International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 4(6), 7-10, June (2015) Int. Res. J. Environment Sci. International Science Congress Association 7 Isolation and Characterization of Halotolerant Bacillus sp with extra Cellular A-Amylase production Potential from Sambhar salt lake, India Archana Gaur Department of Zoology, J.N.V.U., Jodhpur, Rajsthan, INDIA Available online at: www.isca.in, www.isca.me Received 23rd November 2014, revised 8th June 2015, accepted 15th June 2015 Abstract Sambhar lake (27 58 N, 75 55 E) is a halo-alkaline playa with water density varying from 3-27 Be (Baume). The lake was explored for the diversity of Halophilic microbes. The study led to the isolation of a Moderate halophilic bacterial strain from brine samples of the lake. The isolate showed a growth range of 3-15% NaCl in alkaline medium and was designated as SL-3. The isolate was aerobic, gram +ve, spore forming, rod-shaped and motile. It also tested positive for catalase and oxidase. The isolate was tested for exo-enzyme production and found positive for -Amylase and negative for lipase and protease production. The Isolates was not able to utilize citrate as a sole carbon source and failed to produce HS in TSI- medium. Carbohydrate fermentation revealed utilization of glucose, fructose and sucrose. Taxonomic Identification of isolate was confirmed by partial 16s rRNA gene sequence analysis that contained 1197 nucleotides. The phylogenetic study revealed that the isolate matches 100% with Bacillus licheniformis strain PS-6 . Therefore the isolated bacterial strain belongs to phylum Firmicutes with low G+C contents and Genus Bacillus. Gene Bank Accession No. for the isolate is KP172152. Keywords: Bacillus, phylogenetic study, sambhar lake, halophiles, -amylase. Introduction Moderately halophilic bacteria growing optimally in media containing 3–15% (w/v) salts are widely distributed in hypersaline habitats. These organisms are of considerable interest owning to their biotechnological potential in the production of compatible solutes and hydrolytic enzymes. According to Kushner though they are less exciting at first glance than the extreme halophiles, however can be more useful as they own the ability to grow over wide ranges of solute concentrations. Halophilic studies in Indian context are limited to the marine salterns near Bhavnagar, Lonar Lake and Peninsular coast. These studies are mainly focused on aerobic red halophilic archaea. Sambhar salt lake, the largest inland lake of India, has also been explored mainly for halophilic archeal species6,7. The aerobic eubacteria always remained understudied. All the Aerobic, spore-forming, moderately halophilic, Gram- positive rods shaped bacteria were originally assigned to the genus Bacillus, but with the advent of molecular methods and improved phenotypic approaches, they have been reclassified as new genera9-11. Further molecular analyses of 16S rRNA gene sequences12,13 led to assigning large number of species and six phylogenetically distinct groups to the genus Bacillus. Recently the studies on Bacillus gained momentum as they are attracting interest because of their great biotechnological potential. In the process of studying moderate halophiles of Sambhar salt lake, a bacterial strain of Genus Bacillus was isolated and subjected to taxonomic characterization. Sambhar lake ecosystem is still under explored in regard of microbial studies and microbial studies with molecular approach need to be encouraged. Material and Methods Soil and water samples were collected from Jhapok dam area of Sambhar Salt Lake (Rajasthan, 26°58N75°05E) in sterile containers. In order to enrich moderate halophiles Nutrient broth supplemented with 3-15% NaCl was employed. 10 ml sample was added to 250 ml broth and incubated for 5-7 days at 35C. 1 ml of enriched browth was spread cultured on Nutrient Agar with 3-15% NaCl. Bacterial colonies appearing after 7 days of incubation were re-streaked on fresh medium. Halophilic colonies were purified by repeated streaking on complete Salt Nutrient agar. The isolates were stored at 4 ºC and sub-cultured at 15 days intervals. Amylolytic activity of the isolates was screened by plate assay on starch nutrient agar plates containing gL-1: starch 10.0; peptone 5.0; yeast extract 3.0; agar 30.0; NaCl 100.0. The pH tested was from 7.0 to 10.0. After incubation at 30 ºC for 72h, the zone of clearance was determined by flooding the plates with iodine solution. Results and Discussion Results of Microbial and Molecular study: Sambhar Lake is the International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(6), 7-10, June (2015) Int. Res. J. Environment Sci. International Science Congress Association 8 largest single salt source in Rajasthan State. The initial source of brine in the lake is catchment water received by downpour annually, mostly during April to September every year14. Water is the most vital abiotic component of the lake ecosystem15. The total 348 mm rainfall was recorded in year 2013-14. Water temperature and Density fluctuated between 7-37C and 3-27 Be (Baume). The water in Lake Basin turns into brine due to percolation and evaporation. The brine contains high salt concentration even up to saturation level. In nature, salinity is often associated with alkalinity. Alkaline saline lakes represent a unique habitat with a high pH and a variable salt concentration. Sambhar Lake exemplifies a halo-alkaline lake as the salinity and pH ranged from 0.53 to 162.21 ‰ and 6.54 to 8.87 respectively. Hypersaline environments represents typical extreme habitats, where high salt concentration, alkalinity, and low oxygen stress may limit their biodiversity15, yet inhabitance of microorganisms in diverse hypersaline environments such as salt lakes16,17 solar salterns18 , salt mines19 and brine wells20 have been reported. The Sambhar lake brine also seemed lifeless; however in the effort to assess Halophilic microbial diversity from lake brine, strain belongings to 7 different genera were isolated. Out of these genera bacteria of genus Bacillus were the most frequent isolates. These isolates were capable of growing on 3-15% NaCl. One such isolate was further tested for biochemical and taxonomic characterization. Morphological and biochemical observations: The reported isolate showed yellowish, convex colonies with entire margins and glossy surface. It grew over a range of 3-15% salt with optimum growth at 10%. The isolate tested catalase and oxidase positive. Optimum temperature varied between 30- 35C.It gave negative results for HS production and Citrate utilization. The bacterial isolate showed motility and appeared as Gram +ve, rods. Various sugar fermentation tests were carried out in media containing 10% NaCl. The isolate could utilize only glucose, fructose and lactose out of the 10 types of sugar media. Nitrate reduction test gave positive results with the isolate. Plate Assay for Exo-enzymes production: The moderate halophilic isolate was screened for the characteristic halophilic property of exo-enzyme secretion. The bacterial strain was unable to produce lipase and protease. However strong amylolytic activity was shown at 10% NaCl. Taxonomic Characterization: DNA isolation by phenol chloroform Extraction Method: The isolate suspected to be Bacillus was subjected to DNA isolation by phenol-chloroform extraction method. It is a liquid-liquid extraction method that separates mixtures of molecules based on the differential solubility’s of the individual molecules in two different immiscible liquids. Amplification of 16s ribosomal genes: The isolated DNA templates were subjected to 16s r-DNA amplification, using the primers 18F and 1492R as described by Weisburg et al21. Optimized PCR reaction and thermal cycling conditions used for the primers, described by Weisburg et al21 Phylogenetic Identification: The identification of phylogenetic neighbor was initially carried out by the BLAST and mega BLAST programmes. BLAST results of 1197 nucleotides showed 100% similarity with Bacillus licheniformis strain PS-6 . Nucleotide sequence accession number: The 16s rDNA sequence was submitted in Gene Bank and was allotted Accession No. KP172152. Discussion: The above mentioned results showed that the isolated bacterial strain is a members of Genus Bacillus and is moderate halophilic in nature as it showed growth in media with 3-15% salt concentration. Genus Bacillusis comprised of Gram-positive, rod-shaped (bacillus), bacterial species that have the ability to be aerobic or anaerobic. The Genus is a member of phylum Firmicutes that includes low-G+C group. A number of moderately halophilic Bacillus species have been isolated from hypersaline environments22-26. Rahman (2005)14 reported that Sambhar brine mainly contains Bacillus and Halobacterium, and reported Bacillus subtilis, B. amyloliquefaciens, B. sphaericus, B. licheniformis from the lake. The occurrence of Bacillus licheniformis in the lake was reported to 78% by Rahman14. Banat27 reported organic hydrocarbon degrading potential in B. licheniformis. Sambhar lake is a haloalkaline lake and brine samples always showed high pH. Bacillus licheniformis prefers alkaline growth range28. Since the isolated bacillus strain was capable of growing in twin extremities of pH and salinity, it can be categorized as halo-alkaliphilic. Survival strategies, robust biocatalysts and unique metabolic capabilities of these organisms can be a great source of novel commercial applications. Members of the order Bacillales shows great metabolic versatility and ability to grow under physico-chemical extremes, which make them survive in almost every environment on earth from the stratosphere30 to the deep subsurface 31,. This ubiquity is, in part, attributed to their ability to form resilient spores that can be transported over long distances29. Sahoo reported heavy metal tolerance in B. licheniformis32. The Bacillus strain under the study showed amylolytic potential by producing extra-cellular amylase, while it tested negative for protease and lipase production. However Bacillus subtilis growing optimally at 10% NaCl, showing protease activity has been isolated from Sambhar lake31. Bacterial enzymes are the ones that are having wide variety of industrial applications. Among these enzymes, a-amylase has high demand 32. Amylases from Bacillus species have potential worthwhile applications in conversion of starch to sugar syrup, production International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 4(6), 7-10, June (2015) Int. Res. J. Environment Sci. International Science Congress Association 9 of cyclodextrin33 and in other industries such as pharmaceutical, textile etc; therefore, these enzymes account for about 30% of the world’s enzyme production34. Bacillus diposauri, Halobacillus sp, Haloferax mediterranei, Halothermothrix sp are known to be good producer of a amylase. 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