农业废物堆肥氨氧化细菌（AOB）和古菌（AOA）对基质微环境变化的响应特征及驱动氨氧化的关键因子分析

The transfer and transformation characteristics of nitrogen and the related microbial communities during agricultural waste composting have been attracting worldwide attentions. However, the researches on the enzyme expression differences between ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA), as well as the characteristics of dynamic response of AOB and AOA to the variations of micro-environmental conditions and the key driving factors during agricultural waste composting are scarce. In this project, the functional amoA gene encoding a subunit of the ammonia monooxygenase enzyme responsible for the first step of the nitrification process, will be used as study subjects. The in situ difference of amoA gene expression for AOB and AOA, as well as the effects of amoA gene abundance, structure, and in situ amoA gene expression on the potential ammonia oxidation capacity will be investigated through molecular biology techniques combined with conventional microbiological physiology methods. The main point will be focused on the characteristics of dynamic responses of AOB and AOA to the variation of micro-environmental conditions, at the meantime, multivariate analyses were performed to test which factors have significant influence on the communities of the ammonia-oxidizing microorganisms, and provide statistical tests for those correlations. Significant proportion of shared variation can be assigned to particular parameter by variation partitioning analysis after elimination of possible effects due to other composting parameters. The results will provide scientific basis for the research of ammonia oxidation microorganisms and their functions, and instruct new theoretical directions for the safety evaluation and scientific management of nitrogen cycle during agricultural waste composting.

农业废物堆肥化过程中氮素转移转化规律及相关功能微生物种群特性已引起学者广泛关注。然而，针对堆肥化过程中氨氧化细菌和古菌种群在堆肥微环境条件变化的影响下对基质中氨的微生物氧化的功能活性差异特征及关键影响因子还知之甚少。本项目以编码催化氨氧化第一步反应的氨单加氧酶基因（amoA）为研究对象，采用分子生物学和传统微生物生理学技术相结合的方法，研究氨氧化细菌和古菌在农业废物堆肥化不同阶段amoA基因原位表达活性的差异；掌握细菌和古菌amoA基因数量、组成及其原位表达活性对堆肥基质潜在氨氧化能力的影响规律；重点考察氨氧化细菌和古菌种群在堆肥基质微环境变化的影响下驱动氨的氧化规律特征，并通过多元分析方法找出驱动氨氧化细菌和古菌amoA基因动态变化的关键理化因子。本项目有望为堆肥过程中氨氧化微生物种群及其功能特性的研究和氮素循环的科学管理提供理论指导。

农业废物堆肥化过程中氮素转移转化规律及其相关功能微生物种群特性已引起国内外研究者的广泛关注。然而，针对堆肥化过程中氨氧化细菌和古菌种群的活性差异特征，及其在堆肥微环境条件变化和胁迫下对基质中氨的微生物氧化的驱动机制，目前还缺乏相关研究。. 本项目首先使用聚合酶链式反应-变性梯度凝胶电泳技术研究了堆肥化过程中古细菌的种群动态变化特征。通过对种群动态变化特征和氨氧化速率的探究，了解了古细菌种群多样性与与堆肥基质潜在氨氧化活性的关系。. 进而基于生物信息学方法，从NCBI核酸数据库中共下载堆肥化过程中氨氧化古菌AOA的amoA基因序列，利用Muthor、Mega6.0 和ClustalW等生物信息学相关工具，结合已经发表的相关论文，通过统计学相关方法，得出AOA在不同堆肥系统中的分布特征，包括丰度分布和多样性分布特征。. 并通过尝试在堆肥化过程中添加外源白腐真菌（黄孢原毛平革菌，P. chrysosporium），研究不同接种模式下的农业废物堆肥化过程中氨氧化细菌种群的数量与组成变化特征。以编码催化氨氧化第一步反应的氨单加氧酶基因（amoA）为研究对象，重点考察氨氧化细菌种群在不同外源白腐真菌接种体系中微环境变化的影响下驱动氨的氧化规律特征，并通过多元分析方法找出驱动氨氧化细菌amoA基因动态变化的关键理化因子。. 模拟了农业废物好氧堆肥的方法进行堆肥，借助荧光定量PCR分析技术对堆肥过程中的反硝化功能基因nirK、nirS及nosZ的基因丰度进行测定，采用SPSS数据统计软件对堆肥理化参数，如：温度含水率、水溶性有机碳（WSC）、pH、含水率等参数，与反硝化功能基因丰度进行相关性分析，然后根据相关性分析结果挑选出与基因丰度显著相关的环境因子与相对应的基因丰度进行曲线估计，得出最适合的模型。