Scholarly article on topic 'Aquatic Bacillus cereus JD0404 isolated from the muddy sediments of mangrove swamps in Thailand and characterization of its cellulolytic activity'

Aquatic Bacillus cereus JD0404 isolated from the muddy sediments of mangrove swamps in Thailand and characterization of its cellulolytic activity Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Aiya Chantarasiri

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 β-glucosidase activity was 1.778±0.003U/mL, 0.079±0.001U/mL and 0.048±0.002U/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.

Academic research paper on topic "Aquatic Bacillus cereus JD0404 isolated from the muddy sediments of mangrove swamps in Thailand and characterization of its cellulolytic activity"

Egyptian Journal of Aquatic Research (2015) xxx, xxx-xxx

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National Institute of Oceanography and Fisheries Egyptian Journal of Aquatic Research

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Egyptian Journal of Aquatic Research

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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

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-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.

© 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 coastal wetland ecosystems which are found along tropical and subtropical coastlines dominated by halophilic plants in the genera Rhizophora and Avicennia (Mitsch and Gosselink, 2015). The mangrove swamps and neighbouring coastal environments provide many ecological benefits (Ghosh et al., 2010;

* 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.

Sandilyan and Kathiresan, 2014), including coastal protection against natural disasters, storage of organic material, habitats for estuarine organisms and mitigation of the global warming phenomenon. Mangrove ecosystems can store large amounts of organic carbon and are rich in organic carbon in sediments which mainly originated from litter falls and the underground roots of mangrove plants (Yong et al., 2011). The mangrove microbial communities play a vital role in the organic carbon cycle. Cellulolytic microorganisms can perform the degradation of cellulose-based plant litter, resulting in the production of simple-sugar derivatives in the sediments (Soares-Junior et al., 2013). Microbial cellulolytic enzymes, called cellulase, are the complex enzymes that consist of endoglucanases

http://dx.doi.Org/10.1016/j.ejar.2015.08.003

1687-4285 © 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/).

(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 work to hydrolyse the b-1, 4 glycosidic bonds of cellulose. Cellulase have become focal biocatalysts in various green technology industries, such as textile production, detergent composition, food processing, animal feed production, removal of bacterial biofilm and bioconversion for biofuel production (Juturu and Wu, 2014). Most cellulolytic enzymes isolated from mangrove swamps and related aquatic environments are produced from fungi belonging to the genera Cladosporium, Alternaría and Byssochlamys. (Alsheikh-Hussain et al., 2014; Matondkar et al., 1980; Thatoi et al., 2013), and bacteria belonging to the genera Micrococcus, Bacillus, Pseudomonas, Xanthomonas and Brucella (Behera et al., 2014). Interestingly, Thailand is one of the Asian countries that contain large areas of mangrove swamps (Sandilyan and Kathiresan, 2014), but the knowledge of cellulolytic microbes isolated from mangrove swamps is limited (Behera et al., 2014). For this study, the aquatic bacteria demonstrating cellulolytic performance were isolated from muddy sediments of mangrove swamps in Thailand and identified. The purpose was to determine a competent aquatic cellulolytic bacterium for possible use in green technology industries and related biotechnological applications.

Materials and methods

Description of sampling sites

Muddy sediment samples were collected from mangrove swamps in Rayong River (12° 39' 57.40" N, 101° 14' 30.79" E), Rayong Province, Thailand (Fig. 1). Samples were collected twice during the rainy season, once in July 2014 and another in September 2014. Muddy sediments close to the roots of mangrove plants or beneath the decomposed plant litter at a depth of 0-15 cm were taken from sampling sites

at different locations, placed in sterile zip-lock plastic bags and kept at 4 0C.

Isolation and purification of mangrove bacteria

The isolation of mangrove bacteria and media was modified from Dias et al. (2009). The samples were serially diluted with sterile normal saline solution (0.85% NaCl) within 24 h of collection to obtain 1:10,000 dilutions. One hundred microlitres of each diluted sample was spread plated on Tryptone Soya Agar (Himedia, India) supplemented with 1.8% NaCl and incubated at 28 0C, the average sediment temperature of the sampling sites, for 48 h. The agar plates were investigated in terms of colony morphology including shape, margin, elevation and pigmentation. Morphologically dissimilar colonies were selected and streak plated on Tryptone Soya Agar to obtain 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 iodine staining method. Five microlitres of overnight growth culture in the Tryptone Soya Broth (Himedia, India) of each bacterial isolate 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 incubated at 28 0C for 48 h and then flooded with Gram's iodine solution for 10 min. The cellulolytic isolates were detected by the cellulolytic zone around the colonies after Gram's iodine staining. The hydrolysis capacity (HC) value that determined the cellulolytic activity was calculated from the ratio between the diameter of the cellulolytic zone and the diameter of the 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.

non-cellulolytic bacterium, Escherichia coli TISTR073 (Thailand Institute of Scientific and Technological Research, Thailand) and the positive control was the cellulolytic bacterium isolated from bovine faeces, Bacillus methylotrophicus RYC01101 (Chantarasiri, 2014).

Identification of cellulolytic bacteria

The selected cellulolytic isolate was identified by morphological examination, biochemical characterization and molecular genetic analysis. The morphological examination was performed by Gram staining, endospore staining and motility testing. Growth at different parameters including pH, temperature, salinity condition and anaerobic environment was investigated 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 Triple Sugar 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 analyses including PCR amplification, 16S rDNA sequence analysis and homology similarity analysis were carried out by the Thailand 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 h under aeration conditions. Bacterial cells were removed from the culture broth by centrifugation at 4500 xg for 30 min at 4 °C. The cell-free supernatant obtained after centrifugation served as a crude enzyme solution. The crude enzyme solution was partially purified by Amicon® Ultra-15 (10 kDa) centrifugal filter devices (Millipore, Ireland).

Cellulolytic activity assay

The cellulolytic activity assays were modified from the previously described study by incubating the enzyme solution with the substrate and determining the amount of products liberated (Kim et al., 2012). Endoglucanase activity (CMCase activity) was measured by incubating 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 50 °C for 30 min. The reducing sugars liberated were determined by the 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). The enzyme reaction was terminated by adding 3.0 mL of DNS reagent and then boiled for 5 min. The solution was completely cooled and the optical density of the reaction mixture was measured at 540 nm. Exoglucanase activity (Avicelase activity) was measured using 2% Avicel® PH-101 (Sigma-Aldrich, Germany) suspended in 0.1 M sodium phosphate buffer (pH 7.0) as a substrate and incubating it with 0.5 mL of enzyme solution at 50 °C for 1 h. The supernatant of the reaction mixture was collected in order to determine the quantity of reducing sugars by the DNS method. The enzyme activity was calculated using a glucose standard curve. One unit (U) of CMCase and Avicelase

activities is defined as the amount of enzyme required to release 1 imol of reducing sugars as glucose equivalents per minute under standard assay conditions. b-glucosidase activity was measured by incubating 0.5 mL of enzyme solution with 1 mL of 0.1% p-nitrophenyl-b-D-glucopyranoside (pNPG) in 0.1 M sodium phosphate buffer (pH 7.0) at 50 0C for 1 h. The enzyme reaction was terminated by adding 2.0 mL of 1 M Na2CO3 solution and the optical density of the reaction mixture 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 of enzyme required to release 1 imol of p-nitrophenol per minute under standard assay conditions.

Effect of temperature on cellulolytic activity and thermal stability

The effect of temperature on enzyme activity was examined by incubating 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 0C to 85 0C for 30 min. Thermal stability was examined by incubating the enzyme solution in 0.1 M sodium phosphate buffer (pH 7.0) at temperatures ranging from 20 0C to 85 0C for 24 h and the residual activity was monitored using 2% CMC sodium salt as a substrate. The cellulolytic activity of enzymes was assayed using the method described above.

Effect of pH on cellulolytic activity and pH stability

The effect of pH on enzyme activity was measured by incubating 0.5 mL of enzyme solution with 0.5 mL of 2% CMC sodium salt in different pH buffers at 50 0C 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: sodium citrate buffer (pH 3.0-6.0), sodium phosphate buffer (pH 6.0-8.0), Tris-HCl buffer (pH 8.0-9.0) and glycine-NaOH buffer (pH 9.0-11.0). The pH stability was determined by incubating the enzyme solution in the above-mentioned buffers at 50 0C for 24 h and the residual activity was monitored using 2% 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 by incubating 0.5 mL of enzyme solution with metal ions, detergent, 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 + , with the final concentration of metal ion solution at 5 mM following the study of Seo et al. (2013). The effect of detergent on enzyme activity was studied using 1% TWEEN 80® (Sigma-Aldrich, Germany). The result of a chelating agent on enzyme activity using 5 mM EDTA was determined. Residual activity of enzymes was monitored using 2% CMC sodium salt as a substrate after being incubated with additive solutions at 50 0C for 60 min (Annamalai et al., 2013). The effect of eight organic solvents on enzyme activity was examined by incubating the enzyme solution with a 25% concentration of various organic solvents 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.

ethanol, ethyl-ether, methanol, n-hexane and toluene at 50 0C for 4h (Annamalai et al., 2013) and the residual activity of enzymes was measured using 2% CMC sodium salt as a substrate. The cellulolytic activity of enzymes was assayed using the 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 biomass was hydrolysed by a selected aquatic bacterium. Cassava stems, hay, rice straw and peanut shells were used as the carbon 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 0C under aeration conditions for 48 h. The culture medium 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 dissimilarly morphological colonies were isolated from forty-two muddy sediments. The supplement of the Tryptone Soya Agar medium with 1.8% of NaCl ensured the selection of isolated bacteria 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 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 morphological 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 x 4 im, 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 0C and 45 0C 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 Technological 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 (taba 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 and the organic matter degradation process in mangrove ecosystems (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 CMCase activity, 0.079 ± 0.001 U/mL of Avicelase activity and 0.048 ± 0.002 U/mL of b-glucosidase activity. Its cellulolytic activity was compared to other bacteria in the Bacillus genus (Table 2). The comparisons showed that the B. cereus JD0404 is a productive endoglucanase-producing bacterium, but it barely produced exoglucanase and b-glucosidase. This cellulolytic

5 40 0

0 20 25 30 35 40 45 50 55 60 65 70 75 80 85 Temperature (°C)

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.

performance was in agreement with the lack of the complete cellulolytic system of the Bacillus genus (Kim et al., 2012). Most Bacillus enzymes showed primary activity being on CMC with their endoglucanase activity, but hardly degraded crystalline forms of cellulose (Ladeira et al., 2015; Robson and Chambliss, 1984). It could be stated that CMC is the experimentally appropriate carbon source for cellulolytic enzyme production of bacteria. However, over the years, many studies have attempted to isolate the cellulolytic Bacillus bacteria from

2 3 4 5 6 7 8 9 10 11 12

100 ■ ^^ù.

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1 60 .

20 ■ \ o : s-■-■-■-■-»-♦-♦-

2 3 4 5 6 7 8 9 10 11 12

Figure 4 Effect of pH on CMCase activity (a) and stability (b) from B. cereus JD0404. Enzyme activity was measured in sodium citrate buffer (o), sodium phosphate buffer (A), Tris-HCl buffer (□) and glycine-NaOH buffer (•). 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

environments and have reported their resulting enzymes having complete cellulolytic activity (Balasubramanian and Simoes, 2014; Kim et al., 2012; Ladeira et al., 2015).

Characterization of cellulolytic enzyme from aquatic B. cereus JD0404

The optimum temperature for cellulolytic activity (CMCase activity) was found to be 50 0C (Fig. 3a) and this remained stable at up to 60 0C (Fig. 3b). For optimum pH, the B. cereus JD0404 showed optimum activity at pH 7.0 (Fig. 4a) and was stable at pH 5.0-8.0 (Fig. 4b). These pH and temperature characteristics were related to other Bacillus enzymes isolated from different environments. It was found that endoglucanase from Bacillus sp. are active at a temperature range of 50-60 0C and a pH 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 activity from B. cereus JD0404 was greatly enhanced by Mn2 + and slightly inhibited by Hg2 + , K + and Zn2 +. Similarly, bacterial endoglucanase 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 performance by working as a cofactor (Irfan et al., 2012). Based on the increase of catalytic activity by Mn2 + , it could be assumed that this metal ion responds to certain amino acid residues in the active site and promotes the favourable conformation to enzyme activity (Azzeddine et al., 2013). The inactive phenomenon of enzymes caused by Hg2+ could possibly indicate that the active site of the enzyme contained the thiol group (Irfan et al., 2012; Yin et al., 2010). Cellulolytic activity was slightly reduced by TWEEN 80® indicating that B. cereus JD0404 could be used for industries dealing with detergents. The reduction of catalytic performance of cellulolytic enzyme by a chelating agent revealed that the endoglucanase from B. cereus JD0404 could be identified as a metalloenzyme (Annamalai et al., 2013). To further apply B. cereus JD0404 to bioremediation of wastewater contaminated with organic solvents or industries working with organic solvents, the effect of organic solvents on enzyme stability from B. cereus JD0404 was investigated. The results showed that only toluene had critically inactivated enzymatic performance, which was in agreement 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 and lignocellulosic waste by a bioconversion process is a worldwide concern nowadays because they are the prerequisite substrate of biofuel and bio-based products. In this study, the local

agro-residues were bioconverted to reducing sugars by the potential cellulolytic bacterium, B. cereus JD0404. After 48 h of bacterial incubation, cassava stems, hay, rice straw and peanut 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 experiment was higher than the previous study using cellulolytic enzyme 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 during recent years (Wang et al., 2009), because the bioconversion process of lignocellulosic biomass is environmentally friendly and provides several advantages.

Conclusions

The mangrove cellulolytic bacteria play a significant role in the carbon flow and the cycle of cellulosic matter in related aquatic environments. These cellulolytic bacteria have recently been applied to various industrial processes and also minimize the damage from pollution. This study is the first report of cellulolytic bacteria isolated from mangrove swamps in Rayong Province, Thailand. The aquatic bacterium strain JD0404 was isolated, identified and finally designated as B. cereus JD0404. The cellulolytic characteristics of B. cereus JD0404 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 of Technology North Bangkok. Contract No. KMUTNB-GEN-57-53. I am grateful to Dr. Narumon Boonman, Suan Sunandha Rajabhat University for her guidance on this research.

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