Biodiversity and Phylogenetic Analysis of Halophilic Bacteria from Karak Salt Mines

Abstract

Halophiles are extremophile organisms that thrive in environments with very high concentrations of salt. Because of the distinctive physiological structure, hereditary property and metabolic mechanism of halophilic microorganisms; they have fascinated widespread attention of scholars at home and abroad. They are a kind of microbiotic resource with value of academic research and utilization. Our country has large salt deposits, which provide suitable condition for the isolation and classification of halophilic microorganisms. In this study; some saline samples were collected from salt mines of Karak, Khyber Pakhtunkhwa Province. The salt mines of Karak region, is an extremely saline environment and its microbial communities have not yet been explored. Halophilic bacterial strains were divided into three groups on the basis of NaCl concentration; first was slightly halotolerant / halophilic; second, moderately halophilic and third, extreme halophilic bacteria. This thesis reports isolation, identification and characterization of Halophlic/Halotolerant bacteria, especially this represents the first report, to our knowledge, of the novel species of bacteria that requires NaCl for its growth and that can tolerate more than 15%. Forty one slightly, twenty moderately and seventeen extremely halophilic bacteria isolated by enrichment culture, were collected from rock salt, plant root soil and stream water samples. These strains can grow in media with 0-40% NaCl concentrations. Morphological, physiological and biochemical characteristics of these strains were studied by optimizing their growth conditions such as pH range 5-10, NaCl range 0-40 % Biodiversity and Phylogenetic Analysis of Halophilic Bacteria from Karak Salt Mines ix and temperature rang 4-50 0C. A high microbial density was observed at low NaCl concentration. The phylogenetic analysis inferred from 16S rRNA gene sequence of these strains demonstrated that these are closely related to species belonging to different genera: Thallossobacillus, Halobacillus, Halomonas, Brevibacterium, Terribacillus, Jeotgalicoccus, Oceanobacillus, Staphylococcus, Pseudomonas, Escherichia, Gracilibacillus, Kocuria, Virgibacillus, Salinivibrio, Salinicoccus, Planococcus and Bacillus. NCCP-58, NCCP-62, NCCP-71, NCCP-93, NCCP-168, NCCP-176, NCCP-701 and NCCP-705 are candidate novel strains in this study. Based upon phylogenetic analyses, DNA-DNA homology, phenotypic and chemotaxonomic data, the isolated strain NCCP-168 belong to the genus; Bacillus with the highest similarity to Bacillus seohaeanensis BH724T (AY667495; 97.1%), and less than 97% similarity with other closely related taxa (95.6% with B. subtilis subsp. subtilis NCIB3610T; ABQL01000001), has been characterized as Bacillus pakistanensis sp. nov. A moderately halotolerant strain of bacteria (NCCP-168T) was isolated from salt mines. Cells of strain NCCP-168T can grow at 10-40○C (optimum at 30-35○C), in a pH range of 5.0-9.0 (optimum at pH 8.0) and in 0-17% (w/v) NaCl on agar medium. DNA–DNA relatedness between strain NCCP-168T and the type strains of the closely related species was less than 30 %. Chemotaxonomic data [major menaquinone, MK-7; cell wall peptidoglycan type, A1γ (meso-diaminopimelic acid); major fatty acids, iso-C15:0 (29.9%), anteiso-C15:0 (29.3%), iso-C16:0 (11.4%), iso-C14:0 (8.9%) and anteiso-C17:0 (7.0%); major polar lipids, diphosphatidylglycerol, phosphatidylglycerol and Biodiversity and Phylogenetic Analysis of Halophilic Bacteria from Karak Salt Mines x phosphatidylethanolamine] supported the affiliation of strain NCCP-168T with genus Bacillus. Therefore, on the basis of phenotypic, chemotaxonomic and phylogenetic data, strain NCCP-168T distinguished from the closely related taxa and thus represents a novel species in the genus Bacillus, for which the name Bacillus pakistanensis sp. nov. is proposed, with the type strain NCCP-168T (=JCM 18975T = KCTC 13786 T = DSM 24834T). This study showed that the salt mine of Karak are rich in halotolerant / halophilic bacterial population with diverse bacterial communities, which may be utilized in various industrial applications in future.