兼具异养硝化-好氧反硝化和除磷功能菌株的代谢机理研究

Conventional nitrogen and phosphorus removal processes are intrinsically paradoxical because of the different requirements for nitrification, denitrification, phosphorus release and phosphorus uptake, which has become a primary restriction on the development and innovation of biological nutrients removal technologies. Novel strains capable of heterotrophic nitrification-aerobic denitrification and phosphorus removal have been found in our lab recently, in which the nitrification, denitrification and phosphorus uptake could take place simultaneously under permanent aerobic conditions. Therefore, the present study aims to fully understand the metabolic process and mechanism of this special kind of bacteria. On the basis of analyzing the variation of C, N and P in gas, liquid, intracellular and extracellular phases, N and P co-metabolic mechanism will be revealed through respectively investigating the characteristics of phosphorus removal with ammonia nitrogen, nitrite and nitrate as the nitrogen sources; the function of intracellular and extracellular substances will be explained through systematically examing the influence of different carbon sources on the process of simultaneous nitrogen and phosphorus removal. Furthermore, the metabolic process will be affirmed through cloning and sequencing the key functional genes responsible for nitrogen and phosphorus removal. Then, a biochemical metabolic model will be constructed to visually describe the mechanism. These findings will be of particular significance to enriching biological nutrients removal theories and exploiting novel biological nutrients removal technologies.

因参与硝化、反硝化、释磷和吸磷过程微生物作用机制的差异，导致了脱氮与除磷过程存在不可避免的矛盾，成为制约同步脱氮除磷工艺发展的瓶颈问题。申请人前期研究中发现了一类新型的在单一好氧环境下兼具异养硝化-好氧反硝化和除磷功能的菌株，能够实现好氧条件下C、N和P的同时去除。因此，本项目拟在已经拥有纯菌种资源的前提下，通过深入分析C、N、P在水、气、胞内和胞外的变化来研究不同氮源与磷在单一好氧环境下的共代谢特性以及不同碳源对同步脱氮除磷过程的影响，进而阐明N、P的共代谢机理，揭示胞内和胞外物质在同步脱氮除磷过程中的作用机制；在此基础上，结合对脱氮除磷关键功能基因的克隆与测序分析，确定菌株同步脱氮除磷的生化代谢过程，建立代谢模型，为这类细菌在污水脱氮除磷中的应用和创新型同步脱氮除磷工艺的开发提供理论基础和技术支持。

基于传统的生物脱氮除磷理论，生物脱氮与除磷过程存在不可避免的矛盾，已经成为制约同步脱氮除磷工艺发展的瓶颈问题。本项目基于自主筛选培育的能够在单一好氧条件下实现C、N和P同步去除的根癌土壤杆菌（Agrobacterium tumefaciens）LAD9，深入研究了分别以NH4+-N、NO2--N和NO3--N为N源，PO43-为P源时菌株LAD9在单一好氧条件下的氮磷共代谢特性，剖析了非含氮碳源、含氮碳源以及固体碳源对菌株LAD9脱氮除磷过程的影响，构建了菌株LAD9的全基因组精细图，识别了参与氮磷代谢的关键功能基因，在此基础上建立了菌株LAD9同步脱氮除磷的生化代谢模型。研究结果表明：1）三种N源条件下菌株LAD9均可实现高效同步脱氮除磷，且以氨氮为氮源时脱氮除磷效率最高；2）以葡萄糖、乙酸、乙酸钠和丁二酸钠等非含氮易生物降解物质为碳源，均可获得较为满意的脱氮除磷速率；3）菌株LAD9具有较强的氨化作用，能够利用有机含氮碳源，并将氮素转化为氨氮去除；以牛血清蛋白（BSA）为碳源时，TN，BSA和有机氮去除率分别可达53.8%，100%和98.3%；4）以玉米粉为固体碳源，菌株LAD9能够利用玉米粉缓释释放出的COD（主要为还原糖）作为碳源进行生长；5）菌株LAD9全基因组大小为5,931,195 bp，G+C含量为59.12%，含编码基因5729个；6）菌株LAD9对磷的去除是通过细胞的过量吸磷作用实现的，正磷酸在无机磷转运酶pit的作用下转移至胞外聚合物和细胞膜内，细胞膜内的正磷酸盐一部分在聚磷激酶ppx的作用下转化为poly-P，另一部分转化为有机磷共细菌生长；7）菌株LAD9对硝酸盐氮或亚硝酸盐氮的去除是通过好氧反硝化作用实现的，硝酸盐氮在周质硝酸盐还原酶napA的作用下转化为亚硝酸盐氮，经过亚硝酸盐还原酶nirK的作用转化为一氧化氮，经过一氧化氮还原酶（norB和norC）的作用最终转化为氧化亚氮。上述研究成果对开发新型污水脱氮除磷技术，解决传统生物脱氮除磷过程存在的固有矛盾提供了理论基础和有力的技术支撑。