Isolation And Identification Of Petroleum Hydrocarbon-degrading Bacteria At Bohai Bay And Its Genomics Analysis

Marine environment is polluted by oil spills and other pollutants at Bohai Bay. Oil degrading bacteria play a very important role in the bioremediation of oil pollution. In this study, many petroleum hydrocarbon degradation bacteria were isolated from water samples,oil samples and mixed water and sludge samples, all of which were collected from various petroleum-contaminated areas in Bohai Bay. The strains were identified according to their physiological, biochemical and molecular biological characteristics. The bacterial degradation rate of alkanes, cycloalkanes, and aromatics were determined by internal standard method using GC-MS. In these strains, a high-efficient petroleum hydrocarbon-degrading bacterium was chosen to investigate the relationship between bacterium growth and oil degradation trend by determining the oil degradation rate during 3 days,6 days and 9 days cultivation. Genomic DNA was sequenced by Pacbio single-molecule real-time (SMRT) sequencing method. Genomic DNA was obtained by genome-wide denovo assembly. The genome sequence was verified by correcting the obtained template. The genome model was used to predict the genome, and the position information and amino acid sequence of the gene model were obtained. The amino acid sequence of the predicted genomic region was compared with the database of COG and other homologous sequences. The functional description of the gene and the basic information of the later data analysis were obtained, and the degradation gene in the strain was analyzed.The results showed that three bacteria strains possessing high petroleum hydrocarbon degrading capability were screened from 28 isolates. The 99% 16SrDNA sequence similarity were found between Acinetobacter and the three bacteria strains by BLAST.GC-MS analysis showed that all three strains had good degradation ability to petroleum.After degradation for 10 days,the degradation rate of alkane is 74% -98%, thdee gradation rate of aromatic hydrocarbon is 74% -88%,the degradation rate of cyclic hydrocarbon is 63% - 88%. The petroleum hydrocarbon degrading capability of the three bacteria strains was found in descending order: Tust-DM21> Tust-DC12>Tust-DW04. The degradation rate of different oil compositions was observed in descending order: alkanes > aromatics>cycloalkanes.Among the three strains, the strain Tust-DM21 is a highly effective petroleum hydrocarbon degrading marine bacterium,which degradation rate of alkanes (C10-C30)shown up to 98%, and its degradation rate of aromatics and cycloalkanes shown up to 88%after 10 days of culture at 28 ?. And the growth of the strain was positively correlated with the degradation ability, and the degradation rate of the strain reached the highest at the stable stage. The whole genome sequence showed that Acinetobacter sp. Tust-DM21 was composed of two circular genomes and three circular plasmids.There were 168 possible genes involved in petroleum degradation in Acinetobacter sp.Tust-DM21, of which 56 genes were found in genome g1, 95 genes were found in genome g2, 17 genes were found in plasmid 1. Acinetobacter sp. Tust-DM21 includes the major alkane degradation genes AlkB and cytochrome P450, the main aromatics degradation gene catechol 1,2-dioxygenase and a-subunit benzene 1,2-dioxygenase and some other dioxygenase, monooxygenase and hydroxylase. The KEGG metabolic pathway of the gene encoded lactaldehyde dehydrogenase in the genome of Acinetobacter sp. Tust-DM21 was predicted, which suggested that the enzyme participated in degradation of alkanes, alcohols,olefins and naphthalene, and also participated in the biosynthesis of secondary metabolites.It is speculated that lactaldehyde dehydrogenase in Acinetobacter sp. Tust-DM21 plays an important role in the direct dehydrogenation oxidation process for the petroleum hydrocarbons biodegradation.These results suggest that the strain Acinetobacter sp. Tust-DM21 has high- efficent petroleum hydrocarbons degrading capability of alkanes, aromatics and cycloalkanes. It is a petroleum degrading bacteria which will have a bright application prospect and bring obvious social benefit and environmental benefit.