A sterilized loop was dipped into the suspension of desired organism and was streaked on the surface Alectinib of solidified agar plate. The plates were then incubated for 24–48 h to get the individual colonies. Bacteria grows on the surface nutrient agar, and is clearly visible as small colonies. Thermal soil samples were inoculated in anaerobic liquid basal medium consisting of (g/l): NH4Cl 0.5, Yeast extract 5, K2HPO4 0.25, KCl 0.002, MgCl26H2O 0.125, NH4CO3 0.4, Peptone 1, NH4H2PO4 0.4, NaH2PO4 0.5. Trace element 1 ml, vitamin solution 1 ml.20 Sucrose (10 g/l)
was used as a carbon and energy source. All the culture bottles were incubated at 70 °C for 3 days and sub cultured after 3 days of incubation. All the sub cultures and diluted cultures were incubated at 70 °C under atmospheric pressure. Cells were observed under a light microscope and pure isolate was routinely cultivated in anaerobic liquid basal medium. Morphological characteristics were investigated. Gram staining was performed to confirm the gram reaction and spore position. Motility was determined by hanging drop method.19 All isolates were evaluated by conventional tests for catalase, oxidase, indole, urease, methyl red, voges-proskauer, citrate utilization, triple sugar AZD0530 in vivo iron, starch hydrolysis, hydrogen sulphide and oxidative
fermentative carbohydrate utilization.19 Genomic DNA was extracted from the isolate using Pure Fast® Bacterial Genomic DNA isolation kit. 1 μL of genomic DNA was used as template and amplified by PCR using Master Mix Gene kit (HELINI biomolecules Chennai, India) with the aid
of 16S rDNA primers (16S Forward Primer: 5-AGAGTRTGATCMTYGCTWAC-3 16S Reverse Primer: 5-CGYTAMCTTWTTACGRCT-3) with MTMR9 the programme consisted of denaturation at 94 °C for 1 min and subsequent 35 cycles of denaturation at 94 °C for 30 sec, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min followed by final extension at 72 °C for 5 min. Amplified product was sequenced using the Dye Deoxy Terminator Cycle sequencing kit (HELINI biomolecules Chennai, India) as directed in the manufacturer’s protocol. The nucleotide sequencing of 16S rRNA gene of the isolate was compared with other related sequences using FASTA programme. Further, the nucleotide sequences of the isolate was aligned with closely related sequence using CLUSTAL W mega version-5. The hydrogen production by P. stutzeri was analysed for the synthetic sources selected i.e. starch and sucrose. In order to find the effect of starch and sucrose, these sugars were taken 7.5 g in 1500 ml, 5.0 g in 1000 ml, 3.75 g in 750 ml, 2.5 g in 500 ml. Similarly, the amount of hydrogen produced by utilizing the mango juice effluent was also studied. For this study 1500 ml, 1000 ml, 750 ml and 500 ml mango juice effluent (waste water) were used. Mango juice effluent was collected from the Maaza juice production unit located at Krishnagiri, Krishnagiri District, Tamil Nadu.