POLYPHASIC ANALYSIS OF TWO THERMOTOLERANT, AND EXOZYMES PRODUCING GEOBACILLUS SPECIES FROM HOT SPRING OF NEPAL

Abstract

Background and Aim: Nepal’s geothermal areas are considered as uncharted place for microbiological and biotechnological researchers since not many researches has been conducted on these areas. The main aim of this study was to isolate thermophilic bacteria from hostspring of Nepal. Methods: From one of the hottest natural thermal spring of Nepal, Bhurung Tatopani, 20 different bacteria were isolated and Characterized by both culture dependent and independent approaches. Results: Morphological and biochemical tests along with polyphasic analysis of these thermal isolates confirmed that two of these species belongs to Geobacillus sp. The isolates THG1 and THG2 were found to be gram variable and rod shaped with smooth colony. They were found to be osmotolerant up to 2% NaCl and thermotolerant with optimum temperature of 72°C. They showed significant production of various industrially important enzymes like amylase, lipase, protease and cellulase. 16S rRNA amplification was also performed and sequenced which revealed that the isolate THG1 has 99% similarity with Geobacillus kaustophilus and THG2 has 99% similarity with Geobacillus thermoleovorans. THG1 and THG2 16S rRNA sequences are deposited in genebank with accession id KP764939 and KP764940. For the further confirmation and deeper analysis, fatty acid analysis (FAME) was also performed and their fatty acid composition profile was also created. Conclusion: From the Bhurung hotspring two unique Geobacillus species were isolated.