Diversity Analysis Of The Oil-degrading Bacteria And Alkane Hydroxylase From Surface Seawater Of The India Ocean

At present, with the development of marine oil exploitation and transportation, the oil leakage and spill occur frequently. Increasingly serious oil pollution lead to adverse effect on marine environments and ecosystem. By far, biodegradation by various microorganisms is considered as an effective way to reduce and remove marine oil pollution. Thus, the study on the isolation and identification of oil-degrading bacteria and key enzyme for degradation (Alkane hydroxylase) have important significance of theory and application.In this study,22oil-degrading consortia were enriched by a mixture of crude oil and diesel oil (volume ratio of1:1) from surface seawater across the India Ocean to research the diversity of oil-degrading bacteria and alkane hydroxylase.At first, the diversity of culturable strain in oil-degrading consortia was analysed by spread plate method. The results demonstrated that the dominant groups of12oil-degrading consortia were bacteria, while, the dominant groups of10oil-degrading consortia were yeast. After enrichment,91strains were isolated in12oil-degrading consortia. They were consisted of19genera,30species. Phylogenetic analysis based on16S rRNA gene of91bacteria indicated:the majority belonged to Alaphaproteobacteria (40.66%), Gammaproteobacteria (35.16%) and Actinobacteria (17.58%), the minority belonged to Firmicutes (5.49%) and Bacteroidetes (1.1%). The dominant genera included Alcanivorax, Salinicola, Marinobacter, Novosphingobium, Thalassospira and Microbacterium. This result demonstracted that the diversity of culturable oil-degrading bacteria from surface seawater accross the India Ocean was rich. Meanwhile, seven bacteria, Yarrowia lipolytica and Pichia sp and several consortia were sufficient to degrade oil and potential for application.Secondly, the diversity of unculturable bacteria in oil-degrading consortia was analysed by454pyrosequencing. The result showed that the bateria of12oil-degrading consortia belonged to seven classes, the dominant classes were Alaphaproteobacteria (37.10%), Gammaproteobacteria (61.67%). At genus level, the dominant genera in58genera were Alcanivorax (37.03%), Thalassospira (18.50%), Marinobacter (13.77%), Novosphingobium (11.90%), Parvibaculum (4.86%), Salinicola (2.74%), Cobetia (2.63%), Pseudomonas (2.60%) and Pseudoalteromonas (1.41%). The status and role of not dominant taxa in12oil-degrading consortia were studied further. Moreover, the similarities analysis and Principal Component Analysis of12oil-degrading consortia displayed no direct the relationship between their components and their sampling position not exist.At last, the diversity of alkane hydroxylase from12oil-degrading consortia was analysed by clone library.463clones of alkane hydroxylase were obtained and divided into11OPUs based on the threshold of80%amino acid similarity. OPU1and OPU2were as the dominant OPUs, the highest homology sequences between OPUs and reference sequences were respectively Alcanivorax sp. S17-16(ACJ22719) and Uncultured bacterium KL2cO9(AFC90113), the latrer was Marinobacter. These result corresponded with dominant bacteria by454pyrosequencing. OPU11had only66.06%identity with reference sequence and was a novel alkane hydroxylase. In addition, Phylogenetic analysis based on alkane hydroxylase sequences demonstrated that alkane hydroxylase were widespread and had rich diversity.In this report, the diversity of22oil-degrading consortia and alkane hydroxylase of12oil-degrading consortia enriched from the surface water across the India Ocaen were investigated, the oil-degrading bacteria and key genes were acquired. These findings facilitate to understand the distribution and diversity of oil-degrading bacteria from surface seawater and prodive numerous strains and genetic resources for removing oil pollution by bioremediation.