基于厌氧甲烷氧化的水体中高氯酸盐和硝酸盐复合污染生物自养还原机制研究

The combined pollution of perchlorate and nitrate is increasely threatening to the water safety and human health, which has become one of the urgent environmental problems. The denitrification and biological autotrophic reduction of perchlorate based on anaerobic methane oxidation(AMD) had been comfirmed and preliminarily studied, but the related mechanisms are controversial. Therefore, we.will investigate the mechanism of perchlarate and nitrate removal from water by biological autotrophic reduction based on AMD and the response to the changing environmental conditions in membrane biofilm reactor (MBfR). Main research contents include: the coupling mechanism of AMD and biological autotrophic reduction of perchlorate and nitrate in methane-fed MBfR will be investigated deeply. To reveal the cooperative or competitive relationship betwwen them, the main microorganisms will be determined by molecular biology technology. The responses of microbial community structure, gene diversity and metabolic ability to changing environmental conditions will be emphasized. As a result, the process control strategy for biological autotrophic reduction of perchlorate and nitrate based on AMD will be established. The study could provide a theoretical supports for the bioremediation of perchlorate and nitrate pollution in water and exploit a new approach to reduce the emission of methane and mitigate the greenhouse effect.

当前高氯酸盐和硝酸盐复合污染日益严重，已开始威胁水体安全及人类健康，是亟待解决的环境问题。基于厌氧甲烷氧化的反硝化以及高氯酸盐生物自养还原过程均得到了证实和初步研究，但有关机理尚存在争议。为此，本项目拟以甲烷基质MBfR为反应器，开展基于厌氧甲烷氧化的水体中高氯酸盐和硝酸盐复合污染生物自养还原净化机制与变化环境下的响应机制，包括：深入研究甲烷基质MBfR中厌氧甲烷氧化耦合高氯酸盐和硝酸盐自养还原机制，结合系统微生物种群分子生物学检测，揭示主要功能微生物间协同或竞争作用关系；剖析变化环境条件下耦合体系微生物群落结构、基因多样性与代谢功能等响应机制，结合体系中各物质代谢模型的构建，确立体系稳定运行的调控策略。本研究可为水体中高氯酸盐和硝酸盐复合污染的生态修复提供方法参考和理论借鉴，更为甲烷减排缓解温室效应开拓新的途径。

本课题展开了反硝化型厌氧甲烷氧化（DAMO）微生物的厌氧批式培养和序批式反应装置的富集培养研究，并进一步利用移动床生物膜反应器（MBBR）探索厌氧氨氧化甲烷化反硝化耦合脱氮的过程和微生物学机制。在前期DAMO过程的研究基础上，利用膜生物膜反应器（MBfR）探索新型厌氧甲烷氧化耦合高氯酸盐还原过程以及微生物群落特征及代谢机制。.以厌氧混合污泥为接种进行DAMO微生物的厌氧批式培养，结果表明以NO3-为底物的DAMO微生物活性随培养时间而提高，NO3-平均还原速率达到1.022 mgN/g VSS·d-1。DAMO微生物比生长速率与底物NO3-浓度的关系符合莫诺方程；NO3-的最大比去除速率qmax为10.08 mgN/g VSS·d-1，半饱和系数Ks为12.88 mgN/L。然而，以NO2-为培养基的DAMO微生物对NO2-平均还原速率随培养时间降低至0.81 mgN/g VSS﹒d-1。无甲烷供给的厌氧培养对照实验证实，CH4作为碳源和电子供体对于DAMO微生物实现生物脱氮十分关键。.采用MBBR反应器以含Anammox微生物的悬浮填料以及DAMO培养物为接种污泥，NH4+和NO3-为底物，CH4为碳源和电子供体，成功实现厌氧氨氧化甲烷化反硝化脱氮。NO3-的还原速率达83.66 mgN/L·d-1，NH4+的氧化速率达62.67 mgN/L·d-1，反应器内无NO2-积累；Anammox细菌对NO2-的消耗占据NO2-总转化率的比例达到74%以上。荧光原位杂交分析结果确认，MBBR中存在DAMO古菌和大量Anammox细菌、DAMO细菌和甲烷氧化菌的共存生长，是厌氧氨氧化甲烷化反硝化的关键微生物种群。.以甲烷为外碳源和电子供体且高氯酸盐（ClO4-）为电子受体，利用MBfR研究厌氧甲烷氧化耦合高氯酸盐生物还原。结果表明，在MBfR中能够实现厌氧甲烷氧化耦合高氯酸盐的生物还原。当ClO4-浓度为5-10 mg/L时，ClO4-的去除效率达到100%，并保持稳定运行。实时荧光定量PCR分析得知，高氯酸盐生物还原的关键功能酶基因pcrA和nirS丰度以及厌氧甲烷氧化的关键功能酶基因mcrA和pMMO丰度，随着进水高氯酸盐浓度的提高而增长，且呈线性正相关趋势，说明MBfR中高氯酸盐还原菌和甲烷氧化菌通过厌氧甲烷氧化与高氯酸盐的生物还原过程密切耦合的生化代谢活动。