硝酸盐还原条件下厌氧苯降解菌群的互营关系及电子传递机制研究

Benzene is proved to be carcinogenic, teratogenetic and mutagenic and consequently endanger human's health by contaminating underground water during the periods of production, transportation and storage. Anaerobic benzene biodegradation plays a significant role in groundwater remediation due to its higher feasibility and economic efficiency. Syntrophic associations have also suggested in enrichment cultures capable of anaerobic benzene degradation under sulfate-, nitrate- and Fe (III) - reducing conditions. Microbial communities and their interaction responsible for syntrophic benzene are little known and the underlying degradation mechanisms and electron transfer mechanisms are not fully understood. In this study, syntrophic benzene degradation will be studied using anoxic enrichment cultures under nitrate-reducing condition. Benzene degradation characteristics and pathways under nitrate-reducing conditions will be monitored by the analysis of gas chromatography-mass spectrometry (GC-MS), scanning electron microscopy and isotope fractionation. Microbial community composition and their interactions will be elucidated using metagenomics and protein-based stable isotope probe (protein-SIP) approach. Mechanisms of intermediates and electron transfer will be studied by conducting inhibition experiments of intermediate products e.g. hydrogen and acetate and constructing microbial fuel cell. This study will provide a theoretical basis for further understanding of syntrophic benzene degradation, and provide a new strategy for the remediation of benzene contaminated groundwater.

苯是一种具强烈的致癌、致畸和致突变作用的有毒化合物，其在生产、运输和储存中极易进入地下水环境，从而导致地下水的严重污染。因此苯厌氧生物降解在地下水修复过程中具更高的可行性及经济有效性。微生物的互营关系在硫酸盐、Fe3+和硝酸盐还原条件下的苯厌氧降解过程中发挥重要作用。然而对互营苯降解过程中的互营关系及种间电子传递机制的研究还不充分。本研究以硝酸盐还原条件下厌氧苯降解富集物为研究对象，通过质谱分析，扫描电子显微镜观察以及稳定性同位素分馏技术探索硝酸盐还原条件下厌氧苯降解特性及降解途径；利用宏基因组学和稳定性同位素蛋白探针（protein-SIP）方法，阐明厌氧苯降解中互营菌群的动态变化及相互作用关系；通过中间产物抑制实验及构建微生物燃料电池进的方法，探索厌氧苯降解互营菌间的底物和电子传递机制。本研究将为进一步认识厌氧互营苯降解过程提供理论依据，为地下水苯污染修复治理提供新的策略。

本项目利用宏基因组学，蛋白质组学、稳定性同位素蛋白探针（protein-SIP）方法及微生物电化学技术，阐明了厌氧苯降解菌与硝酸盐还原菌之间的互营代谢关系及种间电子传递机制。为研究苯降解特性，对化学计量关系分析发现，苯的降解过程同时耦合硝酸盐还原为亚硝酸以及硝酸盐完全反硝化作用产生氮气两个反应过程；为解析苯降解初始激活机制，采用二维稳定性同位素分析技术（2D-CSIA）对Δδ2H-Δδ13C的相对变化值Λ进行拟合，结果发现该降解体系中的Λ值与初始苯降解激活途径monooxygenase机制的Λ值比较接近，因此推测此培养体系中苯的初始激活是通过monooxygenase完成的；利用宏基因组学和宏蛋白质组分析，追踪13C标记的活性蛋白及活性物种，鉴别出了参与苯降解与硝酸盐还原的活性蛋白及其归属菌株，推测苯的降解首先在硝酸盐还原反应产生微弱氧的情况下激活monooxygenase，然后进入厌氧降解途径，这一过程是通过降解菌Clostridiles，Burkholderiales和Anaerolineales菌以及硝酸盐还原菌Azoarcus协同互营作用进行的，降解菌（Clostridiles，Burkholderiales和Anaerolineales）催化苯氧化产生的中间产物可以进一步被硝酸盐还原菌Azoarcus 利用，从而驱动厌氧苯降解耦合硝酸盐还原互营代谢过程。为解析厌氧苯降解菌与硝酸盐还原菌间的种间电子传递途径，进行了中间产物抑制实验及微生物电化学测量，结果表明硝酸盐还原条件下厌氧苯降解过程中同时存在通过中间代谢产物（氢气和乙酸）以及直接种间电子传递两种机制。本研究对硝酸盐还原条件下的厌氧苯降解互营过程进行了深入研究，为科学的利用这一微生物过程进行苯降解及污染位点的生物修复提供了理论指导。