新型反硝化脱氮中自养/异养菌群稳定同位素探针识别及作用机制

One of the major concerns in biological removal of nitrogen is to construct stable and efficient functional bacteria. Specially, in novel denitrification processes applicable to waste water with low C/N ratio, autotrophic and heterotrophic denitrifying bacteria perform competition and interaction in community balance. However, due to the limitation of traditional isolation/culture and molecular techniques, it is difficult to identify the autotrophic or heterotrophic function of denitrifying bacteria in situ, and is thus hard to reveal the interaction of the autotrophic or heterotrophic bacteria. DNA-based stable isotope probing technology (DNA-SIP), which was developed in the field of microbial ecology, provides effective method for coupling analysis of microbial community characteristics and physiological function. In this study, DNA-SIP is introduced to the field of waste water treatment for the first time. Considering the microbial characteristics of autotrophic and heterotrophic bacteria, an in-situ identification method for autotrophic and heterotrophic functions of denitrifying bacteria is going to be constructed, by using stable isotope probing of H13CO3- and ultracentrifugation of DNA. And then based on a novel Fe-C internal electrolysis denitrification process with inorganic carbon, organic carbon, and nitrate, coupling analysis of microbial function and community structure of autotrophic and heterotrophic denitrifying bacteria will be performed with combination of DNA-SIP and high-throughput sequencing. This study can further reveal the interaction between autotrophic and heterotrophic denitrifying bacteria in various conditions of water quality, and thus provide theoretical and practical basis for microbial community construction and optimization in novel denitrification processes.

构建高效的微生物功能菌群是生物脱氮的技术瓶颈。特别是适用于低碳氮比污水的新型反硝化工艺中，自养/异养反硝化菌间存在平衡、竞争与转化作用，但由于传统分离培养和分子生物技术的局限，难以原位识别反硝化菌的自养/异养功能，无法揭示自养/异养菌群独立与相互作用机制。微生物生态学领域先进的稳定同位素核酸探针技术，为复杂环境中微生物种群特征及其生理功能的耦合分析提供了有力工具。本研究首次将该技术应用于水处理领域，针对自养/异养菌群的微生物学特征，利用13C同位素标记碳酸根和DNA超高速离心分离，建立反硝化菌群自养/异养功能的原位识别方法；进而基于新型铁-碳内电解生物反硝化工艺，综合同位素探针识别和高通量测序，解析在无机碳、有机碳和硝酸盐等共存的复合水质中，自养/异养反硝化菌群的功能特征和群落结构，揭示其自养/异养代谢类型在不同水质中的相互关系，为新型反硝化脱氮工艺种群构建与优化调控奠定理论与实践基础。

传统的异养反硝化以有机物作为能源和碳源，当进水有机物不足时，脱氮效率受到明显影响，需要外加有机碳源。而自养反硝化以无机物为电子供体，利用无机碳源完成反硝化过程，近年来得到了越来越广泛的关注。构建高效的微生物功能菌群是生物脱氮的技术瓶颈，应用于低碳氮比污水的新型自养反硝化工艺中，自养/异养反硝化菌间可能存在竞争或协同作用。然而，利用传统微生物学研究方法难以原位识别反硝化菌的自养/异养功能，无法揭示自养/异养菌群的相互作用机制。本研究首先建立新型铁-碳微电解自养反硝化工艺，利用原电池反应生成H2和Fe2+作为无机电子供体，在无需通电的条件下实现自养反硝化过程；在此基础上，将稳定同位素核酸探针（DNA-SIP）技术应用于污水脱氮领域，针对自养/异养菌群的微生物学特征，利用13C同位素分别标记无机碳源（碳酸氢钠）或有机碳源（乙酸钠），建立反硝化菌群自养/异养功能的原位识别方法；进而解析在无机碳、有机碳和硝酸盐等共存的复合水质中，自养/异养反硝化菌群的功能特征和群落结构。. 本项目在有机碳源不足的水质条件下，获得了高效脱氮效率，在低碳氮比（COD/N<1）的条件下，可稳定达到95%以上的硝酸盐去除率和85%以上的总氮去除率。该工艺解决了传统异养反硝化脱氮工艺中外加有机碳源时，投加量不易控制、运行成本较高等不足，在城市污水处理厂深度脱氮方面具有十分广阔的应用前景，也可扩展应用于地下水硝酸盐去除、地表水富营养化控制等相关领域。此外，本项目将DNA-SIP技术引入新型反硝化脱氮研究中，成功建立了基于稳定同位素13C标记的自养/异养反硝化菌群原位识别方法，实现了对反应器内自养和异养菌群的有效识别；结合高通量测序、实时定量PCR等分子生物学技术，明确了自养反硝化核心菌群特征，包括自养型Thiobacillus、异养型Thauera等，并深入解析了不同COD/N对自养/异养反硝化菌群的影响规律。本研究为新型污水脱氮工艺的发展做出贡献，为反硝化脱氮工艺的种群构建与优化调控奠定了理论与实践基础。