咸淡水水域沉积物硝化反硝化作用释放氧化亚氮（N2O）的微生物适应机制研究

Nitrous oxide (N2O) is an important greenhouse gas (GHG). The microbial conversion of input nitrogen to N2O in aquatic ecosystems is suspected to be one of the largest sources of global N2O emission. The conventional biological nitrogen removal process employs nitrification followed by denitrification. However, hydroxylamine (NH2OH) oxidation, nitrifier denitrification were involved in N2O emission in recent studies. Therefore, NH2OH oxidation, nitrifier denitrification and heterotrophic denitrification are considered as three main biological pathways producing N2O during biological nitrogen removal processes. However, the N2O production from the three pathways was varied from different environments. Therefore, the identification of the key pathways involved with N2O emission is essential for establishing a strategy to mitigate N2O emission in different environments. . The unique gradient environment is formed from freshwater of urban rivers in Ningbo city to saltwater of Zhoushan's coastal area, which is considered as an ideal laboratory to study the relationships between N2O production, microorganisms and environmental factors. Our previous results showed that the interesting differences of N2O production, ammonia-oxidizer and denitrifier diversities in sediments were observed between freshwater, brackish water and saltwater, and the differences were also found between slight pollution areas and heavy pollution areas. However, the real active function microorganism groups and key nitrous oxide production pathways are still not clearly known in the sediments under different area waters. We hypothesize that the different microbial mechanisms of N2O emission exist in the sediments of freshwater and coastal areas. . In this study, spatial and temporal variations of ammonia-oxidizers and denitrifiers based on RNA of in situ collection will be investigated. At the same time, the microcosm experiments will be carried out, using the methods of 15N stable isotope analysis, real-time gas-monitoring analysis and microscale N2O concentration measurements, combing with functional gene transcriptome analysis,454- pyrosequencing. The aims of this project will be: (1) To compare the discrepancies of the contributions to N2O emission between nitrifier denitrification and heterotrophic denitrification; (2) To study the relationships among active denitrifiers, denitrification activity and environmental factors; (3) To get the active gene for denitrifying. Finally, the mechanism of N2O emission with nitrification and denitrification will be clarified in the area of the freshwater and saltwater. The potential of pathways of N2O emission from nitrifier and denitrifier to adapt the environmental transmutations will be analyzed. The highly efficient denitrification microorganism clusters will be also selected. Our results will provide a scientific basis for the control of eutrophic problem of water bodies.

氧化亚氮（N2O）与温室效应和水体富营养化等问题密切相关，对其来源的探索是当前国际研究的热点与难点。近来研究表明，硝化作用（羟胺氧化和硝化菌硝化）与异养反硝化作用一样，也是产生N2O重要途径,但不同环境下两者对N2O释放的贡献不同。项目组研究表明，城市河流及河口近岸海水沉积物中，氨氧化菌和反硝化菌的组成与含量存在明显差异，其N2O释放量也随时空变化存在差异。为探明不同水域沉积物N2O释放分子生态学机制，本项目拟继续就宁波城区内河、甬江及周边海域所构成具有环境梯度水域，通过原位采集结合微宇宙培养试验，综合应用RNA反转录、高通量测序、气相色谱分析和氮同位素示踪等方法，比较淡水和咸水沉积物中硝化与反硝化作用对N2O释放的贡献和活性功能菌群及其对环境因子的响应；揭示沉积物中N2O释放途径、活性功能菌群对环境适应与进化规律，筛选出适合不同水域N2O释放的功能菌群，为治理水体富营养化等问题提供依据。

氮与水域富营养化、温室效应等环境问题密切相关，自然界中的氮素循环是由微生物所驱动，其多样性和功能受环境因子影响。本项目通过原位采集样品、野外小区和室内微宇宙培养等试验相结合，围绕近海水域氮素循环及其相关微生物生态学机理开展研究。应用现代高通量测序、气相色谱等多种分析方法，研究不同时空下咸淡水水域，包括富营养化城市内河和淡水湖、甬江流域、杭州湾入海口、舟山近岸海域及北移大红树林等不同生态系统中在促氮循环过程中含有与硝化与反硝化作用相关功能基因的微生物菌群分布特征及其与环境因子关系。结果表明，不同性质水域的硝化与反硝化作用是由不同的优势功能菌群调控，在氮代谢途径上所注释到与促氮循环相关的同源功能基因和酶丰度不同，对硝化与反硝化作用产生不同影响，并受各环境因子的制约，且根据不同促氮循环功能菌群受环境因子如碳和氮等综合影响可划分为两大类。其中，AOA-amoA，AOB-amoA，norB和nifH基因与总氮显著正相关，但与总碳显著负相关；nirK，nosZ和nxrB基因与总碳显著正相关，但与总氮显著负相关。研究初步探明了反硝化作用释放N2O的分子生态学机理，获得了不同性质水域硝化与反硝化作用的优势功能菌群及活性和环境因子的相关关系。将所建立的技术体系运用于富营养化城市内河生态修复，初步探明因全球气候变暖而北移的大红树生态系统对生物地球化学循环及微生物产生的影响。