稻田厌氧氨氧化的适应性调节机制及其对土壤氮气产生的调控作用研究

Anaerobic ammonium oxidation (anammox) is an important nitrogen removal pathway in aquatic systems. As compared with these reported aquatic systems, the activity and significance of anammox bacteria in terrestrial realm is not well studied. Based on field experiment of paddy-upland rotation, here, we will study the rate and activity of anammox in a typical paddy ecosystem and its response to soil environmental stress using stable isotope labeling experiments, fluorescence quantitative PCR, 13C-PLFA, and high throughput sequencing. Furthermore, the diversity and abundance of anammox bacteria in different fertilizer treatments soils will be investigated over the entire rice growth season. The soil moisture, structure of organic matter and soil chemical properties will be measured to determine the factors influencing the anammox process in paddy rice. The primary objective of the present study is to investigate the relationship between the anammox rate, activity with the characteristics of soil environmental factors in paddy fields. Therefore, the result of our study will give the suggestion how to regulate the soil dinitrogen production of the anammox process. The expected results of this project will supplement the theoretical system of nitrogen cycling and provide a theoretical basis for revealing the circulation pattern and mechanism of nitrogen in rice paddy ecosystem.

目前，厌氧氨氧化过程作为公认的脱氮途径广泛存在缺氧的自然环境中，但它在水稻氮素循环的作用和地位仍缺乏深入了解。本项研究主要以水旱轮作水稻肥料定位试验为基础，联合室内培养，运用同位素标记技术和分子生态学等多种先进技术，研究典型稻田生态系统土壤厌氧氨氧化的速率与活性及其对土壤环境胁迫的反应，探讨长期环境胁迫条件下稻田土壤厌氧氨氧化速率与活性、微生物丰度与群落结构多样性的定向变化规律，解析环境胁迫对稻田土壤厌氧氨氧化过程的影响机制，明确稻田土壤厌氧氨氧化活性对土壤环境胁迫的适应性调节机制，阐明这种适应性调节机制对稻田生态系统土壤氮气产生的调控作用。本项目预期成果将对陆地氮循环理论体系进行补充，也为揭示氮素在水稻生态系统中的循环模式与机制提供理论依据。

本研究以水旱轮作稻田土壤Anammox过程为主线，综合运用15N同位素示踪技术、荧光定量PCR、DNA-SIP、克隆文库和高通量测序等技术，（1）以长期定位肥料试验基础，探明了在水稻和小麦季节的Anammox细菌活性，明确了施有机肥或化肥对Anammox细菌群落结构的影响；通过对AOB和AOA的定量PCR检测，发现AOB和AOA具有提供水稻-小麦轮作体系中Anammox过程所需的NO2-的作用；通过环境因子与功能基因之间的相关性分析，发现土壤环境因子如有机碳和hzs-β基因之间的负相关性。（2）通过室内培养试验，明确了有机碳源的添加减少了土壤活性Anammox细菌的丰度，但对Anammox细菌的群落结构无明显影响；发现外源有机碳后的土壤Anammox速率显著降低，但促进了反硝化活性，反硝化与Anammox竞争来抑制Anammox过程。（3）通过不同水分胁迫培养试验，发现淹水和干湿交替对Anammox活性的激发作用，但水分条件下对Anammox细菌群落结构没有影响，提出可通过改变田间水分条件和水中的溶解氧，对土壤Anammox速率起到一定的调节作用。（4）通过分析PLFA浓度和进一步通过定量PCR确认来评估Anammox的存在和活性，建立了13C-尿素-PLFA的厌氧培养研究厌氧菌微生物群落结构的方法，提出了Anammox的生物标志物13C磷脂脂肪酸。以上结果对完善稻田生态系统中氮循环过程及相关机理提供理论支撑，为优化农业生产施肥模式提供理论支撑。发表论文 12 篇，其中IF5y>10论文4 篇；授理与授权专利7项，培养研究生3名。