Isolation and characterization of bacteria for lipase production and its biotechnological application

Abstract

Lipase is one of the important enzymes can be easily obtained from bacteria and can be used in different important sectors. Water pollution is a big burning issue all over the world. Lipid is an important component of domestic wastes that causes severe environmental pollution. Wastewater with lipid often caused major problems in biological wastewater treatment process. Different microbes producing lipases are used for this wastewater remediation process. The present study was undertaken to isolate potential indigenous lipase producing bacteria from the lipid-rich environment. The isolated bacteria were tested for desired lipase production along with their biotechnological applications. The aerobic heterotrophic bacterial load of the samples ranged in between 4.6×10 3 7 and 1.37×10 3 , 1.65×10 7 and 1.25×10 3 , 1.28×10 and 7 2 1.56×10 , 8.3×10 5 and 2.64×10 cfu/g or cfu/ml in NA, PYG, TBA and LB media, respectively. The highest bacterial count (1.56×10 7 cfu/g) was observed in soil of Dairy Farm from Savar and lowest (8.3×10 2 cfu/ml) in water of The Buriganga River. The highest percentage (94.51%) was found in soil of Edible Oil Mill and the lowest (23.44%) in water of The Buriganga River.

Desired indigenous lipase producing bacteria were isolated from soil and water of lipid-rich environment. On the basis of lipase activity, 30 isolates showed better lipase activity among the total isolates. Result showed that both Gram positive and Gram negative lipase positive bacteria were found to be associated with studied samples. Among them Bacillus, Staphylococcus, Micrococcus and Planococcus were Gram positive while Acinetobacter, Acetobacter, Pseudomonas, Alcaligenes and Serratia were Gram negative bacteria found to be associated in the lipid-rich environments. Qualitatively lipase activity was measured as zone ratio on TBA medium and the zone ratio was found to be ranged in 1.55 – 4.08. Among them 10 better isolates were selected for molecular identification and quantitative lipase production. Among 10 isolates 9 were matched with their conventional identification. Conventionally identified Acetobacter liquifaciens was found to be as Stenotrophomonas maltophilia e-a22 in case of molecular identification. Among 10 isolates, Stenotrophomonas maltophilia e-a22 showed the highest lipase activity (26.89 U/ml) at 72 h of incubation before optimization. Three isolates could produce lipase more than 10 U/ml among the selected isolates. These three isolates viz. Stenotrophomonas maltophilia e-a22, Pseudomonas aeruginosa 12 and Bacillus subtilis 20B were optimized for lipase production. Before optimization these isolates could produce enzyme 26.89, 10.89 and 13.50 U/ml, respectively. Through optimization, enzyme production increased up to 136.47, 96.33 and 84.30 U/ml by the Stenotrophomonas maltophilia e-a22, Pseudomonas aeruginosa 12 and Bacillus subtilis 20B, respectively. Among the indigenous bacterial isolates Stenotrophomonas maltophilia e-a22 found to be potential for lipase production, which could produce a considerable amount of lipase (136.47 U/ml).

Bacterial growth was deterimend by viable cell count using serial dilution technique. After incubation it was found that the highest growth (6.7×10 7 cfu/ml) was obtained at 72 h of incubation in case of Stenotrophomonas maltophilia e-a22 and the cell number decreased over this time of cultivation. The highest lipase activity was found in late exponential phase (72 h). Pseudomonas aeruginosa 12 and Bacillus subtilis 20B showed the highest growth (3.7×10 7 7 and 6.9×10 cfu/ml) at 84 h of incubation and cell number decreased over this time. The result clearly reflected that the lipase activities were the highest in exponential growth phase at 36 h of incubation.

These three bacterial isolates and their consortium were used for biotechnological application with special reference to synthetic wastewater treatment. The initial COD value of synthetic wastewater was 1,200 mg/L. Among three indigenous bacterial isolates Stenotrophomonas maltophilia e-a22 showed better COD removal performance (74.75%) in case of monoculture. As per speculation consortium of three isolates showed better COD removal than that of monoculture and a maximum of 83.33% COD reduction was achieved by the used consortium. Therefore, it could be concluded that consortium of the isolated three indigenous isolates could be useful for lipid-rich wastewater treatment as a seeding material with special reference to lipidrich wastewater. Biotechnologically this indigenous consortium of Stenotrophomonas

maltophilia

e-a22, Pseudomonas aeruginosa 12 and Bacillus subtilis 20B could be useful and play an important role in the water pollution management in Bangladesh.