海洋钻井平台海域微生物资源的石油烃降解机制研究

Oil spill occurred during drilling process jeopardized the marine ecosystem severely. Offshore oil spill has occurred repeatedly in history. Although lots corresponding measure was applied for remeditiation, but the effect was not very satisfactory. Among the oil spill treatments, microbial degradation of petroleum hydrocarbons is low-cost, with minimal side effects and broad prospects. Aiming at the key issue of technology of microbial degradation of hydrocarbons while oil spill, this project will mainly focused on microbial community structure, gene expression cooperation strategy among and within species, degradation rate and mechanism of petroleum hydrocarbon in the process of transformation of petroleum hydrocarbons into metabolic secretory products, cell components and energy. This project will implement the following works: 1. to obtain the hydrocarbon-degrading marine microflora from sea area near offshore oil rigs based on the mix culture system; 2. to determine the community structure by analyzing 16S rRNA with high throughput sequencing technology; 3. To reveal the deep pattern of metabolic gene interaction of marine microbial degradation of petroleum hydrocarbons by analyzing the differences in gene expression of microbial degradation of petroleum hydrocarbons with digital expression profiling; and 4.to illustrate the patheways of degradating petrolem hydrocarbons and the community structure of functional microflora by employing real-time quantitative PCR, metabolitic analysis and fluorescence in situ hybridization. The project will provide theoretical basis for the exploration of offshore oil degradation model and the utilization in microbial treatment technology of oil pollution.

海洋石油平台在钻采过程中发生的原油泄漏，对海洋生态系统危害极大。在历史上发生的多次海上原油泄漏事件中,采取的相应措施，效果都不十分理想。目前对溢油的处理方法中，利用微生物降解石油烃成本低、副作用小，前景广阔。本项目针对海洋微生物降解溢油烃类物质技术的关键问题，重点研究在石油烃转化为代谢分泌产物、细胞组成成分、及参与能量传递循环的过程中，微生物群落组成及功能、物种间及种内基因表达协作策略、石油烃降解率与机制。本项目基于混合培养系统获得海洋钻井平台附近烃类专性菌群，利用高通量测序技术分析菌群16S rRNA，确定烃类专性菌群结构；利用数字表达谱技术分析烃类专性菌群降解石油烃类基因表达差异，揭示海洋微生物降解石油烃类代谢基因间互作深层规律；通过实时定量 PCR、代谢产物分析及荧光原位杂交，阐述石油烃类降解途径和机制。为探索海洋石油降解模式，利用微生物治理石油污染技术提供理论基础。

海上溢油事故时有发生，在众多处理溢油污染的方法中，微生物方法由于具有操作方便、成本低、无污染等优点逐渐受到相关研究者的关注。本项目围绕石油烃类微生物降解技术的关键科学问题，以海上钻井平台附近海域海洋微生物为研究对象，研究烃类降解微生物的群落结构与功能，石油烃降解途径与效率, 降解基因代谢网络调控机制等内容。.通过对海上钻井平台环境微生物群落16SrRNA基因高通量测序，本项目获得了特殊生境中的微生物群落结构数据。本项目完成了海上钻井平台环境微生物群落的宏基因组测序，得到特殊生境中的微生物的基因代谢通路与基因间互作关系。本项目进一步对在人工培养不同条件下的微生物群落进行了宏转录组测序，得到了石油烃降解相关代谢通路的基因表达丰度与基因互作信息。进一步，项目对石油烃降解菌的znuA、phoB、nqrF、rubB、znuB、modA、gltX、ectA等八种主要关键基因（GOI）在十二种条件下多个时间点进行实时定量PCR分析，得到了关键基因对降解反应的调控策略。本项目设计了关键基因的荧光探针，在十二种条件下的多个时间点对培养细胞进行荧光原位杂交，获得功能菌群时空分布规律。此外研究了钻井平台微生物群落对十种常见烃类在人工不同培养条件下多个时间点上的降解情况，得到了微生物群落降解石油烃的反应速率和优化条件。项目还筛选出得到十一株原油降解菌，其中MWD-15 、MWD-18两株为高效原油降解菌，分属于微杆菌属、鞘氨醇单胞菌属。 .项目获得了海上钻井平台附近海域石油烃降解菌落结构、石油烃降解途径与基因代谢网络的系统信息，为微生物处理海上钻井平台溢油事故奠定理论基础。