IDENTIFICATION OF BIOSURFACTANT PRODUCING BACTERIA ASSOCIATED WITH OIL-POLLUTED SOILS OF SOME MECHANIC WORKSHOPS IN AKURE

Abstract

Pollutants from spent fuel disposed in mechanic workshops are often recalcitrant due to non-availability to degrading microbes. The biosurfactant producing activity of degrading bacteria isolated from five mechanic workshops in Akure was investigated in this study. Soil samples were collected from five mechanic workshops. Isolation was done using standard microbiological techniques. Degradative activity of the isolates was assayed on Bushnell Haas broth (BHB) supplemented with 2% petroleum hydrocarbons (petrol, diesel, crude oil and kerosene) monitored with carbon dioxide evolution and spectrophotometric method at 600 nm wavelength. The pH of the medium was monitored for seven days while the amount of oil degraded was determined gravimetrically. Biosurfactant activity was determined using hemolysis, drop collapse, oil spread assay, foaming and emulsification activity (E24). Biosurfactant producing isolates were identified using morphological and biochemical method and further authenticated using molecular methods. Mean of total heterotrophic bacterial count ranged between 10.00±2.65× 105 CFU/g and 25.70±0.03 × 107 CFU/g. The isolates were identified as Pseudomonas sp., Clostridium sp., Bacillus sp., Arthrobacter sp., Aeromonas sp., Staphylococcus sp., Proteus sp. and Micrococcus sp. Five isolates survived on 2% petrol, diesel and crude oil while only three isolates survived on kerosene. The pH of the medium reduced slightly from 7.00. Carbon dioxide evolution ranged between 528 and 2552 mg/L. Bacillus sp. had the highest biosurfactant activity as determined by drop collapse assay, oil spread assay (22.00 mm), foaming property and emulsification index (60%). The biosurfactants produced were soluble in chloroform and alcohol but insoluble in water. Biosurfactant producing strains were molecularly characterized as Bacillus cereus OFOa strain and Clostridium punense OFOb strain with accession numbers MN700654.1 and MN700653.1 respectively. The biosurfactant produced by these hydrocarbon degrading strains can be used as bioemulsifiers in bioremediation.