0–11.0, are defined as – alkalophilic. 2 The temperature range of the organism was 25–45 °C with the optimum temperature of GSI-IX in vitro 30 °C and it could tolerate NaCl up to 10%. It was negative towards citrate utilization, indole test, MR-VP tests, H2S production, urea hydrolysis and could reduce nitrate weakly. The strain was oxidase and catalase positive, capable of hydrolyzing starch, casein and liquefaction of gelatin. Acid production from carbohydrates like glucose, fructose, lactose, sucrose, xylose, mannitol and maltose was negative. The overall biochemical and physiological characteristics

indicate that strain 2b is an alkaliphilic Bacillus belonging to the species agaradhaerens. The organism identified as B. agaradhaerens was further confirmed by Microbial Type Culture Collection Center and Gene Bank (MTCC), Institute of Microbial Technology, (IMTECH), Chandigarh, India and deposited under Accession number MTCC 9416. Many scientists have studied B. agaradhaerens. 1 Nielsen 1 has made considerable revisions of the classification of alkalophilic Bacillus species according to the phylogenetic and phenotypic characterizations and has proposed B. agaradhaerens as one out of the nine new species of alkalophilic Ku-0059436 chemical structure Bacillus. To investigate the taxonomic position of the alkaliphilic Bacillus strain, 16S rRNA gene sequence analysis was

performed. The genotypic characterization of the 16S rRNA gene sequence of the isolate confirmed that it was B. agaradhaerens.

After the Oxymatrine sequence characterization, the sequence was submitted to NCBI under the name B. agaradhaerens strain nandiniphanse5. The GenBank/EMBL/DDBJ Accession number of the sequence deposited in GenBank Database is JN703504.1. Sequences showing a relevant degree of similarity were imported into the CLUSTAL W program16 and multiple sequence alignment was performed. Alignment of 16S rRNA partial gene sequence of different strains of B. agaradhaerens species is shown in Fig. 1. Phylogenetic tree was constructed from 16S rRNA gene sequences of members of genus Bacillus. In the neighbour-joining tree, the sequences form a distinct lineage, with alkaliphilic Bacillus species as the closest relatives. Phylogenetic construction of B. agaradhaerens strain nandiniphanse5 against other species of Bacillus is shown in Fig. 2. The dataset B. agaradhaerens strain nandiniphanse5 consisted of 770 bp (100%) is parsimony informative. The matrix was competently and manually aligned. Coding gaps as binary characters, missing data had no affect on the topology and very affect on branch support. The 100% bootstrap consensus tree is shown ( Fig. 2). To characterize the B. agaradhaerens strain further, a phylogenetic tree, based on its 16S rRNA gene sequence, showing the relationships of the identified alkaliphilic bacterium B. agaradhaerens strain nandiniphanse5 and the type strains of the same species, was constructed ( Fig. 3).