脱卤拟球菌还原性降解地下水中氯代乙烯的过程及机理研究

Chlorinated ethenes have been wildly applied in manufacturing and agriculture, which pose a great potential threat to the water environment and human health. In groundwater environment, Dehalococcoides is the only known bacteria that can reduce chlorinated ethenes to non-toxic ethene. However, the microbial mechanism of Dehalococcoides in the degradation of chlorinated ethenes, as well as the competitive relationship between exogenous Dehalococcoides and in situ microorganisms has not yet clear. In view of these problems, this project aims to study the pathways and mechanisms of the degradation of chlorinated ethenes in the groundwater via the detection of 13C isotopic natural abundance, fluorescence quantitative PCR and cloning/sequencing. Better understanding of the intrinsic relationship between Dehalococcoides, oxygen content, pH, organic carbon content, redox potential and the remediation of chlorinated ethenes, in order to clarify the key biogeochemical indexes that affect the reduction of chlorinated ethenes by Dehalococcoides. To obtain the microbes with high degrading efficiency for in situ bioremediation, selective medium has been applied to screen out the Dehalococcoides with high environmental adaptability. Key reductive dehalogenase have been purified and identified to study its enzymatic properties and degradation mechanism. The culture with high degrading efficiency has been inoculated into the contaminated groundwater site for remediation by active Dehalococcodies, and the high-throughput sequencing is applied to study the change of indigenous microbial community composition. The research results of this project is expected to provide theoretical basis and practical strategy for the application of Dehalococcoides in the bioremediation of groundwater contaminants.

氯代乙烯在工农业中应用广泛，对水环境和人类健康潜在威胁极大。在地下水中，脱卤拟球菌是目前唯一能将氯代乙烯还原成无毒产物乙烯的细菌。但其降解氯代乙烯的微生物机理，以及添加外源脱卤拟球菌进行原位修复时其与土著微生物的竞争关系尚未明确。针对这些问题，项目拟通过13C自然丰度测定、荧光定量PCR、克隆/测序等手段研究地下水中脱卤拟球菌降解氯代乙烯的途径及机理。并阐明脱卤拟球菌、含氧量、pH、有机碳、氧化还原电位与氯代乙烯修复的内在关系，明确影响脱卤拟球菌还原氯代乙烯的关键生物地球化学指标。利用选择性培养基筛选出环境适应力强的脱卤拟球菌为原位修复提供高效降解菌。纯化鉴定关键的还原脱卤酶，研究其酶学特性及降解机理。将扩大培养的高效降解菌接种到氯代乙烯污染地下水区域，利用活性脱卤拟球菌进行修复并通过高通量测序研究土著微生物群落组成变化。项目有望为脱卤拟球菌在地下水污染修复中的应用提供理论依据与实践方法。

氯代乙烯在工农业中应用广泛，对水环境和人类健康潜在威胁极大。在地下水中，脱卤拟球菌是目前唯一能将氯代乙烯还原成无毒产物乙烯的细菌。但其降解氯代乙烯的微生物机理，以及添加外源脱卤拟球菌进行原位修复时其与土著微生物的竞争关系尚未明确。针对这些问题，项目通过荧光定量PCR、新一代高通量测序等手段研究了典型地下水样品中脱卤拟球菌降解氯代乙烯的途径及机理，证实了脱卤拟球菌可作为评估氯代乙烯微生物降解的生物标记。研发成功了一套具有自主知识产权的厌氧培养瓶充气抽真空清洗系统，用于脱卤菌的富集培养和分离纯化。成功分离获得了一株能够将外源有机卤代污染物进行还原脱卤的纯菌菌株DBB。为了进一步强化氯代乙烯污染地下水区域的原位修复效果，解决外源脱卤菌与土著微生物竞争问题，研究了典型脱卤细菌Dehalococcoides、Dehalobacter、Desulfitobacterium与地下水环境中其他微生物代表种群之间可能存在的相互联系和相互作用，从分子层面上阐明了在氯代乙烯还原性脱卤过程中的微生物响应机制。为了优化氯代乙烯污染地下水的生物修复策略，进一步构建了碳基/铁基材料（生物炭、纳米零价铁、石墨烯）和功能厌氧菌相耦合的高效修复体系。揭示了厌氧环境中碳基/铁基材料和三种功能菌（脱卤菌、硫酸盐还原菌、铁还原菌）的耦合优势，发现外源材料的添加可以为脱卤菌的生长和活性创造适宜的环境。脱卤菌、硫酸盐还原菌能和纳米零价铁协同降解氯代乙烯，有利于氯代乙烯污染地下水环境的生物修复。同时深入分析了纳米材料的水环境毒性效应，揭示了高浓度的纳米零价铁和氧化石墨烯对于脱卤菌的生长及其还原性脱卤过程的抑制作用。项目的理论研究结果有助于深入理解脱卤拟球菌还原性降解氯代乙烯这一微生物过程，技术研发成果可为氯代乙烯污染水环境修复提供菌种储备与技术支撑。