Phylogenetic Analysis of Thermophilic Bacteria

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We report the community of thermophilic bacteria cultivated from Tanjung Sakti Hot Spring in South Sumatera Indonesia that has temperature 80 – 91 0C and pH 7 – 8. Based on phylogenetic analysis, the 16 sequences of 16S rRNA gene fragments obtained from the community clustered within four distinct genera as Anoxybacillus, Geobacillus, Brevibacillus, and Bacillus. Two sequences that have 96% similarity with data sequences in GenBank, are potentially as novel species/sub species.

Hot spring is a unique area that characterized by high temperature and has a great diversity of natural environments. Understanding of thermophilic microbial diversity has opened up a lot of information about microbial interactions with the environment. The
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Colonies formed was purified again with inscribed repeated several times in an agar medium.
DNA extraction from microbial culture
Microbial chromosomal DNA was isolated using Protein Purification Kit (Promega). Pellet cells derived from microbial cultures were resuspended in 300 mL cell lysis solution, incubated at room temperature for 10 minutes, then added 300 mL nuclei lysis solution and 2 mL RNase and incubated at 37 ° C for 15 min. Next, add 300 mL of protein precipitation, and centrifuged 13,000 rpm for 10 min at 20 ° C. To the filtrate was added 300 mL of chloroform: isoamilalkohol (24:1), vortex and centrifuged at 16,000 rpm for 30 seconds, the top layer is taken. The treatment is done 2 times. Subsequently, 0.6 volume of isopropanol was added and incubated at room temperature for 60 minutes, centrifuged at 13,000 rpm for 15 minutes. DNA pellet formed was washed with 70% ethanol and then dried. DNA pellet is then dissolved in 50 mL ddH2O.
16S rRNA gene amplification
16S rRNA gene fragment was amplified by PCR using primers 27F and 1492R (Baker, 2003, Frank, 2008). Amplification was performed by 30 cycles (denaturation 95 ° C for 30 s, annealing 55 ° C for 60 s, chain extension 72 ° C for 90 seconds) with an initial denaturation 95 ° C for 3 min and a final chain extension of 72 ° C for 5 min . Taq DNA polymerase enzyme (GoTaq Green Master Mix from Promega) was used for the amplification reaction according to standard usage.

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