Catechol-2,3-dioxygenase and Lipase Activities during Degradation of Crude Oil by Hydrocarbon-degrading Bacteria Isolated from Bitumen-polluted Surface Water in Agbabu, Ondo State

Abstract

Environmental pollution arising from petroleum leakages in storage tanks, spillage during transportation of petroleum products, deliberate discharge of petroleum products and various industrial processes is hazardous to soil and water ecosystems. Bioremediation is regarded as a preferable technology for long term restoration of crude oil contaminated sites. This study evaluates activities of some enzymes during degradation of crude-oil degradation by some hydrocarbon-degrading bacteria. Bacterial isolates recovered from bitumen-contaminated surface water were screened for hydrocarbon- degrading potentials by plating them on minimal salt medium (MSM) supplemented with 2% crude-oil over a period of 10 days. Their growth was monitored by measuring the absorbance (OD600nm) and Total viable count (log10 CFU/ml). The isolates were identified by the amplification and sequencing of the 16S rRNA sequences. Degradation procedure was carried out under optimum growth temperature and pH. Catechol-2,3-dioxygenase activity was determined by spectrophotometric method and the unit activity was calculated from standard curve prepared from varying concentrations of 2-hydroxymuconic aldehyde while lipolytic activity was determined by colorimetric method based on the activity in cleavage of p-nitrophenylpalmitate (p-NPP) at pH 8.0. Five isolates that showed maximum utilization of crude-oil as sole carbon source and were selected for degradative studies. The identities of these isolates as revealed by 16S rRNA sequences were Pseudomonas aeruginosa, Bacillus cereus, Dyadobacter koreensis, Campylobacter hominis and Micrococcus luteus. Optimum Catechol 2, 3-dioxygenase (C23O) and lipase activities was observed on the 7th and 8th day of degradation respectively. Highest C23O activity (20.0U/ml) was observed with Bacillus cereus while Pseudomonas aeruginosa had highest lipase activity (8.0U/ml). The temperature optimum of the reaction rate of Catechol 2,3-dioxygenase (C23O) from the selected isolates was between 40 and 50°C while optimum pH for C23O activity was within pH 6 and 8. Optimum activity of lipase recovered from the isolates was within 40°C to 55°C and pH 7 to 8. The degradative and enzymatic activities of these organisms recommend them as potential tools for the bioremediation of crude oil polluted environments.