ISOLATION, CHARACTERISATION AND BIODEGRADATION ABILITY OF BACTERIA ISOLATED FROM SOIL CONTAMINATED WITH HYDROCARBONS

ABSTRACT

Contamination of land and water bodies by crude oil and refined petroleum products is a major challenge worldwide. Indiscriminate disposal of crankcase oil into the environment has increased hydrocarbon pollution in Nigeria. Microorganisms have been identified as major contributors in fighting pollution. The remediation ability of bacteria isolated from hydrocarbon contaminated organic rich soil has not been fully investigated. This research was designed to study in-situ genera and hydrocarbon degrading ability of bacteria isolated from an organic rich tropical soil deliberately contaminated with a Nigerian crude oil and crankcase used oil Bacterial enrichment for hydrocarbon degradation was carried out by deliberately contaminating garden soil samples collected from the Nursery of the Department of Microbiology, University of Ibadan. Top soil were collected and mixed with Forcados Blend crude and used crankcase oil at a mixed ratio of 5:1. Hydrocarbon degrading bacteria counts were obtained at two week intervals for ten weeks by sub-culturing on mineral salts oil agar supplemented with the hydrocarbons. Isolation was done by randomly selecting colonies of bacteria based on morphological and growth characteristics. Isolated bacteria were screened on sterile Hydrocarbon agar plates and were identified by classical methods. The DNA extraction and amplification of ten selected strains were carried out using molecular technique. Amplified DNA was digested by HaeIII and Rsal restriction enzyme and subjected to Restriction Fragment Length Polymorphism analysis and sequencing of the 16SrRNA.The BLAST search for the obtained sequences were made and phylogenetic tree of amplicons constructed using MEGA4.1. Plasmid presence, sizes and numbers in the isolates were determined. Hydrocarbon degradation rate by the bacteria isolates was determined by gravimetry and Gas Chromatography analysis using Flame Ionisation Detector. Hydrocarbon-utilising bacteria increased from 35 × 104 to 265 × 104 cfu/mL, while total bacteria count decreased from 245 × 104 to 123× 104 cfu/mL between the second to tenth week. Ten out of forty-two hydrocarbon-utilizing bacteria detected showed high crude and crankcase oil degrading ability. Phylogenetic analyses of the isolates showed high sequence identities (75-100%) in amplified genes when compared to those in the GenBank. The isolates belonged to four genera; Bacillus (5), Providencia (3), Proteus (1) and Alcaligenes (1). Utilization of complex hydrocarbons present in crude and crankcase oil by these isolates ranged between 51.9-77.0% and 42.4-75.8% respectively. Four out of the ten bacterial isolates contained plasmids of varying sizes. Bacillus OUE3 and Providencia OCR1 contained two plasmids each of sizes 2.57 kb and 2.0 kb, and 1.3 kb and 1.9 kb respectively, while Bacillus OUE6 and Providencia OCR2 contained a plasmid each. The percentage total degradation for polycyclic aromatic hydrocarbon ranged from 29.64 to 98.45% for crude and crankcase oil. About 25.2 to 91.7% and 98.2 to 99.6% of aliphatic groups were utilised by the isolates in crude and crankcase oil respectively within 20 days. Ten of the isolated bacteria could remediate hydrocarbon pollution from soil environment. Providencia sp. had the highest degradative ability.