典型PAHs厌氧降解菌群的富集及其在土壤污染修复中的应用研究

It has been confirmed that PAHs can be degraded under some micro-aerobic or anaerobic conditions, such as denitrification, sulfate-reducing, iron-reducing and methane generation. However, the relationship between these reducing microorganisms and soil PAHs contamination is still unclear. In this study, based on the field investigation of soil samples with high risk of PAHs pollution and soil microcosm incubation experiment, the responses of activity and abundances of soil reducing microorganisms to PAHs will be investigated. Then, the microbial consortium with the ability of PAHs anaerobic degradation will be gained using the enrichment media containing special electron acceptor with typical PAHs as sole carbon source, and the mechanisms of electron acceptor transformation and PAHs metabolism will be preliminarily studied. Subsequently, the effect of the enriched consortium on the soil PAHs elimination will be verified through a combined method of inner microcosm incubation and pot culture experiment with wheat and rice. The aim of this study is to elucidate the response mechanism of soil denitrifier, sulfate-reducer, iron-reducer, and methane generator to PAHs pollution, identify the contribution of reducing process to the PAHs elimination in soil, and offer some useful information for the application of reducing microorganisms on the remediation of soil PAHs pollution.

研究表明，多环芳烃类污染物能够在多种微氧及厌氧条件下降解。然而，多环芳烃与土壤中上述还原微生物之间的关系尚不明确。本研究拟采用土壤分子生态学方法，将野外土壤调查与室内添加PAHs及相应电子受体的土壤培养实验相结合的手段，综合探讨上述还原微生物对土壤PAHs污染响应的机制。在此基础上，通过以PAHs为单一碳源且添加不同电子受体的富集培养实验，获取能够厌氧代谢典型PAHs的富集菌群，并初步研究PAHs降解与相应电子受体还原过程相偶联的机理。而后，通过室内接种富集菌群的土壤培养及温室盆栽实验（旱地、水田），来考察获取的富集菌群在土壤PAHs污染修复中作用，进一步深入探究电子受体、PAHs及土壤相关还原微生物活性及丰度之间的关系。通过上述研究，可明晰土壤还原微生物对PAHs污染的响应机制，初步明确还原过程在土壤PAHs去除中的作用，并为还原菌群在实际土壤PAHs污染修复中的应用打下基础。

厌氧过程在土壤等生境中的污染物去除中起到不可忽视的作用。因此，本研究首先以潜在PAHs污染的土壤为对象，考察土壤基本性质、PAHs含量与产甲烷活性、铁还原活性及相关功能微生物群落结构的关系；对上述典型样品中的铁还原菌群进行了富集，探索了该菌群联合化学氧化（Fenton）法去除土壤典型PAHs的效果；随后以清洁农田土壤为对象，进一步验证产甲烷过程对PAHs污染的响应机制，并明确其在水田土壤PAHs去除中的贡献；最后，从反硝化降解菌群中分离获取了菲代谢纯菌，通过盆栽实验，考察了接种该菌对土壤菲去除及作物生长的影响。结果表明，页岩气田土壤的产甲烷活性（MPA）和有机碳含量（PAHs）呈显著正相关，这两者也是影响原核生物群落结构的主要因素，且Saccharibacteria和Methanolobus可能在该土壤的复杂有机物代谢及甲烷产生过程中起关键作用。铁还原菌的群落结构是直接影响该气田土壤铁还原活性（IRP）的关键因子，而土壤铵态氮、有效磷可以通过影响铁还原微生物群落结构间接影响IRP，且氨氮还可通过直接影响有效磷来间接作用于IRP。水田土的MPA、DEA（反硝化酶活）均远高于旱地，但它们均对菲污染无显著响应，旱地土壤微生物群落结构对菲污染的响应较水田土壤敏感，且菲污染有增加水田土产甲烷菌丰度的趋势。水田土产甲烷菌丰度与土壤MPA显著正相关，选择性地抑制水田土壤的MPA能同时抑制其菲去除率（PDR），MPA与PDR呈显著正相关，明确了产甲烷过程在水田土菲去除中的潜在贡献；Fenton试剂处理能有效去除土壤菲污染，但显著降低了土壤细菌群落的多样性和丰度，接种铁还原菌群虽然未能显著提升菲去除率，但有利于土壤细菌群落的恢复。获取的菲降解纯菌均为假单胞菌属的细菌，直接接种菌株对土壤菲去除率无显著影响，但以生物炭为载体接种能够显著提高玉米及水稻的生物量。上述结果可为进一步明晰土壤PAHs迁移转化的微生物机制及修复提供理论依据。