茶园土壤自养氨氧化微生物产生氧化亚氮的量化及其分子生态机制

The high flux of nitrous oxide has become a serious problem in tea orchard soil ecosystem, but its biochemical and microbial mechanisms were still not so clear. Our previous study suggested that nitrification was mainly driven by ammonia oxidizing archaea in tea orchard soil. In this study, some representative tea orchard soils were collected, and nitrous oxide produced by autotrophic ammonia oxidizers was quantified by the dual-isotope labelling method.The relationship of nitrous oxide emission to nitrogen fertilizer utilization and soil physic-chemical properties was analysed. Nitrous oxide flux from the oxidation of ammonia to nitrite and the reduction of nitrite to nitrous oxide and nitrogen was investigated respectively. The traditional culture and microbial molecular techniques were used to compare the relative contribution of ammonia oxidizing archaea and bacteria to nitrous oxide emission. Meanwhile, the occurrence of genes encoding homologues of nitrite reductases in ammonia oxidizing archaea and bacteria was evaluated using genomic approaches and PCR amplification. The objective of this project is to understand microbial molecular mechanism of nitrous oxide produced by antotrophic ammonia oxidizers in tea orchard soil, assit in improving fertilizer utilization, decreasing the emssion of greenhouse gas and protecting tea orchard ecosystem.

高通量氧化亚氮排放是茶园土壤系统面临的严重问题，然而其中的生化过程和微生物机理尚不清楚。本项目建立在我们已结题项目成果"氨氧化古菌是茶园土壤硝化作用的主要驱动者"的基础上，拟选取代表性茶园土壤样品, 采用氧氮双同位素标记法对自养氨氧化微生物产生的氧化亚氮进行量化，结合茶园施肥管理和土壤理化性质，区分并明确自养氨氧化微生物在不同生化过程中形成氧化亚氮的具体通量；采用分离培养和微生物分子生态技术等手段，比较茶园土壤氨氧化古菌和细菌对氧化亚氮排放的相对贡献，研究亚硝酸还原酶等功能基因在氨氧化古菌和细菌中的分布情况，明确茶园土壤自养氨氧化微生物产生氧化亚氮的分子生态机制，为正确评估茶园土壤的环境生态效应、降低温室气体排放和提高氮肥利用、促进茶园生态系统良性循环提供科学依据。

茶园土壤通常释放高通量的氧化亚氮。本项目选择代表性茶园土壤样品，应用分离培养、稳定性同位素标记、生物抑制剂、定量PCR、末端限制性片段标记（T-RFLP）、克隆分析和高通量测序等一系列传统和现代分子生物学技术，研究茶园土壤中的自养氨氧化微生物对氧化亚氮排放的相对贡献、相关功能基因的丰度和多样性、以及环境驱动因子。研究表明，随着土壤pH的提高，自养氨氧化作用对氧化亚氮排放的贡献逐渐提高。在pH3.7，5.1，6.2和7.4的土壤中，自养氨氧化贡献百分比为34.3%，66.5%，90.6%和89.8%。不同pH和硝氮处理的茶园土壤nirS基因丰度在1.4E+7到2.5E+7范围内变化，低于nosZ基因丰度的1/10，两者均随土壤pH增加而上升。强酸性茶园土壤中nirS基因序列与Ralstonia eutropha有很高的相似性，而nosZ基因序列与Pseudomonas denitrificans和Paracous denitrificans相似性较高。典型变量分析表明，土壤pH、可溶性碳含量、铵态氮等显著影响nosZ和nirS基因型的分布。进一步大区域尺度分析表明，土壤自养氨氧化微生物受多环境因子共同驱动。土壤pH和底物所解释的变异不到总变异的1/4。应用选择性培养基，分离纯化了自养氨氧化微生物和产高通量氧化亚氮真菌，获得国内第一株氨氧化古菌的纯培养，并分离出多株嗜酸青霉，这些青霉在强酸性条件下可产生高通量氧化亚氮。