Response And Degradation Potential Of Bacterial Communities Against Petroleum Hydrocarbons In The Surface-sediments Of Bohai Sea, China

Petroleum exploitation caused serious pollution to the semi-enclosed Bohai Sea ecosystem. Indigenous microbial populations adapted to the petroleum-polluted environments were thought to have the ability to degrade the hydrocarbons. However, little was known about the diversity of the oil-degraders and their response to the pollutants in this shallow marine surface-sediment ecosystem. Thus, the application of microorganisms in the bioremediation of petroleum contaminated Bohai Sea ecosystem was limited.14 surface-sediment samples were collected from 3 different areas, namely Suizhong oilfield, Jinzhou oilfield and nearshore non-oilfield area in Bohai Sea. The α and β diversity of bacterial communities and their response to aromatic hydrocarbons, (benzene, toluene, ethylbenzene and xylene, BTEX) in the surface-sediment were analyzed using 16S rRNA gene based T-RFLP fingerprints. The results indicated that the a diversity (Shannon index) were negatively correlated with BTEX and TN (total nitrogen) contents (P< 0.05); The β diversity at T-RF level were significantly correlated with both spatial parameters (water depth and latitudes) and environmental factors (TN, AP, AK and pH), while the content of HM, BTEX, AK and pH value were the best predictors for the community variation at phylum/class level. Pearson correlation analysis revealed that Firmicutes and Alphaproteobacteria were enriched as a response to BTEX pollution. Partial mantel tests showed a close correlation between variations in the bacterial communities at T-RF level and geographic distances (P= 0.03) at a small to intermediate scale (1.842-142.196 km) in Liaodong Bay; While at phylum/class level, environment heterogeneity significantly influenced the community structure (P= 0.013).Phylogenetic diversity analysis of alkane hydroxylase (AlkB) and soluble di-iron monooxygenases (SDIMOs) genes showed that:almost 72.61% of the AlkB sequences were related to Gammaproteobacteria, including Alcanivorax and Marinobacter; Phenol-2-like, Mmo-like, ThmA-like, PmoC-like and PrmA-like SDIMOs genes were retrieved in the sediment samples with almost 72% of new SDIMO gene sequences.One BTEX-degrading bacterial consortium was enriched from BTEX polluted nearshore non-oilfield area in Liaodong Bay (3.4% salinity).555.21 mg L-1 of BTEX was biodegraded up to 43.9% over a period of 36h by the consortiums.16S rRNA gene based DGGE combined with clone library were used to clarify the structure of the consortium.6 dominant genus namely, Pseudomonas, Mesorhizobium, Achromobacter, Stenotrophomonas, Sphingobacterium and Saccharothrix were detected.5 genotypes (tollxylAltodC1lxylE1ltodE) coding the key enzymes involved in BTEX degradation pathways were detected by PCR. The result revealed that, the initial oxidative attack consists of direct oxidation of the alkyl side chain via a mono-oxygenase (TOL pathway) and oxidation of the aromatic ring via a dioxygenase (TOD pathway). The initial oxidative attack converting the BTEX compound into a catechol structure and the subsequent cleavage of the catechol structure was catalysed by catechol 2,3-dioxygenase (C23O) (meta-cleavage pathway) producing the respective cleavage products which might further be metabolized to Krebs cycle intermediates, allowing the mineralization of the pollutant.These results provide a theoretical support for the in situ bioremediation of crude oil pollution in Bohai ecosystem and a valuable bacterial consortium for the application of bioaugmentation.