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

HO ST E D BYNational Institute of Oceanography and Fisheries

Egyptian Journal of Aquatic Research

http://ees.elsevier.com/ejarwww.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

* Tel./fax: +66 (0)38 627012.

E-mail address: aiya.c@sciee.kmutnb.ac.th

Peer review under responsibility of National Institute of Oceanography

and Fisheries.

http://dx.doi.org/10.1016/j.ejar.2015.08.0031687-4285 � 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/).

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

Bacillus cereus;

Cellulolytic activity;

Mangrove swamp;

Muddy sediment

Abstract This study aimed to conduct the isolation, screening and identification of bacteria with a

high level of cellulolytic activity from the muddy sediments of mangrove swamps in Thailand. One

hundred and ninety aquatic bacterial isolates were isolated from different muddy sediments and

eighty one isolates were determined to be cellulolytic bacteria. The cellulolytic bacterium identified

as Bacillus cereus JD0404 showed maximum hydrolysis activity on carboxymethylcellulose agar

plates. Its cellulolytic performance for CMCase activity, Avicelase activity and b-glucosidaseactivity 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.� 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

Mangrove swamps or mangrove forests are the unique coastalwetland ecosystems which are found along tropical and sub-

tropical coastlines dominated by halophilic plants in the gen-era Rhizophora and Avicennia (Mitsch and Gosselink, 2015).The mangrove swamps and neighbouring coastal environ-

ments provide many ecological benefits (Ghosh et al., 2010;

Sandilyan and Kathiresan, 2014), including coastal protectionagainst natural disasters, storage of organic material, habitatsfor estuarine organisms and mitigation of the global warming

phenomenon. Mangrove ecosystems can store large amountsof organic carbon and are rich in organic carbon in sedimentswhich mainly originated from litter falls and the undergroundroots of mangrove plants (Yong et al., 2011). The mangrove

microbial communities play a vital role in the organic carboncycle. Cellulolytic microorganisms can perform the degrada-tion of cellulose-based plant litter, resulting in the production

of simple-sugar derivatives in the sediments (Soares-Junioret al., 2013). Microbial cellulolytic enzymes, called cellulase,are the complex enzymes that consist of endoglucanases

258 A. Chantarasiri

(E.C. 3.2.1.4), exoglucanases (E.C. 3.2.1.91, and E.C. 3.2.1.176)and b-glucosidases (E.C. 3.2.1.21) which synergistically workto hydrolyse the b-1, 4 glycosidic bonds of cellulose. Cellulase

have become focal biocatalysts in various green technologyindustries, such as textile production, detergent composition,food processing, animal feed production, removal of bacterial

biofilm and bioconversion for biofuel production (Juturu andWu, 2014). Most cellulolytic enzymes isolated from mangroveswamps and related aquatic environments are produced from

fungi belonging to the genera Cladosporium, Alternaria andByssochlamys. (Alsheikh-Hussain et al., 2014; Matondkaret al., 1980; Thatoi et al., 2013), and bacteria belonging tothe genera Micrococcus, Bacillus, Pseudomonas, Xanthomonas

and Brucella (Behera et al., 2014). Interestingly, Thailand isone of the Asian countries that contain large areas of man-grove swamps (Sandilyan and Kathiresan, 2014), but the

knowledge of cellulolytic microbes isolated from mangroveswamps is limited (Behera et al., 2014). For this study, theaquatic bacteria demonstrating cellulolytic performance were

isolated from muddy sediments of mangrove swamps in Thai-land and identified. The purpose was to determine a competentaquatic cellulolytic bacterium for possible use in green technol-

ogy industries and related biotechnological applications.

Materials and methods

Description of sampling sites

Muddy sediment samples were collected from mangroveswamps in Rayong River (12� 390 57.4000 N, 101� 140 30.7900

E), Rayong Province, Thailand (Fig. 1). Samples were col-lected twice during the rainy season, once in July 2014 and

another in September 2014. Muddy sediments close to theroots of mangrove plants or beneath the decomposed plantlitter at a depth of 0–15 cm were taken from sampling sites

Figure 1 Map of mangrove swamps in Rayong River. The sampling

the Google Maps service.

at different locations, placed in sterile zip-lock plastic bagsand kept at 4 �C.

Isolation and purification of mangrove bacteria

The isolation of mangrove bacteria and media was modifiedfrom Dias et al. (2009). The samples were serially diluted with

sterile normal saline solution (0.85% NaCl) within 24 h of col-lection to obtain 1:10,000 dilutions. One hundred microlitresof each diluted sample was spread plated on Tryptone Soya

Agar (Himedia, India) supplemented with 1.8% NaCl andincubated at 28 �C, the average sediment temperature of thesampling sites, for 48 h. The agar plates were investigated in

terms of colony morphology including shape, margin, eleva-tion and pigmentation. Morphologically dissimilar colonieswere selected and streak plated on Tryptone Soya Agar toobtain pure colonies.

Screening of cellulolytic bacteria

Screening of the cellulolytic bacteria was conducted from that

previously described (Kasana et al., 2008) using car-boxymethylcellulose (CMC) agar plates and Gram’s iodinestaining method. Five microlitres of overnight growth culture

in the Tryptone Soya Broth (Himedia, India) of each bacterialisolate was spot plated on CMC agar (0.2% NaNO3, 0.1%K2HPO4, 0.05%MgSO4, 0.05% KCl, 0.2% CMC sodium salt,0.02% peptone and 1.7% agar). The agar plates were incu-

bated at 28 �C for 48 h and then flooded with Gram’s iodinesolution for 10 min. The cellulolytic isolates were detected bythe cellulolytic zone around the colonies after Gram’s iodine

staining. The hydrolysis capacity (HC) value that determinedthe cellulolytic activity was calculated from the ratio betweenthe diameter of the cellulolytic zone and the diameter of the

bacterial colony. The negative control for screening was the

site covered an area of 75,400 m2. The figure was generated using

Characterization of cellulolytic activity of aquatic Bacillus cereus 259

non-cellulolytic bacterium, Escherichia coli TISTR073 (Thai-land Institute of Scientific and Technological Research, Thai-land) and the positive control was the cellulolytic bacterium

isolated from bovine faeces, Bacillus methylotrophicusRYC01101 (Chantarasiri, 2014).

Identification of cellulolytic bacteria

The selected cellulolytic isolate was identified by morphologi-cal examination, biochemical characterization and molecular

genetic analysis. The morphological examination was per-formed by Gram staining, endospore staining and motilitytesting. Growth at different parameters including pH, temper-

ature, salinity condition and anaerobic environment was inves-tigated at 28 �C for 24 h following Chantarasiri (2014).Biochemical characterization was examined by catalase testing(Gagnon et al., 1959) and oxidase testing (Gordon and

McLeod, 1928). Dextrose, lactose and sucrose fermentation,and hydrogen sulphide production were analysed using TripleSugar Iron Agar (Himedia, India). The PCR amplification and

16S rDNA sequence analysis were described by Yukphan et al.(2004) using a set of primers as follows: forward primer 27F:GAGTTTGATCATGGCTCAG and reverse primer 1492R:

CGGTTACCTTGTTACGACTT. All molecular genetic anal-yses including PCR amplification, 16S rDNA sequence analy-sis and homology similarity analysis were carried out by theThailand Institute of Scientific and Technological Research

(Thailand).

Preparation of cellulolytic enzyme solution

The selected isolate was grown in CMC broth at 28 �C for 48 hunder aeration conditions. Bacterial cells were removed fromthe culture broth by centrifugation at 4500�g for 30 min at

4 �C. The cell-free supernatant obtained after centrifugationserved as a crude enzyme solution. The crude enzyme solutionwas partially purified by Amicon� Ultra-15 (10 kDa) centrifu-

gal filter devices (Millipore, Ireland).

Cellulolytic activity assay

The cellulolytic activity assays were modified from the previ-

ously described study by incubating the enzyme solution withthe substrate and determining the amount of products liber-ated (Kim et al., 2012). Endoglucanase activity (CMCase

activity) was measured by incubating 0.5 mL of enzyme solu-tion with 0.5 mL of 2% CMC sodium salt in 0.1 M sodiumphosphate buffer (pH 7.0) at 50 �C for 30 min. The reducing

sugars liberated were determined by the 3,5-dinitrosalicylicacid (DNS) method (Miller, 1959). The enzyme reaction wasterminated by adding 3.0 mL of DNS reagent and then boiled

for 5 min. The solution was completely cooled and the opticaldensity of the reaction mixture was measured at 540 nm.Exoglucanase activity (Avicelase activity) was measured using2% Avicel� PH-101 (Sigma–Aldrich, Germany) suspended in

0.1 M sodium phosphate buffer (pH 7.0) as a substrate andincubating it with 0.5 mL of enzyme solution at 50 �C for1 h. The supernatant of the reaction mixture was collected in

order to determine the quantity of reducing sugars by theDNS method. The enzyme activity was calculated using a glu-cose standard curve. One unit (U) of CMCase and Avicelase

activities is defined as the amount of enzyme required torelease 1 lmol of reducing sugars as glucose equivalents perminute under standard assay conditions. b-glucosidase activitywas measured by incubating 0.5 mL of enzyme solution with1 mL of 0.1% p-nitrophenyl-b-D-glucopyranoside (pNPG) in0.1 M sodium phosphate buffer (pH 7.0) at 50 �C for 1 h.

The enzyme reaction was terminated by adding 2.0 mL of1 M Na2CO3 solution and the optical density of the reactionmixture was measured at 400 nm. The enzyme activity was

calculated using a p-nitrophenol standard curve. One unit(U) of b-glucosidase activity is defined as the amount ofenzyme required to release 1 lmol of p-nitrophenol per minuteunder standard assay conditions.

Effect of temperature on cellulolytic activity and thermal

stability

The effect of temperature on enzyme activity was examined byincubating 0.5 mL of enzyme solution with 0.5 mL of 2%CMC sodium salt in 0.1 M sodium phosphate buffer (pH

7.0) at various temperatures ranging from 20 �C to 85 �C for30 min. Thermal stability was examined by incubating theenzyme solution in 0.1 M sodium phosphate buffer (pH 7.0)

at temperatures ranging from 20 �C to 85 �C for 24 h andthe residual activity was monitored using 2% CMC sodiumsalt as a substrate. The cellulolytic activity of enzymes wasassayed using the method described above.

Effect of pH on cellulolytic activity and pH stability

The effect of pH on enzyme activity was measured by incubat-

ing 0.5 mL of enzyme solution with 0.5 mL of 2% CMCsodium salt in different pH buffers at 50 �C for 30 min.Enzyme activity was measured at a range of pH values between

3.0 and 11.0 using 0.1 M of the following buffers: sodiumcitrate buffer (pH 3.0–6.0), sodium phosphate buffer (pH6.0–8.0), Tris–HCl buffer (pH 8.0–9.0) and glycine-NaOH

buffer (pH 9.0–11.0). The pH stability was determined by incu-bating the enzyme solution in the above-mentioned buffers at50 �C for 24 h and the residual activity was monitored using2% CMC sodium salt as a substrate. The cellulolytic activity

of enzyme was assayed using the method described above.

Effect of additives on cellulolytic activity

The effect of additives on enzyme activity was investigated byincubating 0.5 mL of enzyme solution with metal ions, deter-gent, a chelating agent and organic solvents. There were twelve

different metal ions used, including Ca2+, Co2+, Cu2+, Fe2+,Hg2+, K+, Mg2+, Mn2+, Ni2+, Pb2+, Sr2+, and Zn2+, withthe final concentration of metal ion solution at 5 mM following

the study of Seo et al. (2013). The effect of detergent on enzymeactivity was studied using 1% TWEEN 80� (Sigma–Aldrich,Germany). The result of a chelating agent on enzyme activityusing 5 mM EDTA was determined. Residual activity of

enzymes was monitored using 2% CMC sodium salt as a sub-strate after being incubated with additive solutions at 50 �Cfor 60 min (Annamalai et al., 2013). The effect of eight organic

solvents on enzyme activity was examined by incubating theenzyme solution with a 25% concentration of various organicsolvents such as benzene, cyclohexane, dichloromethane,

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.

260 A. Chantarasiri

ethanol, ethyl-ether, methanol, n-hexane and toluene at 50 �Cfor 4 h (Annamalai et al., 2013) and the residual activity of

enzymes was measured using 2% CMC sodium salt as a sub-strate. The cellulolytic activity of enzymes was assayed usingthe method described above.

Application on cellulose-based biomass by bioconversion process

The application to the bioconversion process was investigated.

To produce the reducing sugars, the cellulose-based biomasswas hydrolysed by a selected aquatic bacterium. Cassavastems, hay, rice straw and peanut shells were used as the car-bon source of the bacterial culture. The aquatic bacterium

was cultured in a basal medium supplemented with 1%cellulose-based biomass powder (Chantarasiri et al., 2015)

at 28 �C under aeration conditions for 48 h. The culture med-ium was collected for reducing sugars determination by DNS

method.

Results and discussion

Isolation, screening and identification of cellulolytic bacteria

One hundred and ninety aquatic bacteria with dissimilarlymorphological colonies were isolated from forty-two muddysediments. The supplement of the Tryptone Soya Agar

medium with 1.8% of NaCl ensured the selection of isolatedbacteria mainly found in mangrove ecosystems (Dias et al.,2009). Eighty-one bacterial isolates were defined as cellulolytic

Table 2 Cellulolytic performance of B. cereus JD0404 and related bacteria in the Bacillus genus.

Bacteria CMCase activity

(U/mL)

Avicelase activity

(U/mL)

b-Glucosidase activity

(U/mL)

Reference

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’.

Figure 3 Effect of temperature on CMCase activity (a) and

stability (b) from B. cereus JD0404. Error bars represent the

standard deviation of three replicates.

Characterization of cellulolytic activity of aquatic Bacillus cereus 261

bacteria because they exhibited the cellulolytic zone aroundtheir colonies on CMC agar after Gram’s iodine staining.

HC values were calculated and the results are shown in Table 1and Fig. 2. The bacterium strain JD0404 exhibited a maximumhydrolysis 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 andmolecular genetic analysis. Strain JD0404 is a facultative

anaerobe bacterium with white colour, a raised elevation anda 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 andhydrogen sulphide production analyses showed the strainJD0404 could ferment glucose but could not produce hydrogen

sulphide cultured on Triple Sugar Iron Agar. For growth atdifferent parameters, it could grow between a pH of 5.0 and11.0 at temperatures ranging between 20 �C and 45 �C andsalinity tolerance at 6% of NaCl. The 16S rDNA gene

sequencing analysis evidenced that it exhibited the highesthomology 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 theresults, this aquatic bacterium was designated as B. cereusJD0404. B. cereus is a ubiquitously distributed bacterium

found in decaying organic matter, soil, food, fresh and marinewaters, 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 canbe 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 flowand the organic matter degradation process in mangroveecosystems (Ghosh et al., 2010).

Cellulolytic activity of aquatic B. cereus JD0404

B. cereus JD0404 was examined for cellulolytic activity and this

showed that it could yield 1.778 ± 0.003 U/mL of CMCaseactivity, 0.079 ± 0.001 U/mL of Avicelase activity and 0.048± 0.002 U/mL of b-glucosidase activity. Its cellulolytic activitywas compared to other bacteria in the Bacillus genus (Table 2).The comparisons showed that the B. cereus JD0404 is a produc-tive endoglucanase-producing bacterium, but it barelyproduced exoglucanase and b-glucosidase. This cellulolytic

performance was in agreement with the lack of the complete cel-lulolytic system of the Bacillus genus (Kim et al., 2012). Most

Bacillus enzymes showed primary activity being on CMC withtheir endoglucanase activity, but hardly degraded crystallineforms of cellulose (Ladeira et al., 2015; Robson and

Chambliss, 1984). It could be stated that CMC is the experimen-tally appropriate carbon source for cellulolytic enzyme produc-tion of bacteria. However, over the years, many studies haveattempted to isolate the cellulolytic Bacillus bacteria from

Figure 4 Effect of pH on CMCase activity (a) and stability (b)

from B. cereus JD0404. Enzyme activity was measured in sodium

citrate buffer (�), sodium phosphate buffer (4), Tris–HCl buffer

(h) and glycine-NaOH buffer (d). Error bars represent the

standard deviation of three replicates.

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 80� 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

262 A. Chantarasiri

environments and have reported their resulting enzymes

having complete cellulolytic activity (Balasubramanian andSimoes, 2014; Kim et al., 2012; Ladeira et al., 2015).

Characterization of cellulolytic enzyme from aquatic B. cereusJD0404

The optimum temperature for cellulolytic activity (CMCaseactivity) was found to be 50 �C (Fig. 3a) and this remainedstable at up to 60 �C (Fig. 3b). For optimum pH, the B. cereus

JD0404 showed optimum activity at pH 7.0 (Fig. 4a) and wasstable at pH 5.0–8.0 (Fig. 4b). These pH and temperature char-acteristics were related to other Bacillus enzymes isolated from

different environments. It was found that endoglucanase fromBacillus sp. are active at a temperature range of 50–60 �C and apH range of 4.8–11.0 (Sadhu and Maiti, 2013). The effect of

various additives on enzyme activity is shown in Table 3.The result of metal ions revealed that the cellulolytic activityfrom B. cereus JD0404 was greatly enhanced by Mn2+ and

slightly inhibited by Hg2+, K+ and Zn2+. Similarly, bacterialendoglucanase from many studies was also activated by Mn2+

and hindered by Hg2+ (Annamalai et al., 2013;Balasubramanian and Simoes, 2014; Irfan et al., 2012; Kim

et al., 2009; Lin et al., 2012; Trivedi et al., 2011; Yin et al.,2010). These metal ions have a major effect on enzymatic per-formance by working as a cofactor (Irfan et al., 2012). Based

on the increase of catalytic activity by Mn2+, it could beassumed that this metal ion responds to certain amino acidresidues in the active site and promotes the favourable confor-

mation to enzyme activity (Azzeddine et al., 2013). The inac-tive phenomenon of enzymes caused by Hg2+ could possiblyindicate that the active site of the enzyme contained the thiol

group (Irfan et al., 2012; Yin et al., 2010). Cellulolytic activitywas slightly reduced by TWEEN 80� indicating that B. cereusJD0404 could be used for industries dealing with detergents.

The reduction of catalytic performance of cellulolytic enzymeby a chelating agent revealed that the endoglucanase fromB. cereus JD0404 could be identified as a metalloenzyme(Annamalai et al., 2013). To further apply B. cereus JD0404

to bioremediation of wastewater contaminated with organicsolvents or industries working with organic solvents, the effectof organic solvents on enzyme stability from B. cereus JD0404

was investigated. The results showed that only toluene hadcritically inactivated enzymatic performance, which was inagreement with many studies (Annamalai et al., 2013;

Trivedi et al., 2011).

Application on cellulose-based biomass by bioconversion process

Production of reducing sugars from agro-residues andlignocellulosic waste by a bioconversion process is a worldwideconcern nowadays because they are the prerequisite substrateof biofuel and bio-based products. In this study, the local

Characterization of cellulolytic activity of aquatic Bacillus cereus 263

agro-residues were bioconverted to reducing sugars by thepotential cellulolytic bacterium, B. cereus JD0404. After 48 hof bacterial incubation, cassava stems, hay, rice straw and pea-

nut shells were converted to reducing sugars of 9.42 ± 0.04,8.78 ± 0.13, 7.88 ± 0.09 and 7.76 ± 0.00 mg/mL respectively.For rice straw, the amount of reducing sugars from this exper-

iment was higher than the previous study using cellulolyticenzyme of B. cereus BR0302 isolated from coastal wetland soil(Chantarasiri et al., 2015) by 58-fold. Interest in cellulolytic

bacteria and their enzymes has grown considerably duringrecent years (Wang et al., 2009), because the bioconversionprocess of lignocellulosic biomass is environmentally friendlyand provides several advantages.

Conclusions

The mangrove cellulolytic bacteria play a significant role in thecarbon flow and the cycle of cellulosic matter in related aquaticenvironments. These cellulolytic bacteria have recently beenapplied to various industrial processes and also minimize the

damage from pollution. This study is the first report ofcellulolytic bacteria isolated from mangrove swamps inRayong Province, Thailand. The aquatic bacterium strain

JD0404 was isolated, identified and finally designated as B.cereus JD0404. The cellulolytic characteristics of B. cereusJD0404 that were found will make it a proficient candidate

for industrial processes and biotechnological applications.

Acknowledgments

This research was funded by King Mongkut’s University ofTechnology North Bangkok. Contract No. KMUTNB-GEN-57-53. I am grateful to Dr. Narumon Boonman, Suan

Sunandha Rajabhat University for her guidance on thisresearch.

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