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1. Studies on isolation, characterization and optimization of soil bacteria for lipase production A DISSERTATION SUBMITTED TO MITS SCHOOLS OF BIOTECHNOLOGY UTKAL UNIVERSITY ,BHUBANESWAR IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF DEGREE OF MASTER OF SCIENCE IN APPLIED MICROBIOLOGY BY Ms. SANDHYARANI SWAIN ROLL NO.- 14754U224039 UNDER THE GUIDANCE OF DR. DIPTIPRIYA SETHI Assistant Professor DEPARTMENT OF APPLIED MICROBIOLOGY MITS SCHOOLS OF BIOTECHNOLOGY

2. Introduction   Lipase Hydrolysis Glycerol+ fatty acids Lipases are a class of enzymes (extracellular) that catalyze the hydrolysis of fats . Splits fats into fatty acids and glycerols. A subclass of esterase. Lipases perform essential roles in digestion, transport and processing of dietary lipids in most organisms. They are commonly secreted from plants, animals and microorganisms. However, microbial lipases represent the most widely used class of enzymes in biotechnology. Enzymes are biocatalysts present in all living organisms. Some enzymes are of special interest as they can be used as catalysts in various biochemical process and, hence, have a wide application.

3. Objectives Collection of oil contaminated soil sample. Isolation and biochemical characterisation of isolated microbial colonies . Processing and screening of collected soil sample for isolation of true lipase producing bacterial colonies. Optimization of culture media and physiological parameters for the screening and the isolation of best lipase producing bacterial isolates.

4. MATERIALS AND METHODS SAMPLE COLLECTION Oil contaminated soil sample have been collected from a automobile workshop, near Saheednagar , Bhubaneswar,Odisha . PRIMARY SCREENING FOR LIPASE PRODUCING BACTERIA Pure isolated bacterial cultures were screened for lipase activity using spirit blue agar medium. SECONDARY SCREENING All the selected lipase producing bacterial cultures were screened for production of lipase.

5. ISOLATION OF BACTERIA Isolation of microorganism from the oilcontaminated soil samples was carried out by serial dilution, using the spread plate method on NA plates. Fig. 1 Mixed cultures of bacteria on NA plate after Serial dilution Fig. 2 Pure cultures of bacteriaon NA plate after subculture

6. BIOCHEMICAL TEST

7. Biochemical test results

8. RESULTS AND DISCUSSION A total seven number of lipase producing bacteria were isolated using spirit blue agar medium. It was observed that different bacterial colonies were isolated from the soil sample. Colonies varied from circular,irregular margins,rod shaped as well as filamentous in shape.Different colonies were subjected to gram staining and thus cocci and rod shaped bacilli forms were identified.It was found that maximum strains when subjected to microscopic examination revealed that gram positive bacilli were predominant followed by gram positive cocci. All the bacterial isolate were negative for methyl red test except strain L7.

9. Gram staining 0f bacteria under microscope:

10. REFERENCE Gupta R, Gupta N, Rathi P (2004) Bacterial lipases: an overview of production, purification and biochemical properties. Applied Microbiology and Biotechnology 64(6): 763-781  Adetunji AI, Olaniran AO. Optimization of culture conditions for enhanced lipase production by an indigenous Bacillus aryabhattai SE3-PB using response surface methodology. Biotechnol Biotechnol Equip. 2018;32(6):1514–1526. Sharma R, Chisti Y, Banerjee UC. Production, purification, characterization, and application of lipases. Biotechnol Adv. 2001;19(8):627–662. Bharathi D, Rajalakshmi G. Microbial lipases: an overview of screening, production and purification. Biocatal Agric Biotechnol. 2019;22:101368.