2,4-二氯苯氧乙酸 ( 2,4-D ) 彻底厌氧降解的关键步骤及多种群协作机制研究

Accumulation of toxic intermediate product 4-chlorophenol inhibited the anaerobic mineralization of 2,4-dichlorophenoxyacetic acid in deep soil and groundwater environments. 2,4-D and 4-chlorophenol pose significant threat to subsurface ecosystems and clean water supply due to their carcinogenic potentials. However, the microbial processes involved in the anaerobic biodegradation of 4-chlorophenol is largely unknown. Our preliminary results demonstrated that 2,4-D was completely mineralized in an anaerobic enrichment culture inoculated with river sediment. Based on this observation, we propose to establish a series of microcosms to investigate 2,4-D biodegradation activities under anaerobic conditions. Quantitative and qualitative analysis of intermediate 2,4-D biodegradation product using HPLC and mass spectrometry pipeline will be performed to investigate the biotransformation pathway of 2,4-D. Additional microcosms with the intermediate 2,4-D biodegradation products 2,4-dichlorophenol and 4-chlorophenol will be established to unravel the functionality of key microbial populations associated with different 2,4-D biodegradation stages. High-throughput 16S rRNA gene sequencing will elucidate the structure and biodiversity of the microbial communities and key populations responsible for the anaerobic 2,4-D biodegradation and biotransformation. Furthermore, we will establish microcosms with 13C stable isotope labeled 4-chlorophenol as substrate. 13C stable isotope labeled end products CH4 and CO2 will be analyzed using GC-MS, and the biodegradation pathway of 2,4-D will be verified based on mass balance calculation. Moreover, key populations and their synergistic interactions for the anaerobic biodegradation of 4-chlorophenol would be elucidated by meta-genome sequencing and bioinformatics analysis. The overarching goal of this research is to develop efficient and sustainable biotechnology for in-situ bioremediation of 2,4-D contaminated sites.

中间产物4-氯苯酚降解停滞阻碍农药2,4-二氯苯氧乙酸（2,4-D）的彻底厌氧降解，给生态系统和环境安全带来潜在威胁，而目前催化4-氯苯酚脱氯还原的菌株以及与之协作实现彻底降解的多种群协作机制依然不明。因而，本项目通过构建2,4-D厌氧降解微生态系统，定性定量监测2,4-D降解中间产物和终产物的种类和浓度，探索2,4-D彻底厌氧降解转化途径；通过进一步建立以中间产物2,4-二氯苯酚和4-氯苯酚（全部13C标记）为底物的降解体系，分析各降解阶段的降解菌种群结构（16S rRNA高通量测序）；GC-MS法测定13C标记的终产物CH4和CO2的浓度，通过计算物料平衡验证降解途径，并利用宏基因组测序和生物信息学分析技术揭示关键阶段4-氯苯酚降解转化的种群功能和互作关系。全面系统揭示厌氧环境中2,4-D的彻底降解转化过程及微生物学机理，为发展绿色高效的2,4-D原位生物修复技术提供理论支持。

2,4-二氯苯氧乙酸（2,4-D）是具有潜在致癌风险的苯氧羧酸类除草剂，虽然已经被禁用，但近70年大量而广泛的应用导致其成为土壤和地下水中检出率很高的一种有机污染物。以往研究显示2,4-D在厌氧条件下的微生物降解往往不彻底，容易停滞于中间产物4-氯苯酚，氯酚类中间产物的积累给生态系统和地下水水质安全带来新的潜在威胁。本项目构建了2,4-D厌氧降解微生态系统，并发现以2,4-D为唯一底物时，其通过醚键断裂和一系列还原性脱氯反应经由2,4-二氯苯酚、4-氯苯酚被转化为苯酚，苯酚进一步被完全转化为甲烷和二氧化碳。解决了以往研究中2,4-D的不完全降解和有毒氯酚类中间产物的累积问题。本项目进一步聚焦2,4-D厌氧降解的关键步骤，建立以4-氯苯酚为底物的富集培养体系，结合16S rRNA基因高通量测序和qPCR分析等分子生物学手段，揭示关键降解步骤4-氯苯酚到苯酚的还原性脱氯反应，由一株具有全新功能的脱卤杆菌（Dehalobacter）菌株实现。利用宏基因组数据分析，本项目分箱组装出脱卤杆菌4-CP菌株、丁酸互营菌（Syntrophorhabdus）等18个菌株的全基因组，功能注释和进化分析确定脱卤杆菌含有41个脱卤酶，并推测脱卤酶Dhb.peg.2117可能催化4-氯苯酚还原脱氯转化为苯酚。深入的功能注释分析展示富集培养物基因组含有苯酚通过厌氧发酵产乙酸降解通路的完整功能酶基因，这些催化酶大多集中于丁酸互营菌上，同时追踪降解终产物确认，13C标记苯酚全部转化为甲烷和二氧化碳，电子回收率为109%，这些结果揭示互营菌和产甲烷菌互营代谢驱动苯酚的厌氧发酵。进而阐明脱卤杆菌，丁酸互营菌和产甲烷古菌协同完成4-氯苯酚厌氧降解的微生物学机制，最终实现2,4-D的彻底降解转化。本项目揭示了2,4-D在典型生物地球化学条件下的厌氧完全降解过程及降解菌种群的结构与功能，为2,4-D在厌氧环境中的归趋提供新的信息，从而为发展绿色高效的2,4-D原位生物刺激和生物修复技术提供理论支持。