高效石油污染降解的微生物群落修复基因信息动态研究

PAHs(Polycyclic Aromatic Hydrocarbons) in the oil-contaminant soil, has gathered significant concern for human health. The succession of highly effective PAHs-degradation gene in oil-contaminant soil and the mechanism of biodegradation are the two critical parts of improving the technology of bioremediation. On the basis of the knowledge of microbial degradation, it is expected to further the research on the dynamics of degradation gene to explain the mechanism of biodegradation and enhance the efficiency of biodegradation. In the recent years, a new generation of high-throughput functional gene arrays named Geochip has been developed, which can provide enormous genetic information and has become a powerful tool for tracking bioremediation processes. Also, Geochip can provide great information about the ecological processes and the function of microbial community. In this project, we take advantage of high-throughput Geochip analysis method to study the function of microbial community in the process of biodegradation in oil-contaminated soil. By Geochip analysis on the DNA from microbial community, we emphasize on the dynamics of functional gene and microbial community and the changes of microbial functional activity, which is expected to identify the potential of functional gene and dominant degradation gene and their succession under the different PAHs contamination stress in different period. Also, we use Geochip technology to try on the analysis of RNA from microbial community to understand the expression gene. The objectives of this study are to monitor the potential and actual expression of PAHs degradation gene under different environmental stress and explain the microbial activity and ecological function. The result can help understand the response and regulation pattern of microbial community in the process of gene expression and translation. This will be available to aid in the evaluation and formulation of strategies for effective microbial bioremediation in oily wastes contaminated sites.

摘要：石油中存在的多环芳烃（PAHs）对人类健康构成严重威胁，对油污土壤中PAHs高效降解基因的演替及降解机理研究是解决生物修复技术突破的关键环节。这需要获得更多降解基因动态信息来阐释降解机理过程。近年来发展的高通量新型环境基因芯片技术可获得大量基因组学信息，是跟踪生物修复过程的有力工具。 本研究利用含高通量功能信息的功能基因芯片Geochip，对油田区污染土壤的生物修复过程进行微生物生态功能解析。重点利用功能基因芯片分析土壤微生物群落基因组DNA,了解PAHs 降解过程中的功能基因、微生物群落结构和生态功能变化，以及在不同环境压力下潜在功能基因组和优势种群的动态演替过程。 结合反转录mRNA 基因芯片解析环境因子影响下潜在降解基因真正进行表达的能力和水平，及功能基因转录及表达过程中的微生物群落实际微观响应及调控机制，为油田区污染土壤生物修复及生态评价提供理论基础。

石油中存在的多环芳烃（PAHs）等污染毒性物质对人类健康构成严重威胁。石油开采及加工给周边土壤和水体带来严重的污染，导致土地荒芜、水体生态系统损害等重大环境问题。生物修复由于在技术、环境与经济上的优势，逐渐在石油污染的环境修复领域受到广泛关注。本研究以石油烃污染场地（兰州石化污染场）为研究对象，建立了包含化学污染特征分析、污染物的生物可利用性和遗传毒性分析以及微生物群落结构和功能基因解析的综合评定方法，为石油污染场地的微生物降解潜力的研究及生物修复提供理论和方法基础。.本研究采用GC/MS得到污染场地土壤中主要污染物类型为石油烃（烷烃）和单环芳香烃、PAHs、卤代脂肪族化合物、含氧化合物，含油量最高可以达到58458 mg/kg，随着土壤深度增加，低环芳烃（环数≤3）含量逐渐升高（25.3 %<45.2 %<99.6 %），污染物有明显的纵向迁移的现象；利用萘生物传感细胞、烷烃传感细胞和遗传毒性传感细胞，对研究区的石油烃生物可利用性和遗传毒性进行检测与评估，研究表明在生物可利用率低、遗传毒性较强、微生物降解潜力低的石油污染土壤中遗传毒性污染物从表层到深层的垂直迁移现象明显；运用Illumina高通量测序对土壤DNA进行微生物群落解析，识别不同门类微生物群落随污染水平变化的演替过程，同时结合实时定量PCR方法设计开发烷烃降解基因、环羟基化双加氧酶基因、苯系物和萘等降解酶基因，对石油烃污染物典型生物降解功能基因进行了定量化检测及动力学分析，研究发现一定含量石油烃会对微生物生长起刺激作用，污染物的压力过大会对微生物的种类和功能进行选择，导致优势菌的富集，功能基因含量增多，微生物多样性逐渐降低。本研究的目的是解析环境因子影响下潜在降解基因真正进行表达的能力和水平，及功能基因转录及表达过程中的微生物群落实际微观响应及调控机制，为油田区污染土壤生物修复及生态评价提供理论基础。