典型PAHs的土壤反硝化降解机制研究

The degradation of PAHs under denitrification in soil had been recongnized widely, but the coupling and microbial mechanisms of which were still unclear. Therefore, in this project, the typical PAHs (napthalene and pyrene) would be selected, and clean and PAHs contaminated soil samples would be colllected. Through the combination of anaerobic soil and liquid enrichment culture experiments, the material transformation and expression of the functional genes involved in the PAHs anaerobic degradation and denitrification would be studied.Subsquently,the coupling mechanism of anaerobic PAHs metabolism under denitrification would be discussed based on the material transformation and gene expression individually. Finally, through the soil incubation experiment added with carbon-13 labelled PAHs, the DNA-SIP and 454 pyrosequencing would be used to study the denitrifiers and bacterial population participated in the PAHs degradation under denitrification. Simultaniously,the contribution of anaerobic PAHs degradation under denitrification to the total PAHs removal in the soil also would be discussed. This study could provide some theoretical basis for the deeper understanding of the PAHs transformation in soil and the bioremediation of PAHs polluted soil.

PAHs在土壤中的反硝化降解已为人们所认识，但其厌氧降解与反硝化过程的偶联及微生物机理尚不明确。本项目拟选择萘和芘两种典型的PAHs，以清洁土壤和PAHs污染土壤为研究对象，结合土壤厌氧培养和液体富集培养实验，研究反硝化条件下PAHs代谢过程中的物质转化及相关功能基因（PAHs厌氧降解及反硝化功能基因）表达量的动态变化，分别从物质转化和基因表达来探讨PAHs厌氧代谢与反硝化过程偶联的机理；之后，通过稳定同位素标记的室外土壤培养实验，采用DNA-SIP、454测序等手段，来探究土壤中直接参与PAHs反硝化代谢的细菌及反硝化菌的类群，初步探讨土壤PAHs反硝化降解在自然土壤PAHs去除中的贡献率，为进一步明确PAHs在土壤中的转化过程提供理论基础，为土壤PAHs污染治理新思路的创立提供一定的理论依据。

作为土壤氮素循环重要环节的反硝化已经被证实能与多环芳烃（PAHs）的代谢相偶联。因此，研究土壤反硝化过程对PAHs响应及反其与PAHs代谢相偶联的机制具有重要意义。本项目以具有50多年油气开发史的油田区土壤为研究对象，研究土壤微生物对PAHs污染的响应机制。通过添加电子受体的厌氧培养、富集培养及添加硝化抑制剂（DMPP）的室内培养实验，进一步探讨典型PAHs的降解与土壤反硝化微生物活性及丰度之间的关系，探究土壤PAHs反硝化降解的微生物机制。结果表明，该土壤中，仅细菌多样性与PAHs污染呈显著负相关，PAHs能显著影响土壤细菌及真菌群落组成，而古菌对PAHs污染不敏感。该区域土壤中含有bssA（benzylsuccinate synthase gene）基因的主要类群与Geobacter、Thauera和Azoarcus具有较近的亲缘关系，可通过硝酸盐、硫酸盐及铁还原代谢过程降解土壤PAHs。土壤nirK及nirS基因的丰度均与土壤PAHs含量呈显著负相关（nirK: R2 = 0.54, P < 0.05; nirS: R2 = 0.58, P < 0.05），但nirS型假单胞菌的丰度与土壤PAHs含量呈正相关，表明该菌属可能在土壤PAHs的反硝化代谢中起到重要作用。添加硝酸根能增强土壤的反硝化活性，但对土壤芘和蒽的厌氧降解均无显著的促进作用，同时添加硝酸根和硫酸根能够显著促进土壤芘的厌氧降解。芘对土壤细菌群落结构无显著影响，而蒽能明显改变土壤细菌的群落组成。获取到能够在反硝化条件下有效降解萘和菲的富集菌群，它们对萘和菲的降解率均接近50%。两种富集物中的优势菌属均含有Pseudomonas，再次表明该菌在土壤PAHs的反硝化代谢中具备重要的潜在作用。对富集培养液中萘降解过程中各物质量变化的研究表明，反硝化过程中NO3-还原和随后的NO2-还原过程均能与萘的降解相偶联。在菲污染土壤中，DMPP可以通过抑制AOB（ammonia-oxidizing bacteria）的生长来减弱土壤氨氧化活性，DMPP可显著抑制不添加尿素处理中菲的降解，且能抑制土壤中反硝化相关功能基因的丰度，间接证实反硝化过程可能在土壤菲降解中的作用。本项目的研究结果将有助于全面认识反硝化过程对土壤PAHs污染的响应机制，并为PAHs污染的土壤修复提供一定的理论依据。