African Journal of Microbiology Research Vol. 7(3), pp. 164-173, 15 January, 2013 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR12.1004 ISSN 1996 0808 ©2013 Academic Journals Full Length Research Paper Isolation, characterization and antimicrobial activity of halophilic bacteria in foreshore soils Aarzoo Irshad1, Irshad Ahmad2* and Seung Bum Kim1 1Department of Microbiology and Molecular Biology, Chungnam National University, 220 Gung-dong, Yuseong, Daejeon 305-764, Korea. 2Chemical Resources Laboratory, Bio-Resources Division, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Tokyo, Japan. Accepted 8 January, 2013 Halophilic bacteria are commonly found in natural environments containing significant concentration of NaCl. Two hundred halophilic bacteria were isolated. Among which, 63 strains were tested for the antimicrobial activity against 10 pathogenic bacteria, three yeast and nine pathogenic fungi. Of these isolates, only 12 strains showed activity against the test microorganisms, namely Pseudomonas aeruginosa, Serratia marcescens, Enterobacter cloacae, Staphylococcus aureus, Salmonella typhi, Klebsiella pneumoniae, Micrococcus luteus, Escherichia coli, Bacillus subtilis, Candida albicans, Colletotrichum gloeosporioides, Fusarium solani and Alternaria alternata. These 12 active isolates were identified to be of the genera, Streptomyces (seven strains), Microbacterium (one strain), Micrococcus (one strain), Bacillus (one strain), Planococcus (one strain) and Marinobacter (one strain). Seven strains were active against pathogenic bacteria and five strains against pathogenic fungi. Two strains SC-2 and SC-13 were found to have a broad spectrum of activity against all tested pathogenic bacteria and yeasts but not against pathogenic fungi. The strains SC-2 and SC-13 were closely related to Microbacterium oxydans (99%) and Streptomyces fradiae (99%) respectively. These findings show that the foreshore soil of Daecheon Beach and Saemangeum Sea of Korea represents an untapped source of bacterial biodiversity, and also that most actinobacterial isolates are capable of antibacterial and antifungal metabolite production. Key words: Culturable diversity, halophilic bacteria, foreshore soil. INTRODUCTION Halophilic bacteria are adapted to high osmolarity and and Nagata, 2000; Carrasco et al., 2006; Yuan et al., can grow in high saline environments. These bacteria are 2007; Fukushima et al., 2007). The ecological charac- categorized as slightly, moderately and extremely halo- teristics (diversity, distribution and composition) of philes, according to the extent of their halotolerant cha- halophic bacteria in saline and hypersaline habitats have racteristics (Larsen, 1986; Ventosa et al., 1998, Ventosa, been extensively investigated (Jiang et al., 2007; 2006). These bacteria were isolated from saline environ- Mancinelli, 2005; Oren, 2002a, b). Halophilic bacteria are ments such as ocean, salt lakes and salt fields (Mimura able to grow at higher saline environments because they synthesize compatible solutes in cells or possess the transporters that take them up in the medium. Such osmoregulatory solutes as potassium ion, glutamate, *Corresponding author. E-mail: [email protected]. Tel: proline, ectoine, betaine in bacteria have been reported +81-45-924-5274. Fax: +81-45-924-5274. (Galinski, 1993). In foreshore soils, the microorganisms Irshad et al. 165 Table 1. Samples used in this study. Province, samples were collected from the foreshore soil of Saemangeum Sea (Table 1). The samples were stored at 4°C until Code Sample Site they reached the laboratory. A Foreshore soil Deacheon, Chungnam B Foreshore soil Saemangeum, Jeonbuk Media and isolation of halophilic bacteria The procedure of heat treatment used was previously described by are adapted to live in extreme saline environ- Seong et al. (1993). For the isolation, 10 ml of sterile 1/4 strength ment. Foreshore soil is enriched with salts and nutrients Ringer’s solution (2.25 g sodium chloride, 0.105 g potassium chloride, 0.12 g calcium chloride and 0.05 g sodium bicarbonate in which provides a conducive environment for the growth of 1 L distilled water) was added to 1 g soil sample. The tenfold halophilic microorganisms. Therefore, in this study, sam- diluents were shaken on reciprocal shaker for 20 min and then ples were collected from the foreshore soil in order to incubated at 50°C for 15 min. All media contained Difco Bacto agar determine the halophilic bacterial diversity associated (18 g l -1) at pH 7.0. Aliquots (0.2 ml) of each dilution were spread with marine environment. on media plates using 6 selected media (Table 2) and were Halophilic bacteria provide a high potential for bio- incubated at 30°C for two to four weeks. Distinct colonies were selected and purified by repeated streaking on the same media. technological applications for at least two reasons: (1) The purified isolates were stored in 20% glycerol at -80°C. their activities in natural environments with regard to their participation in biogeochemical processes of C, N, S and P, the formation and dissolution of carbonates, the Cultivation of strains immobilization of phosphate and the production of growth Sixty three (63) strains were selected using the morphological factors and nutrients (Rodriguez-Valer, 1993); and (2) differences based on visible examination of the growth their nutritional requirements are simple. Majority of them characteristics (aerial mycelium, substrate mycelium and diffusible can use a broad range of compounds as their sole figments). For DNA extraction, the selected strains were cultured in carbon and energy source. Most of them can grow at Bennett’s Broth and M1-M4 (Broth) media for 7 days at 30°C. high salt concentrations, minimizing the risk of conta- mination (Birbir and Ilgaz, 1996). Moreover, several ge- DNA extraction netic tools developed for the nonhalophilic bacteria can be applied to the halophiles, and hence their genetic ma- Total DNA was extracted from one week-old cultures using the nipulation seems feasible (Ventosa et al., 1998). Halo- method described previously (Cho et al., 2006). Cells were taken philic bacteria are used for the production of compatible into Eppendorf tube containing 200 μl of STES buffer (500 mM solutes, hydrolytic enzymes, exopolysaccharides, osmo- NaCl, 200 mM Tris-HCl (pH 7.6), 10 mM EDTA, 1% SDS) and glass protectants, stabilizers of enzymes and cells (Margesin beads. The mixture was vortexed for 15 min by TOMY micro tube mixer (TOMY, Japan). The suspension was supplemented with 100 and Schinner, 2001; Lee et al., 2003; Birbir and Ilgaz, μl lysozyme (50 mg/ml) and incubated for 2 h at 37°C, then lysed 1996; Onishi, 1970; Onishi et al., 1983, 1991; Oren, completely by adding 100 μl TE buffer (pH 8.0). An equal volume of 1999). Some of them are used for the degradation of po- phenol/chloroform/isoamyl-alcohol (24:25:1, v/v) was added to the lluting industrial residues or toxic chemicals and for en- supernatant and centrifuged at 5,000 rpm for 15 min. After hancing oil-recovery processes (Ventosa and Nieto, repetition of the above step, it was treated with 3 μl of RNase A (10 1995; Birbir and Ilgaz, 1996). mg/ml). Total DNA in the supernatant was precipitated by adding 0.6 volume of isopropanol, collected by centrifugation and the In this study, the halophilic bacteria diversity of precipitate was washed twice with 70% aqueous ethanol, then dried foreshore soil (Daecheon Beach, Chungnam Province in air for 20 min and dissolved in 50 μl distilled water for 1 h at and Saemangeum Sea, Jeonbuk Province in Korea) and 65°C. The size and amount of extracted DNA were determined by antimicrobial activity of halophilic bacterial isolates were Agarose gel (1%). Extracted DNA was stored at -20°C until needed. examined. The aim of this study was to evaluate halophilic PCR amplification and sequencing of 16S rDNA actinobacterial diversity in foreshore soil and to test the isolates for antagonistic activity. To our knowledge, this is PCR amplification of 16S rDNA was carried out using two universal the first extensive report on halophilic actinobacteria primers, 27f (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492r isolation, exhibiting antimicrobial activity from foreshore (5′-GGY TAC CTT GTT ACG ACT T-3′), which were also used for soil, Korea. sequencing. The PCR consisted of an initial denaturation step at 95°C for 3 min, which was followed by 30 cycles of 95°C for 1 min, 55°C for 40 min and 72°C for 1 min. Sequencing was performed MATERIALS AND METHODS using the service of Solgent Co. (Korea). Collection of samples Phylogenetic analysis The sampling sites are located in Chungnam and Jeonbuk Provinces in Korea. In Chungnam Province, the sample was The 16S rDNA sequences of the isolates were compared with the collected from the foreshore soil of Daecheon Beach. In Jeonbuk sequences available by the BLAST search in the NCBI, GenBank 166 Afr. J. Microbiol. Res. Table 2. Composition of six different media. Media Composition Reference M1 6 mL 100% glycerol, 1 g arginine, 1 g K2HPO4, 0.5 g MgSO4, 18 g agar, and 1 L of natural seawater (Mincer et al., 2002) M2 0.1 g L-asparagine, 0.5 g K2HPO4, 0.001 g FeSO4, 0.1 g MgSO4, 2 g peptone, 4 g sodium propionate, 20 (Zhang et al., 2006) g NaCl, 18 g agar, and 1 L of water M3 2 g peptone, 0.1 g asparagine, 4 g sodium propionate, 0.5 g K2HPO4, 0.1 g MgSO4, 0.001 g FeSO4, 5 g (Webster et al., 2001) glycerol, 20 g NaCl, 18 g agar, and 1 L of water M4 4 g yeast extract, 15 g soluble starch, 1 g K2HPO4, 0.5 g MgSO4, 20 g NaCl, 18 g agar, and 1 L of water (Webster et al., 2001) SCA 10 g soluble starch, 0.3 g vitamin-free casein, 2.0 g potassium nitrate, 2.0 g NaCl, 0.05 g MgSO4, 0.02 g This study CaCO3, 0.01 g ferric sulphate, 5.45 g potassium phosphate, 18 g agar and 1 L of water R2A 0.5 g yeast extract, 0.5 g proteose peptone, 0.5 g casamino acids, 0.5 g dextose, 0.5 g soluble starch, 0.3 This study g sodium pyruvate, 0.3 g dipotassium phosphate, 0.05 g magnesium sulphate, 15.0 g agar and 1 L of water database (http://www.ncbi.nlm.nih.gov).