Molecular Identification of Crude Oil-Degrading Bacteria and Screening for Catechol 2, 3 Dioxygenase (C23O) Gene

Abstract

Aims: To identify crude oil-degrading bacteria isolated from polluted soils and waters and screen the presence of catechol 2, 3 dioxygenase (C23O) gene encoding oil-degradation in the strains with the highest degradative activity. Study Design: Laboratory-experimental design was used in this study. Place and Duration of Study: Crude oil polluted soils and waters were collected from Awoye, Mese and Oluwa villages in Ondo State, Nigeria and three different flow stations (Agbada-Aluu shell, Obite, and Bonny) in Rivers State, Nigeria. Methodology: The identities of the isolates were confirmed by extracting their total genomic DNA using standard DNA protocols while a portion of 16S bacterial gene of their DNA was amplified by polymerase chain reaction (PCR) using the primers E9F and U1510R and sequenced using Sanger method. Degenerate primers were used to isolate and identify the gene encoding C23O, responsible for the degradation of oil. Molecular cloning of the gene was done by transforming into Escherichia coli DHα. The correct inserts from the selected clones were performed by colony PCR. The isolated gene was sequenced with a Dye terminator sequencing kit and the product was analyzed with Prism DNA sequencer. Results: The results obtained from the conserved sequence of the 16S rRNA coupled with the nucleotide sequence revealed ten (10) crude oil-degrading bacteria, with CFfab 14 and CFfab 12 having the highest and lowest degrading activity of  78.92 ± 0.9 Unit/mL/h and 43.89 ± 1.3 Unit/mL/h on day 3 respectively. Conclusion: The gene C23O responsible for the production of catechol 2, 3 dioxygenase was isolated from strains CFfab 5, CFfab 14 and CFfab 15. The nucleotide base sequence of the gene was determined to be 238 bp. It is expected that in bioremediation, indigenous microorganisms from polluted environments should be screened for the possible existence of this unique gene sequence for effectiveness. Further studies could be conducted on the possibility of cloning this C23O gene into other bacteria for more efficiency and effectiveness in the bioremediation process.