Molecular Characterization and Biotechnological Potential of Thermostable Alkaline Lipase-Producing Bacillus Subtilis RSMLLP01 Isolated from Lonar Crater Lake

Abstract

Lonar Crater Lake represents a unique haloalkaline ecosystem containing extremophilic microorganisms with significant industrial relevance. The present study aimed to isolate, identify, and characterize alkaline thermostable lipase-producing bacteria from Lonar Lake and evaluate their potential biotechnological applications. Water and sediment samples were subjected to alkaline enrichment and selective screening using olive oil agar medium. Four bacterial isolates were obtained, among which RSMLLP01 showed maximum lipase activity with a hydrolysis zone diameter of 28.4 ± 1.2 mm. Morphological and biochemical analyses identified the isolate as a Gram-positive rod-shaped bacterium with thermotolerant properties. Molecular identification through 16S rRNA sequencing revealed ≥99% similarity with Bacillus subtilis. Partial purification of lipase by ammonium sulfate precipitation demonstrated progressive hydrolytic activity with zone diameters increasing from 8.2 ± 0.4 mm to 24.5 ± 1.1 mm after incubation. UV-visible spectroscopic analysis showed a characteristic absorbance peak at 210 nm confirming the proteinaceous nature of the enzyme preparation. The findings indicate that Bacillus subtilis RSMLLP01 may serve as a promising source of alkaline thermostable lipase with potential applications in detergents, biodiesel production, leather processing, and wastewater treatment. However, further studies involving enzyme kinetics and process optimization are required.