Isolation and Characterization of Halophilic Bacteria from Sundarban Soil
Halophiles, the most predominant organisms found in the mangrove forest, include varied genera of halophilic bacteria in different environment such as salt lakes, saline soils and salted food. The majority of halophilic microorganisms studied so far produce compounds with great potential in industrial process and they have physiological properties which facilitate its use with commercial aims. In this study of Sunderban soil, focus has been made on the isolation of halophilic organisms and their characterisation. Soils were collected from four different places of Sunderban and were used for physiochemical and microbiological analysis. Qualitative screenings of the isolates were done and three among them was selected having moderately good growth which was further optimised in different growth media. Characterisation of the isolates were done and based on 16S rRNA gene sequencing phylogenetic tree were constructed. The isolates showed multiple heavy metal tolerance and antibiotic resistance. The organisms were further tested for EPS as well as various extracellular enzyme productions thereby exploring their usage for various biotechnological purposes. Key wordsSundarban, Halophiles, EPS, NaCl IJLSSR INTRODUCTION Mangroves are highly productive marine ecosystem where bacteria actively take part in bio mineralization and biotransformation of minerals [1] Halophilic and halo tolerant microorganisms are able to thrive and grow in saline and hypersaline environments [2] These microorganisms are being the object of basic studies in relation to the origin of life in our planet and the molecular mechanisms of adaptation to saline and hyper saline conditions. Mangroves are typically tropical fragile coastal ecosystems of inter tidal zones of river deltas and back water areas. They are mostly moderately saline and fragile ecosystems. In spite of that they are highly productive and biologically diversified habitats. Because of richness in minerals and other nutrient content, the mangrove ecosystem harbours diverse microbial communities which can adapt to the saline condition of this ecosystem. The bacterial communities in saline environment would be halophilic and halotolerant bacteria of various functionalities i.e sulphur oxidisation, phosphate solubilization, cellulose degradation, antibiotic and enzymes production etc [3] Access this article online Quick Response Code: Website: www.ijlssr.com DOI: 10.21276/ijlssr.2016.2.4.19 Many of these microbes possess unique capability to tolerate the hyper saline condition as well as various heavy metals and metalloids. Heavy metals are increasingly found in microbial habitats due to natural and industrial processes, for which microbes have evolved several mechanisms to tolerate the presence of heavy metals. Due to their high stress tolerance capacity these micro organisms are very useful for biotechnological applications in terms of bioremediation and biomineralization. Therefore, there is a need to study the micro organisms of these soils. Bacterial growth is often accompanied by the production of extracellular polymeric substance (EPS) which have important ecological and physiological functions. Increasing interest is being generated in the study of these molecules because of their wide applications in food, pharmaceutical, petroleum and other industries. In the present study, diverse bacterial genera having different degree of sodium chloride tolerance were isolated from the soils collected from Sunderban and were characterised in details. Attempts have been made to delineate their properties for biotechnological purposes. MATERIALS AND METHODS Study area of Sundarban delta Sundarban Mangrove forest that is located geographically in between 21deg31 N and 22deg30 N and longitude 88deg10 E and 89deg51 E along the North East coast of Bay of Bengal, India. Four soil samples collected during June December month of 2014 by Microbial biodiversity and Environmental toxicology laboratory, department of Research Article (Open access) Int. J. Life. Sci. Scienti. Res. VOL 2, ISSUE 4 http:/ijlssr.com IJLSSR (c) 2015 All rights are reserved Page 443 Microbiology from predominant area namely Sudhanyakhali, Sajnekhali, Burirdabri, and Dobanki. Soil sample collection Four soil samples were collected from four different places namely, Sudhanyakhali, Sajnekhali, Burirdabri, and Dobanki, of the Sunderbans aseptically at a depth of 10 cm from top layer using a hand held soil borer. The samples were collected in sterilized polythene containers and transported to the laboratory. Chemicals used Nutrient Broth (NB) Luria Broth (LB) Luria agar (LA) Starch agar, KCl, CdCl2, MnCl2, K2Cr2O7, CuCl2, CoCl2.6H2O, NiCl2.6H2O, ZnCl2, HgCl2, Pb(NO3)2, Crystal violet. All chemicals used were of analytical grade. Physical characteristics of soil The soil samples were dissolved in 1N KCl in the ratio of 1:2.5, and the mixture was allowed to shake for 1hr; the pH temperature was estimated using digital pH meter. Soil EC was determined by suspending the air dried sample in the ratio of 1:2 after shaking the mixture overnight, it was filtered and the filtrate was analysed using digital conductivity meter (304, Systronics) Isolation of bacteria One gram of soil was suspended in 9 ml sterile distilled water; diluted logarithmically up to 10 5 level. By taking 1ml soil suspension from dilution 10 1, 10 3, 10 5 pour plates were made in NaCl (5% supplemented nutrient agar and incubated at 37degC for 72 hrs. The colony forming units (CFU) were then enumerated. The selected colonies were transferred and maintained on nutrient agar slants containing NaCl (5% Qualitative screening The isolates were tested for their sodium chloride (NaCl) tolerance by growth in nutrient agar plates containing various concentration of NaCl (5% 10% 15% and 20% The NaCl incorporated plates were streaked with freshly grown culture of the isolates and incubated at 37degC for 2 5 days. Growth of the isolates was determined visually as positive or negative. Growth performance in different media The isolates showing moderate growth at 20% NaCl incorporated NA medium were tested for their performance in different types of media such as Luria Bertani Agar, S Agar, Sg Agar SLEV Agar [4] and Basal medium [5] for their optimum growth. Relative growth of selected isolates Degrees of tolerance of selected isolates were evaluated in Slev broth containing 5% 10% 15% and 20% NaCl. Growth of the isolates in Slev broth was determined by measuring the optical density at 600nm using the uninoculated broth as blank. Relative growths of the isolates were expressed as the percentage of those obtained in untreated control which was taken as 100% Characterization and identification of microorganism The potent NaCl tolerant isolates were characterized both morphologically and biochemically (standard methods) 16S rRNA sequence homology was determined for tentative identification of the organism. Genomic DNA was extracted from the culture by conventional method and 16S rRNA amplification was carried out with 8F and 1492R universal primer sequences. The capillary sequencing was done by ABi 3500 Genetic Analyzer machine as per manufacture's information (GCC Biotech Kolkata) Heavy metal tolerance Heavy metal tolerance of the isolates were determined by streaking on Luria Agar plates supplemented with varying concentrations (25, 50 100, 200 mg/ml for Cd, Mn, Cr, Cu, Co, Ni, Zn; while 5, 10, 20, 40 mg/ml for Hg and Pb) of metals. Exopolysaccharide (EPS) production Extracellular polymeric substances (EPS) produced by selected isolates were extracted from mid log phase culture (10hr old) using standard method [6] EPS was extracted from cell pellets and the amounts of polysaccharide, protein, DNA, RNA were measured by standard methods. Antibiotic sensitivity Antibiotic sensitivity of the organism was determined by using antibiotic impregnated discs on nutrient agar incorporated plates (Octadiscs G VIII plus and G I minus, Himedia) RESULTS AND DISCUSSION Physical characteristics of soil Mangroves are saline coastal ecosystem and rich in nutrient content. They harbour a large number of microbial populations of unique nature. Areas of sample collection are shown in Fig. 1. The pH and temperature of soil samples (S1, S2, S3 S4) was around 7 and 22degC respectively. Electrical conductance of sample S1 was 0.58, S2 was 0.66, S3 was 0.49, S4 was 0.68 (Table I) Int. J. Life. Sci. Scienti. Res. VOL 2, ISSUE 4 http:/ijlssr.com IJLSSR (c) 2015 All rights are reserved Page 444 Fig. 1: Map of Sunderban area showing the sample collection sites Table I: Physical and microbiological characteristics of soil samples from Sunderbans Isolation of bacteria Halophilic bacteria were isolated on nutrient agar plates supplemented with sodium chloride. The CFU/gm of soil for samples S1, S2, S3 and S4 were 18x10 3, 5x10 6, 22x10 3, 26x10 3 respectively (Table I) Screening for NaCl tolerance Halophilic bacteria have been categorized as slightly, moderately and extremely halophilic on the basis of tolerance to different concentration of NaCl (Fig. 2) Fig. 2: Qualitative screening of isolates for NaCl tolerance From the relative growth it is evident that the growth of the isolates has decreased gradually with the increase in concentration of NaCl. 40 50% relative growth was observed in medium supplemented with 15% NaCl; the growth was further reduced to around 30% at the maximum concentration of NaCl (20% used. However as the organisms under study exhibited fair growth upto 20% NaCl they are categorised as moderate halophiles and were used for detailed study (Table II) Soil Sample pH Temperature(degC) Conductance(mS) CFU/g of soil S1 7.94 22 0.58 18x10 3 S2 8.39 22 0.66 5x10 6 S3 8.27 22 0.49 22x10 3 S4 8.23 22 0.68 26x10 3 Int. J. 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