酸性农田土壤氨氧化古菌的生态位分异研究

The discovery of Ammonia-oxidizing archaea (AOA) is a major breakthrough in the global nitrogen cycling research. AOA are ubiquitous in terrestrial ecosystems, have unique mechanisms for nitrification, better adaptation to low-pH pressures, and strikingly lower ammonia requirements compared with ammonia-oxidizing bacteria. Recent studies of AOA suggested their significant contributions to global nitrogen cycling in acidic soils. However, the response and adaptation of different AOA populations (Group 1.1a-associated, Group 1.1b and Group 1.1a clusters) to environmental factors (e.g. substrate concentration, pH) is complex, and the niche differentiation of AOA in acidic soils still remained mysterious. Four long-term fertilization soils (amended with mineral fertilizers) and four unfertilized soils will be chosen in this study. Based on in situ investigation and laboratory simulation incubation, DNA-stable isotope probing method and 454 high-throughput pyrosequencing technology will be employed to analysis the DNA sequences of archaeal amoA and 16S rRNA genes. The objective is to investigate the influence of long-term fertilization on acidic soil AOA communities (especial on functional active AOA populations) at the high-throughput level. A microcosm experiment will also be established to analysis the growth and nitrification function of different AOA populations in acidic or in neutral environments. The objective of this study is to explore the adaption of different AOA populations to pH. Results of this study will be beneficial to understand the niche differentiation (e.g. community distribution, physiological metabolism, environmental adaptation) of AOA in acidic farmland ecosystems and to realize the global nitrogen cycling driven by microorganisms.

氨氧化古菌（AOA）的发现是全球氮循环研究的重大突破，研究表明AOA在酸性土壤硝化作用中发挥重要作用。然而，Group 1.1a-associated、Group 1.1b等不同AOA类群对底物浓度、pH等环境因子的响应和适应性明显不同，其生态位分异也较为复杂。本研究拟选取长期施无机肥处理和不施肥处理的酸性土壤为研究材料，基于原位调查和实验室模拟微域培养，采用稳定同位素核酸探针和454高通量测序技术分析古菌amoA和16S RNA基因序列，从高通量水平上研究长期施无机肥处理对酸性土壤AOA群落（特别是功能“活跃的”AOA类群）的影响；本研究还设置酸性和中性环境的微域培养实验，分析不同AOA类群在酸性和中性环境中的生长及功能活性，研究AOA对pH环境的选择适应性。该研究结果有望推进对酸性农田土壤AOA群落分布、生理代谢、环境适应性等生态位特征的认识，深化对酸性土壤微生物驱动氮素循环的认知。

氨氧化古菌（AOA）的发现是全球氮循环研究的重大突破，研究表明AOA在酸性土壤硝化作用中发挥重要作用。然而，Nitrosotalea、Nitrososphaera等不同AOA类群对底物浓度、pH等环境因子的响应和适应性明显不同，其生态位分异也较为复杂。本研究选择长期定位施无机肥的酸性旱地和稻田土壤为研究材料，基于土壤原位调查和实验室模拟微域培养，联用稳定同位素核酸探针技术、DNA高通量测序技术等前沿分子生态学研究技术，研究长期施无机肥对“功能活跃”的氨氧化微生物群落的影响，从生态功能和宏基因组角度明确不同的“功能活跃”氨氧化古菌类群（Nitrosotalea和Nitrososphaera）在不同施肥处理稻田土壤中的生态位分异及其对pH环境的选择适应性。本研究主要研究结果如下：（1）长期施肥后土壤总氮含量的提升显著刺激自养硝化活性；以Nitrososphaera为主的AOA在酸性旱地土壤硝化作用中发挥了重要作用，施有机肥土壤pH上升及C/N下降刺激了Nitrosospira（AOB）生长，从而改变了酸性旱地土壤中活跃的自养硝化微生物类群。（2）13C-稳定同位素核酸探针技术研究发现长期施肥改变了酸性旱地土壤中“功能活跃”的氨氧化微生物类群，CK处理中AOA和AOB均有活性；近30年的氮肥施加（N和NPK）土壤中AOA活性占据主导、AOB活性消失；而施有机肥处理（OM）土壤中AOB活性占据主导、AOA活性消失；且隶属Nitrosotalea的AOA和隶属于Nitrosospira Cluster 3、Cluster 9的AOB是酸性旱地土壤中“活跃的”氨氧化微生物类群。（3）稻田土壤中“功能活跃”的氨氧化微生物群落对于长期施肥的响应与旱地土壤不同。长期施化肥没有显著影响酸性稻田土壤硝化活性及AOB活性，但改变了“活跃的”AOA类群。（4）田间原位实验发现Nitrososphaera在酸性和中性旱地土壤中都能发挥主导作用，且室内模拟实验也证明上述现象，即Nitrosotalea类群营严格嗜酸性生长；而Nitrososphaera类群既能嗜酸性生长，也能嗜中性生长。上述研究结果打破了研究者对于AOA群落分布及生态功能的一贯认识，也明确了驱动酸性土壤微生物群落变化的关键环境因子，拓展了人们对土壤氨氧化微生物类群生态位特征的认识。