Identification of cellulolytic thermophiles based on 16S rDNA gene amplification analysis

Thermophiles are microorganisms that inhabit at extremely high temperature environment. Thermoenzymes from thermophilic microorganisms are heat-stable, able to withstand denaturants of extremely acidic and alkaline conditions, and thus, have a considerable potential for many industrial applications. The aim of this study was to isolate and characterize cellulolytic thermophiles. Five isolates of cellulase producing thermophiles namely M7K1T55C3, M7K1T55C4, M7K2T55C1, M8K1T55C1 and M8K1T55C2 were isolated previously from Dusun Tua Hot Spring, Selangor, Malaysia. The pH of the hot springs within the area was recorded to be slightly alkaline with temperature ranging from 55 ˚C to 70 ˚C. Two media were used to inoculate these samples, which were general enrichment medium, and selective medium that contains carboxymethylcellulose (CMC) sodium salt as their carbon source. Colonies with white abundant growth, irregular margin and glossy smooth were obtained from both samples. These isolates were subjected to gene amplification by PCR using a pair of universal primer to obtain their 16S rDNA gene. Obtained gene was sequenced and analyzed using bioinformatics tools. The isolate of M7K2T55C1 was found to be from the genus Brevibacillus, spore-forming type, aerobic, rod shaped and capable of producing cellulase. Meanwhile, isolates M7K1T55C3, M7KIT55C4, M8K1T55C1 and M8K1T55C2 were found to be from the genus Aneurinibacillus. It is also capable of producing cellulase. Sequence alignment done through BLASTn, shows that isolate M7K1T55C1 had the highest homology with Brevibacillus bonstelensis with a 99% score. Meanwhile, isolates M7K2T55C2, M7K1T55CP, M8K1T55C3 and M8K2T55C4 shared the highest homology with Aneurinibacillus thermophilus with 99% score.