辽河口氮循环关键过程的微生物驱动机制研究

The excessive input of inorganic nitrogen is one of the main reason for eutrophication. As the most active factor in the nitrogen cycle, the function of microorganisms has been a research hotspot nowadays. However, the driving mechanism of microorganisms in the nitrogen cycle is still unclear, and what we knew was only above the theoretical level. The project team has already found that there was a positive correlation between nitrification/denitrification rate and the abundance of nitrifying/denitrifying bacteria. Estuaries are major conduits for the transport of anthropogenically derived nitrogen from land to sea, so we plan to choose Liaohe estuary to carry out field investigation combined with laboratory culture experiments to reveal the characteristics of nitrification and denitrification by using 15N isotope labeling methods. Changes of community structure of ammonia-oxidizing and denitrifying microorganisms will be analyzed by the methods of High-throughput sequencing and establishing the clone library, and abundance of different microbial groups and related Genes will be determined by real-time quantitative PCR method, and then illuminate their contribution to nitrification and denitrification. Real-time quantitative reverse transcription PCR will be utilized to detect the expression of functional genes, and then to explore the expression of microbial functional genes. In addition, we will try to illuminate its effect on the nitrogen cycle by analyzing the response of microorganisms to environmental factors and the relationship between their community structure composition and the nitrification and denitrification rate. The project aims to provide both scientific theroy and experiment data basis for elucidating the microbe-driving mechanism of key nitrogen cycle processes in estuary.

无机氮的过量输入是导致近海富营养化的主要原因之一，微生物作为氮循环过程中最为活跃的因素，其在氮循环中的作用已成为目前的研究热点，然而微生物在该过程中的驱动机制尚未明确。项目组在前期研究中发现硝化细菌和反硝化细菌的丰度与硝化和反硝化速率呈显著正相关。河口是陆源氮进入海洋的主要渠道，本项目以辽河口为研究区域，开展现场调查并结合室内培养实验，采用15N同位素示踪法揭示河口区域硝化和反硝化作用特征；采用高通量测序和克隆文库的方法研究微生物的群落结构变化；采用实时定量PCR方法测定微生物及功能基因的丰度，分析不同类群微生物在硝化和反硝化过程中的贡献；采用反转录实时定量PCR方法，检测微生物功能基因的诱导表达情况；通过探究微生物对环境因子的响应及其群落结构组成与硝化和反硝化速率的关系，阐明其对氮循环的驱动作用。本研究将为阐明河口氮循环关键过程的微生物驱动机制这一科学问题提供理论和数据依据。

无机氮的过量输入是导致近海富营养化的主要原因之一，微生物作为氮循环过程中最为活跃的因素，其在氮循环中的作用已成为目前的研究热点。项目依照研究计划与方案实施各项既定研究任务，完成了辽河口4个航次和渤海1个航次样品的收集，同步分析了环境参数和理化因子。调查了近岸海域沉积物中的氮反应速率、功能基因丰度、功能微生物群落结构以及对氮循环的贡献等，获得了宝贵的微生物信息。采用现场调查结合室内培养的方法，分析了辽河口沉积物中硝化速率、反硝化速率和厌氧氨氧化速率；采用高通量测序和克隆文库构建的方法，分析了渤海和辽河口沉积物中硝化和反硝化关键过程功能微生物的群落结构变化；采用实时定量PCR技术测定微生物及功能基因的丰度，分析了不同类群微生物在硝化和反硝化过程中的贡献；采用反转录实时定量PCR技术，解析了微生物功能基因的转录表达情况；通过探究微生物对环境因子的响应、群落结构组成与硝化和反硝化速率的关系，阐明了微生物对氮循环关键过程的驱动作用。项目的完成丰富了河口生物地球化学循环中氮循环的理论，对河口富营养化治理具有重要的科学意义。项目共培养硕士生4名，发表学术论文11篇，其中SCI收录5篇， EI收录1篇，中文期刊收录5篇，主要发表在《海洋学报》、《Marine Pollution Bulletin》、《Geomicrobiology Journal》、《Acta Oceanologica Sinica》等国内外海洋领域重要刊物上。项目执行期间，共17人次参加国内外学术会议交流，丰富了该领域的理论认知。