CANON过程外加N2H4抑制N2O产生的微生物代谢机制研究

Completely autotrophic ammonium removal over nitrite (CANON) process has the advantages of energy conservation and consumption reduction and with the simple process. But the effective controlling of N2O production, which is the strong green house gas, restricts the engineering application. A more effective process control and a more comprehensive analysis for N2O metabolism are key ways to resolve this difficult problem.It is noted that trace N2H4 addition promoted the nitrogen removal efficiency and inhibited N2O production simultaneously. In order to expound metabolism of key enzymes and related bacteria, the following research are intended to carry out:firstly, building CANON process system, by controlling concentrations of N2H4 added to optimize the process with high-efficiency nitrogen removal and low N2O emission; secondly, under high-throughput sequencing method, Heatmap analysis is used for the change of structure and function diversity of flora before and after N2H4 addition in the system, and combined with metagenome sequencing analysis, through KEGG metabolic pathway annotation and technical means are to reveal genetic diversity and identify unknown enzymes which inhibit N2O production in the system before and after N2H4 addition; and finally, based on above results, further study by metatranscriptome sequencing analysis through gene expression profile analysis and technical means are to clear key enzymes that inhibit N2O production and the related metabolic mechanism. The research results will complement the mechanism of N2O formation of CANON process, and provide theoretical support for the realization of the synergetic CANON process with high-efficiency nitrogen removal and low N2O emission.

完全自养脱氮（CANON）工艺具有节能减耗、工艺流程简捷等优势，但过程中强温室气体N2O的有效减排一直是阻碍其工程应用的瓶颈，更有效的工艺控制和更全面的N2O代谢机理解析是攻克此难题的关键。申请人前期发现外加微量N2H4提高CANON工艺脱氮效率同时显著抑制N2O产生。为揭示该过程的关键酶及相关菌群代谢机制，拟开展以下研究：构建CANON工艺系统，通过调控外加N2H4浓度，得到高脱氮率、低N2O排放的优化工艺；采用高通量测序手段，经热图分析等解析外加N2H4前后系统中菌群结构变化与功能多样性，结合宏基因组测序分析，经KEGG代谢通路注释等揭示基因多样性，辨识抑制N2O产生的未知酶系；基于以上结果，利用宏转录组学手段，经基因表达谱分析等明晰抑制N2O产生的关键酶及与之相关菌群的代谢机制。研究成果将完善CANON过程的N2O生成机制，为实现高效脱氮和N2O减排协同的CANON工艺提供理论支撑。

完全自养脱氮(CANON)工艺具有节能减耗、工艺流程简捷等优势，但过程中强温室气体一氧化二氮(N2O)的有效减排一直是阻碍其工程应用的瓶颈，更有效的工艺控制和更全面的N2O产生及释放机制解析是攻克此难题的关键。研究以基于亚硝化和厌氧氨氧化分步启动的CANON-SBR系统为对象，具体解析了外源联氨(N2H4)对亚硝化、厌氧氨氧化过程中N2O产生及释放机制、微生物菌群特性开展研究，目前获得以下重要结果结论：（1）外源N2H4对CANON过程好氧亚硝化途径的N2O产生存在显著抑制，尽其管对N2O产生量有减缓作用，但同时也对好氧氨氧化速率存在一定影响。CANON过程中亚硝化阶段的N2O产生释放，需要同时考虑羟胺(NH2OH)对亚硝化效率的影响；（2）开展NH2OH影响亚硝化过程的影响，明晰了NH2OH作用规律，即外源NH2OH通过强化好氧氨氧化活性（保持AOB稳定富集以及促进异养硝化-好氧反硝化（HN-AD）菌生长富集）、抑制亚硝酸盐氧化菌生长及活性，实现高效亚硝化过程；（3）进一步考察HN-AD菌对亚硝化阶段N2O产生的影响，发现其对亚硝化阶段的N2O产生具有一定减缓作用；(4)接种厌氧氨氧化（ANAMMOX）工程菌泥，成功启动运行ANAMMOX颗粒SBR工艺，外源N2H4对ANAMMOX过程的N2O产生存在短期添加强化和长期添加抑制的相异作用;（5）宏基因组分析表明，在ANAMMOX工艺系统中，部分菌能在厌氧条件下产生N2O，外加N2H4更有利于抑制N2O的产生，加入微量N2H4可有效促进Anammox过程的蛋白质合成与利用，进而加快氮代谢过程，减少N2O产生;（6）总结现有阶段性结果发现，在自养系统中共存HN-AD菌、且其对自N2O产生具有减缓作用。为长效发挥外加N2H4的强化作用，针对CANON工艺启动及稳定运行周期较长的问题、以及聚焦HN-AD菌相关特性进行初探，目前成功构建了不同供氧模式下的新型膜曝气生物膜反应器（MABR）并获得稳定的具有HN-AD功能的MABR系统。

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