Isolation, Molecular Identification, and Detections of AlkB and C23O Genes of Crude Oil-Degrading Bacteria Isolated from Automotive Service Station Soil of Gondar City, Northwest Ethiopia

Abstract

Engine oil is one of the most significant sources of energy on the planet. As the usage of petroleum hydrocarbon products increases, soil contamination with petroleum oil is becoming one of the major environmental problems. As a result of this, global interest in the microbial biodegradation of petroleum pollutants has grown recently. The aim of this study was isolation and molecular characterizations of engine oil degrading bacteria isolates from engine oil-contaminated soil in Gondar city, Ethiopia. Soil bacteria were isolated by an enrichment culture technique using minimal salt medium with 1% engine oil as a carbon source, and Screening of potential engine oil degrading bacterial isolates were performed by using hole plate diffusion techniques. Engine oil percentage degradation was determined spectrophotometrically as the relative decrease of absorbance wavelength of the medium before and after treatment at OD420 nm. The potential bacterial isolates were molecularly identified using 16S rRNA gene sequencing. A total of 41 bacterial cultures were isolated. Out of 41 bacterial isolates seventeen isolates had able to form a clear zone around the hole on 1% oil medium, but only three Gram-negative bacterial isolates Ar3A, Az2B, and Ar2B showed a better clear zone on 1% engine oil medium with diameters of 36.33±306 mm, 26.0±2.000 mm, and 22.67±306 mm, respectively. Bacterial isolate Ar3A was found to be the best oil degrading isolate in this study with 70.033±1.155% degradation after a 21day incubation period while 65.923±0.414% and 64.39±1.507% degradation was observed using isolate Az2B and Ar2B, respectively. The 16S rRNA gene sequences percent identity of Ar3A was 94.5 % with Stenotrophomonas acidaminiphila, Az2B was 91.53% with Citrobacter amalonaticus and Ar2B was 92.9% with Klebsiella pneumoniae. PCR experiments with specific primers for catabolic genes of AlkB and C23O suggested that only Stenotrophomonas acidaminiphila strain Ar3A can possess both genes with 660 bp and 500 pb, respectively. In conclusion, the three bacteria isolates should be considered as good prospects for their application in the bioremediation of engine oil sludges. However, Further studies could be conducted on the possibility of cloning AlkB and C23O genes of Stenotrophomonas acidaminiphila into other bacteria for more efficiency and effectiveness in the bioremediation process. Keywords: AlkB gene, Bioremediation, C23O gene, and engine oil-degrading bacteria 1 1. INTRODUCTION 1.1. Background of the Study The global environmental problem of engine oil contamination and its derivatives is a