cellulolytic microorganisms can perform the degrada-
* Tel./fax: +66 (0)38 627012. tion of cellulose-based plant litter, resulting in the production
E-mail address: aiya.c@sciee.kmutnb.ac.th of simple-sugar derivatives in the sediments (Soares-Júnior
Peer review under responsibility of National Institute of Oceanography et al., 2013). Microbial cellulolytic enzymes, called cellulase,
and Fisheries. are the complex enzymes that consist of endoglucanases
http://dx.doi.org/10.1016/j.ejar.2015.08.003
1687-4285 Ó 2015 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
258 A. Chantarasiri

(E.C. 3.2.1.4), exoglucanases (E.C. 3.2.1.91, and E.C. 3.2.1.176) at different locations, placed in sterile zip-lock plastic bags
and b-glucosidases (E.C. 3.2.1.21) which synergistically work and kept at 4 °C.
to hydrolyse the b-1, 4 glycosidic bonds of cellulose. Cellulase
have become focal biocatalysts in various green technology Isolation and purification of mangrove bacteria
industries, such as textile production, detergent composition,
food processing, animal feed production, removal of bacterial The isolation of mangrove bacteria and media was modified
biofilm and bioconversion for biofuel production (Juturu and from Dias et al. (2009). The samples were serially diluted with
Wu, 2014). Most cellulolytic enzymes isolated from mangrove sterile normal saline solution (0.85% NaCl) within 24 h of col-
swamps and related aquatic environments are produced from lection to obtain 1:10,000 dilutions. One hundred microlitres
fungi belonging to the genera Cladosporium, Alternaria and of each diluted sample was spread plated on Tryptone Soya
Byssochlamys. (Alsheikh-Hussain et al., 2014; Matondkar Agar (Himedia, India) supplemented with 1.8% NaCl and
et al., 1980; Thatoi et al., 2013), and bacteria belonging to incubated at 28 °C, the average sediment temperature of the
the genera Micrococcus, Bacillus, Pseudomonas, Xanthomonas sampling sites, for 48 h. The agar plates were investigated in
and Brucella (Behera et al., 2014). Interestingly, Thailand is terms of colony morphology including shape, margin, eleva-
one of the Asian countries that contain large areas of man- tion and pigmentation. Morphologically dissimilar colonies
grove swamps (Sandilyan and Kathiresan, 2014), but the were selected and streak plated on Tryptone Soya Agar to
knowledge of cellulolytic microbes isolated from mangrove obtain pure colonies.
swamps is limited (Behera et al., 2014). For this study, the
aquatic bacteria demonstrating cellulolytic performance were Screening of cellulolytic bacteria
isolated from muddy sediments of mangrove swamps in Thai-
land and identified. The purpose was to determine a competent
Screening of the cellulolytic bacteria was conducted from that
aquatic cellulolytic bacterium for possible use in green technol-
previously described (Kasana et al., 2008) using car-
ogy industries and related biotechnological applications.
boxymethylcellulose (CMC) agar plates and Gram’s iodine
staining method. Five microlitres of overnight growth culture
Materials and methods
in the Tryptone Soya Broth (Himedia, India) of each bacterial
isolate was spot plated on CMC agar (0.2% NaNO3, 0.1%
Description of sampling sites K2HPO4, 0.05% MgSO4, 0.05% KCl, 0.2% CMC sodium salt,
0.02% peptone and 1.7% agar). The agar plates were incu-
Muddy sediment samples were collected from mangrove bated at 28 °C for 48 h and then flooded with Gram’s iodine
swamps in Rayong River (12° 390 57.4000 N, 101° 140 30.7900 solution for 10 min. The cellulolytic isolates were detected by
E), Rayong Province, Thailand (Fig. 1). Samples were col- the cellulolytic zone around the colonies after Gram’s iodine
lected twice during the rainy season, once in July 2014 and staining. The hydrolysis capacity (HC) value that determined
another in September 2014. Muddy sediments close to the the cellulolytic activity was calculated from the ratio between
roots of mangrove plants or beneath the decomposed plant the diameter of the cellulolytic zone and the diameter of the
litter at a depth of 0–15 cm were taken from sampling sites bacterial colony. The negative control for screening was the

Figure 1 Map of mangrove swamps in Rayong River. The sampling site covered an area of 75,400 m2. The figure was generated using
the Google Maps service.
Characterization of cellulolytic activity of aquatic Bacillus cereus 259

non-cellulolytic bacterium, Escherichia coli TISTR073 (Thai- activities is defined as the amount of enzyme required to
land Institute of Scientific and Technological Research, Thai- release 1 lmol of reducing sugars as glucose equivalents per
land) and the positive control was the cellulolytic bacterium minute under standard assay conditions. b-glucosidase activity
isolated from bovine faeces, Bacillus methylotrophicus was measured by incubating 0.5 mL of enzyme solution with
RYC01101 (Chantarasiri, 2014). 1 mL of 0.1% p-nitrophenyl-b-D-glucopyranoside (pNPG) in
0.1 M sodium phosphate buffer (pH 7.0) at 50 °C for 1 h.
Identification of cellulolytic bacteria The enzyme reaction was terminated by adding 2.0 mL of
1 M Na2CO3 solution and the optical density of the reaction
The selected cellulolytic isolate was identified by morphologi- mixture was measured at 400 nm. The enzyme activity was
cal examination, biochemical characterization and molecular calculated using a p-nitrophenol standard curve. One unit
genetic analysis. The morphological examination was per- (U) of b-glucosidase activity is defined as the amount of
formed by Gram staining, endospore staining and motility enzyme required to release 1 lmol of p-nitrophenol per minute
testing. Growth at different parameters including pH, temper- under standard assay conditions.
ature, salinity condition and anaerobic environment was inves-
tigated at 28 °C for 24 h following Chantarasiri (2014). Effect of temperature on cellulolytic activity and thermal
Biochemical characterization was examined by catalase testing stability
(Gagnon et al., 1959) and oxidase testing (Gordon and
McLeod, 1928). Dextrose, lactose and sucrose fermentation, The effect of temperature on enzyme activity was examined by
and hydrogen sulphide production were analysed using Triple incubating 0.5 mL of enzyme solution with 0.5 mL of 2%
Sugar Iron Agar (Himedia, India). The PCR amplification and CMC sodium salt in 0.1 M sodium phosphate buffer (pH
16S rDNA sequence analysis were described by Yukphan et al. 7.0) at various temperatures ranging from 20 °C to 85 °C for
(2004) using a set of primers as follows: forward primer 27F: 30 min. Thermal stability was examined by incubating the
GAGTTTGATCATGGCTCAG and reverse primer 1492R: enzyme solution in 0.1 M sodium phosphate buffer (pH 7.0)
CGGTTACCTTGTTACGACTT. All molecular genetic anal- at temperatures ranging from 20 °C to 85 °C for 24 h and
yses including PCR amplification, 16S rDNA sequence analy- the residual activity was monitored using 2% CMC sodium
sis and homology similarity analysis were carried out by the salt as a substrate. The cellulolytic activity of enzymes was
Thailand Institute of Scientific and Technological Research assayed using the method described above.
(Thailand).
Effect of pH on cellulolytic activity and pH stability
Preparation of cellulolytic enzyme solution
The effect of pH on enzyme activity was measured by incubat-
The selected isolate was grown in CMC broth at 28 °C for 48 h ing 0.5 mL of enzyme solution with 0.5 mL of 2% CMC
under aeration conditions. Bacterial cells were removed from sodium salt in different pH buffers at 50 °C for 30 min.
the culture broth by centrifugation at 4500g for 30 min at Enzyme activity was measured at a range of pH values between
4 °C. The cell-free supernatant obtained after centrifugation 3.0 and 11.0 using 0.1 M of the following buffers: sodium
served as a crude enzyme solution. The crude enzyme solution citrate buffer (pH 3.0–6.0), sodium phosphate buffer (pH
was partially purified by AmiconÒ Ultra-15 (10 kDa) centrifu- 6.0–8.0), Tris–HCl buffer (pH 8.0–9.0) and glycine-NaOH
gal filter devices (Millipore, Ireland). buffer (pH 9.0–11.0). The pH stability was determined by incu-
bating the enzyme solution in the above-mentioned buffers at
Cellulolytic activity assay 50 °C for 24 h and the residual activity was monitored using
2% CMC sodium salt as a substrate. The cellulolytic activity
The cellulolytic activity assays were modified from the previ- of enzyme was assayed using the method described above.
ously described study by incubating the enzyme solution with
the substrate and determining the amount of products liber- Effect of additives on cellulolytic activity
ated (Kim et al., 2012). Endoglucanase activity (CMCase
activity) was measured by incubating 0.5 mL of enzyme solu- The effect of additives on enzyme activity was investigated by
tion with 0.5 mL of 2% CMC sodium salt in 0.1 M sodium incubating 0.5 mL of enzyme solution with metal ions, deter-
phosphate buffer (pH 7.0) at 50 °C for 30 min. The reducing gent, a chelating agent and organic solvents. There were twelve
sugars liberated were determined by the 3,5-dinitrosalicylic different metal ions used, including Ca2+, Co2+, Cu2+, Fe2+,
acid (DNS) method (Miller, 1959). The enzyme reaction was Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, Sr2+, and Zn2+, with
terminated by adding 3.0 mL of DNS reagent and then boiled the final concentration of metal ion solution at 5 mM following
for 5 min. The solution was completely cooled and the optical the study of Seo et al. (2013). The effect of detergent on enzyme
density of the reaction mixture was measured at 540 nm. activity was studied using 1% TWEEN 80Ò (Sigma–Aldrich,
Exoglucanase activity (Avicelase activity) was measured using Germany). The result of a chelating agent on enzyme activity
2% AvicelÒ PH-101 (Sigma–Aldrich, Germany) suspended in using 5 mM EDTA was determined. Residual activity of
0.1 M sodium phosphate buffer (pH 7.0) as a substrate and enzymes was monitored using 2% CMC sodium salt as a sub-
incubating it with 0.5 mL of enzyme solution at 50 °C for strate after being incubated with additive solutions at 50 °C
1 h. The supernatant of the reaction mixture was collected in for 60 min (Annamalai et al., 2013). The effect of eight organic
order to determine the quantity of reducing sugars by the solvents on enzyme activity was examined by incubating the
DNS method. The enzyme activity was calculated using a glu- enzyme solution with a 25% concentration of various organic
cose standard curve. One unit (U) of CMCase and Avicelase solvents such as benzene, cyclohexane, dichloromethane,
260 A. Chantarasiri

Table 1 Colony morphology and hydrolysis capacity (HC) values of cellulolytic bacteria.
Bacteria strain Shape Margin Elevation Pigmentation HC value
JD0404 Circular Curled Raised White 4.47 ± 0.30
JD0402 Circular Entire Raised Yellow 3.90 ± 0.23
JD1304 Circular Entire Convex White 3.89 ± 0.24
JD1701 Irregular Undulate Raised White 3.85 ± 0.25
JD0803 Circular Entire Convex White 3.83 ± 0.31
JD1603 Circular Curled Convex White 3.67 ± 0.29
JD1803 Irregular Curled Raised White 3.66 ± 0.32
JD1202 Circular Curled Convex White 3.64 ± 0.14
JD1703 Circular Curled Umbonate White 3.63 ± 0.17
JD1003 Irregular Undulate Raised White 3.51 ± 0.10
JD1101 Irregular Undulate Raised White 3.49 ± 0.13
JD2103 Circular Entire Convex White 3.48 ± 0.33
PS0102 Circular Entire Convex Pale brown 3.43 ± 0.17
JD1502 Circular Curled Convex White 3.39 ± 0.08
PS1504 Irregular Undulate Flat White 3.32 ± 0.13
PS1704 Irregular Curled Raised White 3.27 ± 0.08
JD2102 Irregular Undulate Umbonate Cream 3.24 ± 0.18
PS2006 Circular Entire Convex White 3.24 ± 0.46
JD0502 Circular Entire Convex Cream 3.22 ± 0.11
JD0504 Circular Entire Convex Pale yellow 3.15 ± 0.64
PS1804 Irregular Curled Raised White 3.10 ± 0.32
PS0902 Circular Curled Convex Pale brown 3.10 ± 0.15
Positive control Circular Entire Convex White 3.09 ± 0.39
Positive control was B. methylotrophicus RYC01101 and the HC values of any isolates less than the positive control are not shown.

Figure 2 Cellulolytic zone around the bacterial colonies on CMC agar plates after Gram’s iodine staining.

ethanol, ethyl-ether, methanol, n-hexane and toluene at 50 °C at 28 °C under aeration conditions for 48 h. The culture med-
for 4 h (Annamalai et al., 2013) and the residual activity of ium was collected for reducing sugars determination by DNS
enzymes was measured using 2% CMC sodium salt as a sub- method.
strate. The cellulolytic activity of enzymes was assayed using
the method described above.
Results and discussion
Application on cellulose-based biomass by bioconversion process
Isolation, screening and identification of cellulolytic bacteria
The application to the bioconversion process was investigated.
To produce the reducing sugars, the cellulose-based biomass One hundred and ninety aquatic bacteria with dissimilarly
was hydrolysed by a selected aquatic bacterium. Cassava morphological colonies were isolated from forty-two muddy
stems, hay, rice straw and peanut shells were used as the car- sediments. The supplement of the Tryptone Soya Agar
bon source of the bacterial culture. The aquatic bacterium medium with 1.8% of NaCl ensured the selection of isolated
was cultured in a basal medium supplemented with 1% bacteria mainly found in mangrove ecosystems (Dias et al.,
cellulose-based biomass powder (Chantarasiri et al., 2015) 2009). Eighty-one bacterial isolates were defined as cellulolytic
Characterization of cellulolytic activity of aquatic Bacillus cereus 261

Table 2 Cellulolytic performance of B. cereus JD0404 and related bacteria in the Bacillus genus.
Bacteria CMCase activity Avicelase activity b-Glucosidase activity Reference
(U/mL) (U/mL) (U/mL)
Bacillus sp. SMIA-2 0.29 0.83 ND Ladeira et al. (2015)
B. cereus BR0302 0.12 ND ND Chantarasiri et al. (2015)
B. licheniformis JK7 0.75 ND 0.63 Seo et al. (2013)
B. methylotrophicus RYC01101 0.23 ND ND Chantarasiri (2014)
B. pumilus EB3 0.08 ND 0.04 Ariffin et al. (2006)
B. subtilis AS3 0.07 ND ND Deka et al. (2011)
B. subtilis SL9–9 0.90 0.32 No activity Kim et al. (2012)
B. cereus JD0404 1.78 0.08 0.05 This study
ND denotes ‘not determined’.

bacteria because they exhibited the cellulolytic zone around


their colonies on CMC agar after Gram’s iodine staining.
HC values were calculated and the results are shown in Table 1
and Fig. 2. The bacterium strain JD0404 exhibited a maximum
hydrolysis capacity of 4.47 ± 0.30, greater than the positive
control (B. methylotrophicus RYC01101) by a factor of 1.45.
The identification of strain JD0404 was determined using mor-
phological examination, biochemical characterization and
molecular genetic analysis. Strain JD0404 is a facultative
anaerobe bacterium with white colour, a raised elevation and
a curled margin colony. Bacterial cells were 1  4 lm, rod-
shaped, Gram-positive, endospore-forming and motile. Cata-
lase and oxidase tests were positive. Sugar fermentation and
hydrogen sulphide production analyses showed the strain
JD0404 could ferment glucose but could not produce hydrogen
sulphide cultured on Triple Sugar Iron Agar. For growth at
different parameters, it could grow between a pH of 5.0 and
11.0 at temperatures ranging between 20 °C and 45 °C and
salinity tolerance at 6% of NaCl. The 16S rDNA gene
sequencing analysis evidenced that it exhibited the highest
homology to B. cereus ATCC 14579 with 99.81% similarity
(Certification of Thailand Institute of Scientific and Techno-
logical Research, Request No. 2558/3-063). Based on the
results, this aquatic bacterium was designated as B. cereus
JD0404. B. cereus is a ubiquitously distributed bacterium
found in decaying organic matter, soil, food, fresh and marine
waters, and the intestinal tract of invertebrates (Bottone,
2010). It can be used to biosynthesize numerous hydrolysis
enzymes and has been used for biotechnological applications
(Łaba et al., 2015). According to other studies, B. cereus can
be isolated from mangrove swamps and related environments
(Dias et al., 2009; Tabao and Monsalud, 2010; Thatoi et al.,
2013) because it has an important role in the carbon flow Figure 3 Effect of temperature on CMCase activity (a) and
and the organic matter degradation process in mangrove stability (b) from B. cereus JD0404. Error bars represent the
ecosystems (Ghosh et al., 2010). standard deviation of three replicates.

Cellulolytic activity of aquatic B. cereus JD0404


performance was in agreement with the lack of the complete cel-
B. cereus JD0404 was examined for cellulolytic activity and this lulolytic system of the Bacillus genus (Kim et al., 2012). Most
showed that it could yield 1.778 ± 0.003 U/mL of CMCase Bacillus enzymes showed primary activity being on CMC with
activity, 0.079 ± 0.001 U/mL of Avicelase activity and 0.048 their endoglucanase activity, but hardly degraded crystalline
± 0.002 U/mL of b-glucosidase activity. Its cellulolytic activity forms of cellulose (Ladeira et al., 2015; Robson and
was compared to other bacteria in the Bacillus genus (Table 2). Chambliss, 1984). It could be stated that CMC is the experimen-
The comparisons showed that the B. cereus JD0404 is a produc- tally appropriate carbon source for cellulolytic enzyme produc-
tive endoglucanase-producing bacterium, but it barely tion of bacteria. However, over the years, many studies have
produced exoglucanase and b-glucosidase. This cellulolytic attempted to isolate the cellulolytic Bacillus bacteria from
262 A. Chantarasiri

Table 3 Effect of various additives on CMCase activity from


B. cereus JD0404.
Residual activity (%)
Metal ions
Ca2+ 124.15 ± 1.38
Co2+ 182.84 ± 1.37
Cu2+ 142.90 ± 0.85
Fe2+ 138.72 ± 2.55
Hg2+ 96.22 ± 1.15
K+ 95.41 ± 1.14
Mg2+ 136.15 ± 2.98
Mn2+ 302.92 ± 3.82
Ni2+ 161.79 ± 1.79
Pb2+ 121.59 ± 0.42
Sr2+ 103.91 ± 0.18
Zn2+ 97.57 ± 0.00
Detergent
TWEEN 80Ò 95.28 ± 0.56
Chelating agent
EDTA 78.41 ± 1.49
Organic solvents
Benzene 91.64 ± 0.02
Cyclohexane 91.91 ± 1.51
Dichloromethane 95.01 ± 0.58
Ethanol 95.96 ± 6.10
Ethyl-ether 96.23 ± 3.05
Methanol 90.83 ± 7.61
n-Hexane 98.92 ± 1.14
Toluene 72.21 ± 0.43

et al., 2009; Lin et al., 2012; Trivedi et al., 2011; Yin et al.,
Figure 4 Effect of pH on CMCase activity (a) and stability (b) 2010). These metal ions have a major effect on enzymatic per-
from B. cereus JD0404. Enzyme activity was measured in sodium formance by working as a cofactor (Irfan et al., 2012). Based
citrate buffer (), sodium phosphate buffer (4), Tris–HCl buffer on the increase of catalytic activity by Mn2+, it could be
(h) and glycine-NaOH buffer (d). Error bars represent the assumed that this metal ion responds to certain amino acid
standard deviation of three replicates. residues in the active site and promotes the favourable confor-
mation to enzyme activity (Azzeddine et al., 2013). The inac-
environments and have reported their resulting enzymes tive phenomenon of enzymes caused by Hg2+ could possibly
having complete cellulolytic activity (Balasubramanian and indicate that the active site of the enzyme contained the thiol
Simões, 2014; Kim et al., 2012; Ladeira et al., 2015). group (Irfan et al., 2012; Yin et al., 2010). Cellulolytic activity
was slightly reduced by TWEEN 80Ò indicating that B. cereus
Characterization of cellulolytic enzyme from aquatic B. cereus JD0404 could be used for industries dealing with detergents.
JD0404 The reduction of catalytic performance of cellulolytic enzyme
by a chelating agent revealed that the endoglucanase from
The optimum temperature for cellulolytic activity (CMCase B. cereus JD0404 could be identified as a metalloenzyme
activity) was found to be 50 °C (Fig. 3a) and this remained (Annamalai et al., 2013). To further apply B. cereus JD0404
stable at up to 60 °C (Fig. 3b). For optimum pH, the B. cereus to bioremediation of wastewater contaminated with organic
JD0404 showed optimum activity at pH 7.0 (Fig. 4a) and was solvents or industries working with organic solvents, the effect
stable at pH 5.0–8.0 (Fig. 4b). These pH and temperature char- of organic solvents on enzyme stability from B. cereus JD0404
acteristics were related to other Bacillus enzymes isolated from was investigated. The results showed that only toluene had
different environments. It was found that endoglucanase from critically inactivated enzymatic performance, which was in
Bacillus sp. are active at a temperature range of 50–60 °C and a agreement with many studies (Annamalai et al., 2013;
pH range of 4.8–11.0 (Sadhu and Maiti, 2013). The effect of Trivedi et al., 2011).
various additives on enzyme activity is shown in Table 3.
The result of metal ions revealed that the cellulolytic activity Application on cellulose-based biomass by bioconversion process
from B. cereus JD0404 was greatly enhanced by Mn2+ and
slightly inhibited by Hg2+, K+ and Zn2+. Similarly, bacterial Production of reducing sugars from agro-residues and
endoglucanase from many studies was also activated by Mn2+ lignocellulosic waste by a bioconversion process is a worldwide
and hindered by Hg2+ (Annamalai et al., 2013; concern nowadays because they are the prerequisite substrate
Balasubramanian and Simões, 2014; Irfan et al., 2012; Kim of biofuel and bio-based products. In this study, the local
Characterization of cellulolytic activity of aquatic Bacillus cereus 263

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Acknowledgments
Kasana, R.C., Salwan, R., Dhar, H., Dutt, S., Gulati, A., 2008. A
rapid and easy method for the detection of microbial cellulases on
This research was funded by King Mongkut’s University of agar plates using Gram’s iodine. Curr. Microbiol. 57, 503–507.
Technology North Bangkok. Contract No. KMUTNB- Kim, B.K., Lee, B.H., Lee, Y.J., Jin, I.H., Chung, C.H., Lee, J.W.,
GEN-57-53. I am grateful to Dr. Narumon Boonman, Suan 2009. Purification and characterization of carboxymethylcellulase
Sunandha Rajabhat University for her guidance on this isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-
research. 53. Enzyme Microb. Technol. 44, 411–416.
Kim, Y.K., Lee, S.C., Cho, Y.Y., Oh, H.J., Ko, Y.H., 2012. Isolation
of cellulolytic Bacillus subtilis strains from agricultural environ-
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