菜地土壤硝化反硝化过程及N2O排放的微生物响应机制

The residual soil nitrate content during the fallow season in the vegetable soils is usually higher, while the ammonium is the often used fertilizer form during the growing seasons in the other hand; therefore the relative intensity of nitrification and de-nitrification during the different seasons may vary in the vegetable soils. We use the combination methods of filed sampling, 15N labeled cultivation experiments, quantitative PCR of functional gene, high throughput sequencing and the followed multivariate methods to do the study. The following aspects would be investigated in this project: i) The size and community structure, the change of de-nitrifying bacteria (nirS and nirK) and its relationship with denitrification intensity and the relationship between nosZ gene and N2O emission when residual soil nitrate is higher in the vegetable soil. ii) The size, abundance, dominant strains and their change of ammonia oxidizing bacteria and ammonia oxidizing archaebacteria, also the related N2O emission flux with the associated proceeding of nitrification just after ammonia fertilizer is applied; The relationship of size and community structure and their change of denitrifying bacteria with the proceeding of nitrification-denitrification processes after ammonia is applied in the vegetable soil. iii) The impact of acidification on the size and community structure of nitrifying microrganisms and denitrifying bacteria and their relationships with the nitrification and denitrification processes and N2O emission. This project aims to reveal the microbial mechanisms of nitrification and denitrification in order to provide the scientific base for improving the nitrogen management and decreasing the nitrogen loss and N2O emission from vegetable soils.

菜地土壤非生长季的土壤硝态氮残留高，而在蔬菜生长季铵态氮肥施用量又很高，因此不同时期菜地土壤中反硝化作用和硝化作用的强度、消长及其相应的N2O排放来源有差异。本申请项目主要采用田间取样、15N标记培养实验、功能基因定量PCR、高通量测序及微生物分子生态学分析等方法研究在硝态氮高含量期菜地土壤nirS型、nirK型反硝化菌数量和组成的变化与反硝化强度的关系及nosZ型反硝化菌与反硝化过程中N2O排放的相关性；施肥后铵态氮高含量期氨氧化细菌和氨氧化古菌的数量、丰富度、优势种等的变化与硝化作用进程和N2O排放的关系及随后的反硝化微生物数量和组成的变化与硝化反硝化进程间的消长关系；并探索酸化菜地土壤硝化微生物和反硝化微生物在数量和组成方面的变化与相应的硝化反硝化过程与N2O排放的关系，以揭示菜地土壤硝化反硝化作用的微生物机制，为改进菜地土壤氮素管理、减少菜地土壤氮素损失及N2O排放提供科学依据。

本项目主要研究集约化蔬菜地在土壤无机氮以铵态氮为主期和硝态氮为主期不同N2O产生途径对土壤N2O排放的各自贡献及其参与硝化作用和反硝化作用的主要微生物在数量、多样性、组成结构方面的变化，以阐明菜地土壤N2O排放的生物学过程及其微生物学机理。.主要结论如下：.①土壤中铵态氮含量较高期是菜地土壤中N2O排放的主要时期；硝态氮较高期的N2O排放占N2O总排放量的比例较低，该比例在石灰性菜地和酸性菜地分别为7.2%和28.0%(实际绝对值很低)。.②总体来讲，不同利用方式的土壤中，2种硝化微生物AOB（氨氧化细菌）和AOA（氨氧化古菌）以及3种反硝化微生物(nirS、nirK、nosZ)的数量均为菜地土壤的拷贝数高于典型玉米地，且菜地的AOB多样性指数也高于典型玉米地；此外，不同利用类型土壤中功能微生物的优势种组成及占比有所区别。.③施肥后石灰性菜地中AOB的优势属分别为Nitrosomonas(占比34.5-41.8%)、Nitrosospira (占比49.1-52.0%)和Nitrosomondaceae(占比8.0-13.2%)；酸性菜地中AOB的优势属分别为 Nitrosospira(占比69.2-75.5%)和Nitrosomondaceae (23.0-28.3%;而可鉴别的反硝化微生物的优势属或种在玉米地和菜地中没有显著差别。.④菜地土壤AOB与硝化作用中N2O排放贡献的一致性主要体现在不同时期的优势属所占比例不同：石灰性菜地中Nitrosomonas为优势属，并与来自自养硝化作用和硝化细菌反硝化作用的N2O较高排放出现在同期；而酸性菜地中则以Nitrosospira和Nitrosomonadaceae为优势属，并与其来自自养硝化作用N2O排放较低(占比5.9%)而来自包括异养作用在内的其它过程产生的N2O排放占比略高(占比21.5%)相对应，且这些AOB可能对低铵和高硝环境有一定的适应作用，使得来自硝化细菌的反硝化作用对N2O排放有不可忽视的贡献(占比37.9%)。.⑤反硝化微生物与反硝化作用N2O排放的相关性主要体现在不同反硝化微生物的数量及多样性方面：石灰性菜地来自反硝化作用的N2O排放主要与nirS型和nirK型反硝化微生物的数量及nirS型反硝化微生物的多样性增加有关；酸性菜地中反硝化作用的N2O排放与nirS型和nosZ型微生物的数量增加相一致。