Isolation of bacteria from soil sample Sample Collection: The soil samples were collected from Shahrasti upazilla of Chandpur district. Soil, sediment and ground water in the area have been contaminated with arsenic for many years. Soil samples were collected aseptically from the sub-surface (from 0 to 15 cm in depth from ground) in sterile sample bottles and kept in 4°C until further analysis. For isolation of bacteria firstly 2 g soil sample were mixed with 50 ml autoclaved distilled water. Strain isolation: One gram of each soil sample was taken in 100 ml of 0.1 M phosphate buffer solution and mixed well by vortexing for 3 minutes. Each Sample was serially diluted with sterile saline water (0.85%) and plated on Yeast Extract Mannitol (YEM) agar medium {D-Mannitol (Qualikems, India), 10 g; K2HPO4 (Scharlau, Spain), 0.5 g; MgSO4. 7H2O (MERCK, India), 0.2 g; CaCl2 (Scharlau, Spain), 0.1 g; yeast extract (TM MEDIA, India), 0.5 g; phosphate buffer solution, up to 1000 ml, pH 7.0. The plates were then incubated at 37°C for 48 hours. Pure cultures of different bacterial strains were obtained by successive isolation of morphologically different colonies through repeated cross streaking (Bergey et al. 1984). Identification of bacterial isolates: After single colony isolation, the morphological, biochemical and molecular characteristics of the isolated bacterial strains were evaluated. Color, gram stain and cell morphology were investigated under microscope (Optika B350) (1000X magnification). Biochemical properties of the selected bacterium were tested according to Bergey’s Manual of Systemic Bacteriology (Bergey, et al., 1984). The following biochemical tests were performed: Methyl Red (MR) test, Voges-Proskaur (VP) test, catalase test, glucose, fructose, sucrose, maltose, mannitol fermentation test, indole, citrate, gelatin and nitrate reduction test (Cappucino, et al., 2001). Molecular characterization was done by 16S rRNA sequence analysis. Genomic DNA was extracted by automated DNA extractor (Model: Maxwell 16, Origin: Promega, USA) and qualified by Nanodrop Spectrophotometer (Model: ND2000, Origin: Thermo Scientific, USA). The 16S rDNA from the extracted DNA was amplified by PCR using 27F (5’- AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-ACGGCTACCTTGTTACGACTT3’) primers. The reaction mixture composed of 12.5 µl Hot Start Master Mix (Origin: Promega, USA), 1 µl extracted genomic DNA, 1 µl 37F primer (concentration 10 pMol), 1 µl 1492R primer (concentration 10 pMol), 9.5 µl nuclease free water. PCR was done by Gene Atlas (Model: G2, Origin: Astec, Japan). PCR profile was as follows: initial denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation (95°C, 30 seconds), annealing (48°C, 30 seconds), extension (72°C, 1 minute) and final extension at 72°C for 5 minutes. To confirm amplification gel electrophoresis was done using agarose, 100 bp DNA ladder, ethidium bromide, TAE buffer, all from Promega, USA. Gel documentation was done by Alpha Imager (Model: Mini, Origin: Protein Sample, USA). Sequencing was performed by ABI 3700 Genetic Analyzer, 1st Base Laboratory SdnBhd, Malaysia. The 16S rDNA gene sequence was BLAST searched against GenBank database (http://www.ncbi.nlm.nin.gov/) and phylogenetic analysis was performed using Mega6- tree explorer after multiple alignment by BioEdit software. Growth at different temperatures: In order to study the effect of temperature on bacterial growth, cultivation temperature of the selected isolate was adjusted to 30oC, 35oC, 37oC, 40oC and 47oC. After 16 hour of incubation the absorbance was measured at 660 nm. Growth at different pH levels: In order to determine the appropriate pH on bacterial growth, the pH of the medium was adjusted to different pH level (pH 4 to 10). After 16 hour of incubation the absorbance was measured at 660 nm.
Growth at different salinities: To determine the appropriate salt concentration for the growth of B. aryabhattai, LB medium with different concentration of salt (1% to 7%) having pH 7 were prepared. After 16 hour of incubation the absorbance was measured at 660 nm. Amylolytic activity assay: Bacterial colonies first grown on 1% starch plate {beef extract 3 g/L, starch 10 g/L, agar 12 g/L pH 7.5±0.2 (at 25°C)}. Amylase activity can be detected by flooding the plates of 48 hours old culture on starch agar with iodine complex. Amylase positive organisms show clear zone around colony while development of blue to purple zone indicates starch is not hydrolyzed. Size of the clear zone is directly proportional to the starch hydrolyzing activity of the strain (Islam et al., 2017). Proteolytic activity assay: Skim milk agar plates (SMA) are used for providing nutrition and for the detection of proteolytic activity of microorganisms. SMA is composed of skim milk powder (28 g/L), casein enzymichydrolysate (5 g/L), Yeast extract (2.5 g/L), dextrose (1 g/L), agar 15 (g /L) with final pH 7. The plates are inoculated with a population of microorganisms to test for the ability to digest casein hydrolysis (Rahman et al., 2018).