盐度对EGSB反应器同时产甲烷反硝化影响及其分子生态学机理研究

With the development of traditional anaerobic-anoxic-aerobic technology, simultaneous methanogenesis and denitrification (SMD) have become a new type process which can achieve synchronous removal of nitrate nitrogen and carbon in a single anaerobic reactor. It has attracted extensive concern because of its some advantages such as saving carbon source, simple process, strong ability to resist loading. However, few concerns currently focus on the integration of SMD in a single reactor, and the effect laws of the salinity, environmental conditions and process parameters are not clear, especially the corresponding molecular ecological mechanisms remain unknown. To attain the application of the coupled reaction in wastewater treatment, studies have to be conducted to reveal the microbiological mechanisms of the coupling reaction, as well as the microbial diversity and functions in the coupling reaction systems. This project, based on isolation of methanogenesis and denitrification bacteria, characterization of carbon/nitrate nitrogen removal efficiency, and qualitative and quantitative analysis of their functional genes, aims to investigate the effects of various environmental and operational parameters on performance of SMD in an expanded granular sludge bed (EGSB) reactor and the responses of microbial community structure and methanogenesis/denitrification genes abundance to the changing salinity/factors/parameters. Approaches like high-throughput sequencing including Pyrosequencing and Illumian, metagenomics analysis, and real time polymerase chain reaction will also be adopted to reveal not only the relationship between methanogenesis and denitrification bacterial communities, but also the relationship between methanogenesis/denitrofication genes distribution and microbial community structure during the start and running of the reactors, so as to explore the potential molecular ecological mechanisms underlying the coupled reaction of SMD. This project wishes to provide new theoretical and practical supports for the efficient and economical treatment of high salinity and high nitrate wastewater.

同时产甲烷反硝化工艺是在传统厌氧-缺氧-好氧工艺基础上发展起来的一种新型脱碳除氮工艺，因具有节省碳源、工艺简单、抗负荷能力强等优点引起广泛关注。目前国内外对于单一反应器中同时产甲烷反硝化耦合工艺处于起步阶段，其盐度和工艺条件等影响规律不明确，尤其是分子生态学机理尚不清楚。本项目拟从产甲烷菌和反硝化菌富集和筛选、脱碳除氮性能表征和功能基因定性/定量检测等方面研究不同盐度、环境条件和工艺参数对EGSB反应器中同时产甲烷反硝化反应的影响，分析菌群结构和功能基因丰度对盐度、环境条件和工艺参数变化的响应机制；同时应用Illumina与Pyrosequencing高通量测序、宏基因组学分析、实时定量PCR 等方法研究反应器启动和运行过程中产甲烷菌群和反硝化菌群之间的关系，以及功能基因分布与菌群结构之间关系，探索产甲烷和反硝化耦合的潜在分子生态学机理，为高盐高硝酸盐有机废水生物处理提供理论与技术支撑。

同时产甲烷反硝化工艺是在传统厌氧-缺氧-好氧工艺基础上发展起来的一种新型脱碳除氮工艺，因其具有节省碳源、工艺简单、抗负荷能力强等优点引起被人们广泛关注。本项目主要研究了不同接种污泥、不同反应温度、不同碳源、不同硝酸盐浓度与不同盐度对膨胀颗粒污泥床反应器（EGSB反应器）同时产甲烷反硝化耦合的影响，以及通过应用 Illumina 高通量测序、宏基因组学等研究EGSB反应器启动和运行过程中产甲烷菌群和反硝化菌群之间的关系，以及功能基因分布与菌群结构之间关系，探索了产甲烷和反硝化耦合潜在分子生态学机理，为高盐高硝酸盐有机废水生物处理提供新的技术与理论支撑。其主要研究结果如下：.通过不同接种污泥对EGSB反应器启动的研究发现，采用厌氧污泥接种启动较快；EGSB反应器同时产甲烷反硝化耦合的适应反应温度为15-35 ℃；在HRT为18 h、pH值为7.2±0.1、Vup为3.0 m/h，COD/NO3--N比值大于等于2时，其同时产甲烷反硝化耦合效果较好。而碳源对同时产甲烷反硝化的影响不是非常明显，但相对来说乙酸钠为碳效果较好，通过高通量测序与宏基因组学分析发现不同的碳源其微生物多样性与微生物菌群结构多样性以乙酸钠为碳源的污泥中微生物多样性最丰富，其优势菌门为变形菌门（Proteobacteria），其占比达到63.43%，其次是葡萄糖为碳源，其优势菌门变形菌门（Proteobacteria），其占比达到47.69%；以乙酸钠与葡萄糖为碳源时，其主要功能基因为：napA, narG，其浓度分别达到785ppm、624ppm与448ppm、372ppm。.通过对进水硝态氮负荷的研究发现：当硝态氮容积负荷为0.93-1.45 kg/m3•d时，EGSB反应器内同时产甲烷反硝化工艺运行效果最好。并通过高通量测序分析发现：当硝态氮容积负荷为1.45 kg/m3•d时，EGSB反应器内的微生物多样性最丰富。在菌门水平上分析发现对同时产甲烷反硝化起主要作用的优势菌门为变形菌门（Proteobacteria），其占比都达到了50%以上；在菌属水平上分析发现：反硝化的优势菌属为：Pseudomonas（28.37%），Halomonas（20.71%），Thauera（13.28%），产甲烷的优势菌属为：Methanosaeta（0.21%），Methanobacterium（0.09%）。