异养氨氧化细菌中氮代谢新途径的研究

Ammonia oxidation is the rate-limiting step of nitrification within the nitrogen biogeochemical cycle and plays an important role in the denitrification treatment of wastewater. It is reported that some heterotrophic ammonia-oxidizing bacteria process novel inorganic nitrogen metabolism properities, directly transformation of hydroxylamines or ammonia to nitrogen gas via hydroxylamines; do not involve the reduction of nitrite and/or nitrate as the traditional pathway of denitrification. However, up to date, studies on this new pathway of heterotrophic ammonia oxidizing bacteria are rare; its characterizations, intermediates, metabolic pathway, mechanisms and so on are still remained unclear. A novel heterotrophic ammonia-oxidizing bacterium, strain NNA3, capable of transforming hydroxylamine directly to nitrogen gas, was isolated previously in our laboratory. In this proposal, we will analysis the distribution of intermediates in the novel hydroxylamine metabolism network using 15N stable isotope tracer assay and metabolic flux analysis. Meanwhile, we will also study the regulation mechanism of the novel pathway based on metabolic control analysis of the related environmental factors and time nod. Based on these results, the metabolism pathway and its regulation mechanisms about NH3→NH2OH→N2 in strain NNA3 will be studied thoroughly. It is of great significance to deeply and completely understand of nitrogen biogeochemical cycle; as well as explore effective nitrogen microorganism species for further application.

氨氧化是细菌无机氮代谢中的限速步骤，在氮循环及生物脱氮过程中具有重要意义。研究发现，一些异养氨氧化细菌具有独特的氨氧化机制，能将氨氧化为羟胺后直接转化为氮气，不涉及亚硝酸盐、硝酸盐还原的反硝化过程，目前这一代谢途径的报道很少，所涉及的中间代谢产物、代谢通路及代谢调控机制等很多方面还有待深入研究。本实验室筛选到一株异养氨氧化细菌NNA3，前期研究发现该菌株也具有相类似的氮代谢特性，可能是一种新的氮代谢途径。本项目以菌株NNA3为对象，采用15N稳定同位素示踪技术结合代谢通量分析方法，分析、鉴定、定量中间代谢产物及其在无机氮代谢网络中的分布情况；并基于代谢控制分析原理，研究相关环境因子及时间节点控制对羟胺代谢途径的影响，从而深入研究菌株NNA3从 NH3→NH2OH→N2这一代谢途径的氮代谢过程及其调控机制。本研究有助于深入了解氮素代谢循环及发掘高效脱氮微生物菌种资源。

羟胺是微生物在氨氧化过程中的中间代谢产物，有研究报道一些异养硝化细菌可能存在羟胺代谢途径，能利用羟胺为底物直接生成氮气，或将氨氧化为羟胺后直接转化为氮气，目前对这一代谢途径相关研究还非常少。对硝化细菌羟胺代谢途径的研究，有助于深化人们对氮素循环的认识。本项目以两株具有新颖氮代谢特点的菌株为研究对象，开展了以下研究：1）异养硝化-好氧反硝化菌NNA4的氮代谢研究，发现菌株NNA4菌株能利用羟胺为唯一氮源生长，能将羟胺转化为气态氮化物，菌体中检测到羟胺氧化还原酶（HAO）的酶活，这些结果为菌株NNA4可能具有羟胺代谢途径提供了部分证据；2）全程硝化-反硝化菌HO-1的氮代谢特性研究，发现菌株HO-1能利用氨氮为底物进行彻底的硝化和反硝化，生成终产物氮气，而不能利用亚硝酸盐、硝酸盐进行反硝化产生氮气；3）菌株HO-1的氮代谢通量研究，采用15N稳定同位素示踪技术结合代谢通量分析方法，精确定量了底物氮、中间代谢产物及气态终产物在无机氮代谢网络中的分布情况；4）菌株HO-1氮代谢功能基因研究，对菌株进行了基因组测序及Fosmid文库构建，筛选获得具有氨氧化功能的阳性克隆子，对克隆子的功能基因进行了截短，成功将功能基因定位在三个基因片段上，实现了在E.coli中只表达三个基因，就可完成NH3→NH2OH→N2的工作。通过以上工作，本项目深入研究了硝化菌株从NH3→NH2OH→N2这一途径的氮代谢过程及其相关基因，初步明确了菌株的羟胺代谢通路。本研究有助于深入了解氮素代谢循环及发掘高效脱氮微生物菌种资源。