สาขาวิทยาศาสตร์และพันธุวิศวกรรม การประชุมทางวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 56

A Newly Isolated Halophilic Anaerobic Bacterium That Producing β-Glucosidase, Halanaerobium praevalens Strain SP3-14 from

Sobroney Heng1, Chakrit Tachaapaikoon1,2*, Patthra Pason1,2, Rattiya Waeonukul1,2, Sirilak Baramee1,2, Akihiko Kosugi3, Prattana Ketbot2,Sreyneang Nhim2,Waraporn Apiwatanapiwat4 and Khanok Ratanakhanokchai1

ABSTRACT The halophilic are excellence sources of enzymes that active and tolerant with condition. In this study, screening and isolation of extremely halophilic anaerobic bacterium was performed. In total, 200 isolates were screened and isolated from 56 soil samples from salt evaporation ponds in Thailand. Among of them, a candidate given namely SP3-14 showed quickly and the highest growth at 30% (w/v) NaCl when compared with other candidates. This strain could not growth without NaCl (at least 10% NaCl). The strain was Gram-stain negative, non-motility and rod-shaped with a cell size of 0.6 x 1-3 µm. Optima growth of strain SP3-14 occurred at pH 7.0 and 37oC under anaerobic condition. A phylogenetic analysis based on 16S rRNA showed that the strain SP3-14 belonged within the genus Halanaerobium and was closely related to Halanaerobium praevalens DSM 2228T, and H. alcaliphilum GSLST with 99.6 and 98.2% sequence similarity, respectively. The isolated SP3-14 should be classified as a specie of genus Halanaerobium, for which the name H. praevalens strain SP3-14. Furthermore, the β-glucosidase of the strain SP3-14 was characterized some properties. It revealed optimum pH and temperature as 4.0 and 70oC, respectively. This is the first report of β-glucosidase from H. praevalens species. Therefore, the newly isolated SP3-14 strain probable has the potential as an enzyme resource for applicable lignocellulose degradation under salt tolerant.

Key words: β-glucosidase, Halanaerobium pravalens, halophilic anaerobic bacterium, saltern ponds *Corresponding author; e-mail address:[email protected]

1School of Bioresources and Technology, King Mongkut’s Universityof Technology Thonburi, Bangkok10150, Thailand 2Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Bangkok 10150, Thailand 3Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki, 305-8686, Japan 4Kasetsart Agricultural and Agro-Industrial Product Improvement Institute, Kasetsart University, Bangkok 10900, Thailand

88 การประชุมทางวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 56 สาขาวิทยาศาสตร์และพันธุวิศวกรรม

INTRODUCTION are the microorganisms that can tolerate to high salt concentration. They can be found among all kingdoms of life such as bacteria, eukarya, and (Joo and Kim, 2005). It has been isolated from saltern crystalized ponds, the , solar lakes and hypersaline lakes (Oren, 2002). Halophilic bacteria require salt to generate intracellular ion concentrations to maintain the balance of osmotic pressure in hypersaline environment. Hence, the halophiles are an excellence source of enzymes that are stable and active in the presence of high salt concentration (Yonezawa et al., 2007). According to the salt concentration required for optimal growth, halophiles have been classified into three types, slightly halophiles, moderately halophiles, and extremely halophiles. Slightly , moderately halophile, and extremely halophiles were demonstrated to grow at the salt concentration of 2-5%, 5-20%, and 20-30% NaCl, respectively (Joo and Kim, 2005; and Ventosa et al., 1998). Interestingly, amylase, protease, and lipase producing from halophilic anaerobic bacteria have been reported (Patel et al., 1993; Ryu et al., 1994). However, cellulolytic-producing halophilic anaerobic bacteria were seldom reported. In the realistic of saccharification process is mostly require high temperature that at least 37ºC to hydrolyze lignocellulosic material (Li et al., 2009). Therefore, a moderate or extremely with high temperature of active enzymes are attractively performed to practical uses. Cellulolytic enzyme system is a family of at least 3 groups of enzyme, consist of endo-(1,4)-β-D- glucanase (EC 3.2.1.4), exo-(1,4)-β-D-glucanase (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21). Among of them, β-glucosidase is play important role to hydrolyze cellobiose to glucose. β-glucosidase is generally responsible for the regulation of the whole cellulolytic process and is a rate-limiting factor during enzymatic hydrolysis of cellulose, as both endo-glucanase and exo-glucanase activities are often inhibited by cellobiose (Harhangi et al., 2002). Thus, β-glucosidase not only produces glucose from cellobiose, but also reduces cellobiose inhibition, allowing endo-glucanase and exo-glucanase enzymes to function more efficiently. In this study, the extremely halophilic anaerobic bacteria were screened and isolated from man-made environment. Subsequently, the β-glucosidase of the isolated strain was characterized.

MATERIALS AND METHODS 1. Sampling and medium Soil samples were collected from saltern (salt evaporation) ponds at Samut Sakhon Province, Thailand. These samples were enriched in modified basal medium (BM) (Tachaapaikoon et al., 2012) containing (g/L) NaCl 300.0; KH2PO4 1.5; K2HPO4 2.9; Urea 2.1; yeast extraction 4.5; Cysteine 0.5; resazurin 0.0005; 0.2 ml of mineral solution. The mineral solution contained (per liter) 250 g of . . . MgCl2 6H2O, 37.5 g of CaCl2 2H2O, and 0.3 g of FeSO4 6H2O. The medium was supplemented with

89 สาขาวิทยาศาสตร์และพันธุวิศวกรรม การประชุมทางวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 56 glucose as the sole carbon source. The pH of the medium was adjusted to 7.0 and bubbling with nitrogen gas for made its anaerobically. The solid medium contained 15 g of agar per liter.

2. Screening and isolation of halophilic anaerobic bacteria Fifty six of soil samples were put in the BM broth and incubated at 37oC for several days. Afterward, the grown cultures were enriched at the same fresh BM broth and repeatable to 6 times for enrichment culture. All stable enrichment cultures were used for isolation. The isolation of single colony was done by using Hungate roll tube technique. The stable enrichments were put and rolled in the tube for making thin film, then incubated at 37oC for several days. The colonies were appeared on the tube and then isolated single colony by needle into fresh BM broth again. Totally 200 isolates were obtained in this step. The isolate strain namely SP3-14 grew well under 30% NaCl within 24 hours when compared with other isolates, therefore this strain was used in the next step.

3. Phenotypic and biochemical characteristics of the strain SP3-14 To examine cell shape and spore formation, SP3-14was grown in the BM medium at 37oC for 48 hours. Traditional Gram staining was performed as described previously (Singleton, 2004) and spore formation was examined using the Schaeffer-Fulton staining method (Schaeffer and Fulton, 1993). The microscopic analysis was performed using an Olympus (CH20BIMF200) light microscope. The scanning electron microscopy (SEM) was used to study the morphology of the isolate SP3-14. SEM was performed using a SU8000 scanning electron microscope (Hitachi, Japan). Detection of motility was performed using semi-solid agar. To determine the optimum growth temperature, the isolates SP3- 14 was cultured in BM and incubated at difference temperature in the range 25-60oC in incubators. For observation of optimum pH range for growth, the pH was adjusted with 2M HCl or 1M NaOH to an acidic or alkaline pH, respectively. To determine the optimum of salt concentration on growth was examined by adding 0-30% (w/v) NaCl into BM medium.

4. 16S rRNA gene analysis and phylogenetic analysis of the strain SP3-14 For identification of strains by 16s rRNA gene sequencing analysis, the genomic DNA was extracted by Phenol-Chloroform Isoamyl Alcohol (PCI) DNA Extraction method. The 16s rRNA gene was amplified by PCR using the following primers: 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) (Weisburg et al., 1991). PCR products were purified using the QIAquick PCR purification kit (QIAGEN, Germany). The 16S rRNA gene sequence was obtained by using MicroSEQ®16S rRNA gene sequencing kit containing universal primers (Applied Biosystems Inc., CA, and USA). The identification of phylogenetic neighbors was initially carried out by the BLAST programs against the database of type strains with published prokaryotic names. A phylogenetic tree

90 การประชุมทางวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 56 สาขาวิทยาศาสตร์และพันธุวิศวกรรม was constructed by the neighbor-joining method (Saitou and Nei, 1987) conducted in MEGA6.0 software (Tamara et al., 2013).

5. Enzyme production Strain SP3-14 was grown in BM medium containing glucose as the sole carbon source. The cultures were incubated at 37oC until the late exponential growth phase and harvested by centrifugation (8,000 rpm and 15minutes) at 4oC. The cell pellet was collected and washed with the sodium phosphate buffer (pH 7.0) for several times. After that, the cell pellet was breaking with ultrasonic sonicator. Intracellular supernatant was used as the crude enzyme.

6. β-Glucosidase enzyme assay β-glucosidase was assayed using p-nitrophenyl-β-D-glucopyranoside (pNPG) (Tachaapaikoon et al., 2012). A reaction mixture (100 µl) containing 50µl of enzyme, 50 µl of pNPG (10mM) as substrate and sodium phosphate buffer (50 mM, pH 6.0) was incubated at 50ºC for 30 min, the reaction was terminated by addition of 100 µl of sodium carbonatesolution (0.4 M, pH 10.8). The absorbance was read at 405 nm using microplate reader (MULTISKAN; Labsystems). The amount of p-nitrophenol released was quantified using the pNP standard. One unit of β-glucosidase activity was expressed as the amount of enzyme required to release 1 µmole of pNP per minute under the assay condition.

7. Effect of pH and temperature on enzyme activity The optimum pH of enzyme was measured at various pH(s) under the standard assay conditions as described above. The reaction pH was adjusted with 50 mM various buffers such as acetate buffer (pH 3.0 to 6.0), phosphate buffer (pH 6.0-7.0) and Tris-HCl buffer (pH 7.0 to 9.0). The optimum temperature for β-glucosidase activity was determined by incubating the enzyme at different temperatures (20–80°C).

RESULTS AND DISCUSSION 1. Screening and Isolation a newly extremely halophilic anaerobic bacterium Total 56 soil samples were taken from several places especially of salt evaporation ponds in Samut Sakhon province, Thailand. The halophiles anaerobic bacterium has been reported that can be isolated from different marine and hypersaline environments such as Sehline Sebkha in Tunisia, Great , and Retba Lake in Senegal (Abdeljabbaret al., 2013). Hence, the present study was carried out to screen and isolated halophilic anaerobic bacteria from the various evaporation ponds. The result showed that among of soil samples, 6 (11%) could be grow in 30% of NaCl (data not shown). Six

91 สาขาวิทยาศาสตร์และพันธุวิศวกรรม การประชุมทางวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 56 enrichment cultures were done single colony isolation. Subsequently, 200 isolates were performed from 6 enrichment cultures. Interestingly, a candidate given namely SP3-14 showed the highest and quickly growth on the BM broth in 30% NaCl. Thus, this isolate was characterized in this study.

2. Phenotypic and biochemical characteristics Strain SP3-14 was anaerobic, Gram-negative, non-motile, rod shaped (0.6 x1-3 μm) bacterium, and the endospore were observed. Colonies on BM agar in Hungate tube were glowing slightly white, 1.0-2.0 mm in diameter. Growth was inhibited without NaCl. It needs at least 10% NaCl for growth. The strain SP3-14 utilized glucose, galactose, lactose, maltose, sucrose, xylose and ethanol. Arabinose, fructose and raffinose could not utilized. Optimal growth was obtained at 37oC, and no growth occurred at 60oC. Moreover, the isolate strain SP3-14 might be differentiated from the other related Halanaerobium species by means of some phenotypic characteristics (Table 1) such as the strain SP3- 14 was able to growth at 55oC, pH 5.0, while related type could not growth. The SP3-14 could not use fructose as carbon source, but other related Halanaerobium species can be utilization. Besides, the isolates grew in the salt concentration range from 10-30% NaCl, with an optimum 20% NaCl. The result indicated that this isolate is extremely halophile according to classification by Joo and Kim, 2005.

Table 1 Differentiation of strain SP3-14 from related members in genus Halanaerobium. Strains: 1, SP3-14; 2, H. praevalens DSM 2228; 3,H. alcaliphilum GSLS (DSM 8275) Characteristic 1* 2$ 3$ Morphology Rods Rods Rods Cell size (μm) 0.6 x 1 -3 0.5 -1 x 2 -4 0.5 x 2 - 3 Gram stain reaction Negative Negative Negative Motility Negative Negative Positive NaCl concentration range (%) 10-30 4-24 7.5-34 Optimum NaCl (%) 20 10 18-20 Temperature range (oC) 25-55 20-45 20-50 Optimum Temperature(oC) 37 42 40 pH range 5.5-10 6.3-8.5 ND Optimum pH 7.0 7.0 7.0 Isolation source Salt evaporation Oil well Retba Lake ND = not determined * Data from this study; $Data from Hadi et al., 2013

3. Phylogenic analysis

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Phylogenetic analysis of the nucleotide sequence of 16S rRNA gene revealed that strain SP3-14 belonged to genus Halanaerobium (Figure 1). Trees conducted by the neighbor-joining methods. The strain SP3-14 showed high phylogenetic relatedness with H. praevalens DSM 2228 (99.6% similarity), H. alcaliphilum GSLS (98.2% similarity). Therefore, the isolate is H. praevalens strain SP3-14.

4. Characterization of β-glucosidase β-glucosidase is an important key of cellulolytic enzyme system for biomass degradation and has been in several reports. H. praevalens DSM 2228 was reported in 1983 (Zeikus et al., 1983), after that complete genome sequence of the extremely halophilic H. praevalens was done and reported (Ivanova et al., 2011). Surprisingly, no have report about glycoside hydrolase enzyme especially β- glucosidase from H. praevalens. To make certain of this assumption, whole genome sequencing of H. praevalens was investigated. It revealed that some genes in the genome are predicted involve with glycoside hydrolase function. Therefore, β-glucosidase of the isolate SP3-14 was considered and characterized. The β-glucosidase of strain SP3-14 was intracellular enzyme. The enzyme from strain SP3-14 presented its optimum activity at pH 4.0 (Figure 2A). The optimum temperature was found to be 70oC, although activity was exhibited across a board temperature range from 40 to 80oC with more than 70% relative activity of the optimum value (Figure 2B).

Figure 1 Phylogenetic relationship of H. praevalens strain SP3-14, with other member in genus Halanaerobium. The phylogenetic tree is based on the comparison of nearly complete 16S rRNA gene sequences (1500bp) and was conducted using the neighbor-joining method.

93 สาขาวิทยาศาสตร์และพันธุวิศวกรรม การประชุมทางวิชาการของมหาวิทยาลัยเกษตรศาสตร์ ครั้งที่ 56

100 (A) (B) 90 100 80 90 80 70 70 60 60 50 50 40 40 30 30 20 20 Relative Activity Activity (%)Relative 10 Activity (%)Relative 10 0 0 30 40 50 60 70 80 90 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Temperature (oC) pH

Figure 2 Optimum pH (A) and temperature (B) of β-glucosidase from H. praevalens strain SP3-14.

CONCLUSION Extremely halophilic anaerobic bacterium, strain SP3-14 was successful isolated from saltern ponds. The isolated strain SP3-14 was identified as H. pravalens. Cells are rod shaped (0.6x1-3 µm), Gram-negative and non-motility. Growth occurs between 25 to 55oC, with the optimum 37oC and no growth at 60oC. The pH range for growth is 5.5 to 10.0, with the optimum growth at 7.0. The isolated strain no growth in the medium without NaCl. H. praevalens strain SP3-14 produced intracellular thermostable β-glucosidase. In addition, this is the first report of the β-glucosidase from H. praevalens specie. ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support provided by King Mongkut’s university of Technology Thonburi through the “KMUTT 55th anniversary Commenmorative Fund”, and Japan International Research Center for Agricultural Sciences.

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