Screening And Identification Of Oil-degrading Marine Bacteria From Spilled Oil In The Bohai Sea And Analysis Of Functional Genes

The ocean occupies three quarters of the earth’s surface and provides plenty ofsources which plays a quite important role in the existence and development of humansociety. However, as the increasingly intensive exploiting of marine sources, thepollution of ocean becomes more and more serious and does more harm to humanbeings. Among the countless pollution sources, oil becomes so essential that can notbe ignored. The oil spill of Penglai19-3oil field in Bohai Sea happened in2011resulted in a total of Grade IV water area of870square kilometers.Biodegradation is the most promising method in removing spilled oil because ofits easy operation, low cost and no pollution. Bacteria with highly efficient oildegradation activities are the key organisms to the bioremediation process. Alkanehydroxylases, such as AlkB, P450and AlmA, are responsible for the oxidation ofalkanes. In order to obtain bacteria with efficient oil degradation activity and relatedhydroxylase genes, we have screened the sediment of the Bohai Sea oil contaminatedareas, and18bacteria and6fungi were isolated. A preliminary experiment was carriedout to determine the oil degradation rate of isolates from Bohai Sea and MarineCulture Collection, Ocean University of China (MCCO), from where eleven and fivebacteria with high alkane degradation activity were obtained, respectively.16S rDNAsequences of strains with better oil degradation activity were identified, and90%isolates from Bohai belonged to γ-Proteobacteria whereas one strain belonged toFirmicutes. Marinobacter goseongensis WSL01and Bacillus halodurans WH072were found to show efficient oil degradation activity for the first time. Alkanehydroxylase genes, including alkB, p450and almA, were analyzed from theseidentified strains. All the three genes were detected in WSL01, WSL14, WH069andWH072, the oil degradation rates of which were all above20%. It was confirmed that genes alkB, p450and almA were closely related with the oil degradation activity. Theamino acid sequences of these genes were then used to build a phylogenetic tree withthe cloest protein sequences from NCBI. The results showed that the phylogeny ofgene P450was in consistence with that of16s rRNA gene. The ORF and flankingsequences of alkB, p450and almA in WSL01and WH072were obtained usinghigh-efficiency thermal asymmetric interlaced PCR (hiTAIL PCR). According to theBLSATP results and the phyloneanic analysis, alkB and almA in WSL01and WH072might encode new alkane hydroxylases and need further studies. The p450gene ofWSL01was heterologous expressed in Escherichia coli BL21and the P450proteinwas observed in the recombination strain.In this study, we have screened the sediment of the Bohai Sea oil contaminatedareas in order to obtain highly efficient oil-degrading bacteria and analyze theirrelated genes. This could provide an extensive application prospect of theoil-degrading bacteria in the future.