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。

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.