嗜热芽胞杆菌K5基于关键酶与基因的同步硝化反硝化机制研究

Nitrogen is a critical pollutant leading to eutrophication,and the denitrifying technic and it’s appication, especially biological denitrifying technic based on simultaneous nitrification and denitrification, has attracted a wide attention.However, up to date, the mechnaism of simultaneous nitrification and denitrification has not been clarified completely because of too many kinds of heterotrophic nitrificans. The present study will take a newly isolated thermophilic bacillus K5 as an object. Fistly, the key enzymes including ammonia monooxygenase(AMO),hydroxylamine oxygenase (HAO) and nitrite reductase (NIR) are isolated and purified to confirm the feasibility that K5 could carry out simultaneous nitrification and denitrification. Then, using the real-time fluorescence quantitative PCR, the effects of micromolecule such as inorganic nitrogen and oxygen on the expression of key genes (amoA、hao and nirS/K) are analyzed deeply. Finally, utilizing the transposition mutagenesis and isotopic tracing technologies, the pathway for SND is investigated in detail, as well as the status and effect for the metabolic branch probably existing in SND are disscused. This study not only clarifies the mechanism for SND in K5 under conditions of the pure culture, thereby to enrich and develop the biological denitrifying theory of SND, but also provides solid theoretical basis for the application of K5 for the practical project.

氮是造成水体富营养化的重要元素，除氮技术的研究和应用尤其是基于同步硝化反硝化的生物脱氮工艺引起了人们的广泛关注。然而，由于异养硝化菌种类繁多，目前同步硝化反硝化的机理尚未完全阐明。本课题拟以嗜热芽孢杆菌K5为研究对象，首先对其同步硝化反硝化中的关键酶——氨单加氧酶（AMO）、羟胺氧化酶（HAO）和亚硝酸还原酶（NIR）进行分离纯化，以此确证K5发生同步硝化反硝化的可行性。然后，通过实时荧光定量PCR，深入分析底物等小分子物质如无机氮、氧等对关键基因（amoA、hao和nirS/K）表达的影响规律。最后，利用转座诱变和同位素示踪技术深入剖析K5在纯种培养下的同步硝化反硝化的详细代谢途径，并探讨该途径中其可能发生的代谢支路的地位和作用。本课题的开展不仅可以阐明K5在纯种培养条件下同步硝化反硝化的机制，从而丰富和发展同步硝化反硝化生物脱氮理论，也可为K5在实际工程中的应用提供坚实的理论基础。

基于同步硝化反硝化的生物脱氮工艺引起了人们的广泛关注。然而，由于异养硝化菌种类繁多,目前同步硝化反硝化的机理尚未完全阐明。本项目从酶、基因和代谢途径等三个层面深入研究了芽孢杆菌K5的同步硝化反硝化机制，包括：关键酶的分离纯化及特性分析、关键基因的表达规律分析及氮素在细胞内的迁移途径等。经过系统研究，成功纯化出羟胺氧化酶、亚硝酸还原酶等关键酶，获得了详细的酶学性质；掌握了氨单加氧酶基因、亚硝酸还原酶基因等关键基因的表达规律，发现了影响基因表达的重要因子；探索了氮素的代谢途径，进而阐明了芽孢杆菌K5发生同步硝化反硝的机理。本项目研究不仅进一步丰富了生物脱氮理论，也为该菌株在实际中的应用提供了坚实的理论基础。