基于DNA-SIP技术的污水氨氧化微生物系统发育与生理代谢功能关系的研究

氨氧化是生物硝化的限速步骤。申请者对"50个不同污水生物脱氮工艺中AOA和AOB的种群数量和组成特征"（51078007）进行了系统研究，但是发现现代分子生物学技术无法解决AOA和AOB系统发育与生理代谢功能之间的耦合这一关键问题。因此拟利用稳定同位素核酸探针技术（DNA/RNA-SIP）研究原位状态下AOA、AOB系统发育与生理代谢功能之间的关系。对典型实际污水厂，以DNA-SIP技术为核心，结合RNA-SIP、稳定同位素示踪和FISH技术，探究原位条件下AOA和AOB的氨氮和碳的代谢途径、营养类型、活性、时空异质性、系统发育与贡献的关系，考察环境因素和工艺条件对两者系统发育和代谢功能关系的影响，研究两者在全程脱氮、短程脱氮、同时硝化反硝化及Anammox工艺中的相互作用机制。为充分利用AOA和AOB，开发新型高效的污水脱氮工艺提供微生物学基础和技术指导。

Microbial ammonia oxidation is the first and rate-limiting step of nitrification. For more than 100 years it was believed that Ammonia-Oxidizing Bacteria (AOB) were the only group responsible for the oxidation of ammonia. However, recent findings expanded this pathway also to Ammonia-Oxidizing Archaea (AOA). In our studies (NSFC. 51078007), the abundance and composition of AOA and AOB of 50 full-scale wastewater treatment plants (WWTPs) in China have been investigated using quantitative real-time polymerase chain reaction (PCR), cloning and sequencing approaches based on monooxygenase subunit A (amoA) genes. However, the methods and results only give us the phylogeny information of AOA and AOB in wastewater treatment, they do not necessarily enable us to infer their functions. One of the biggest challenges that microbial ecologists face is "Who eats what, where and when?". New technologies like DNA/RNA based Stable Isotope Probing (DNA/RNA-SIP), is increasingly being used in attempts to link the identity of microorganisms to their functions. So, in this project, the linking between phylogenies of AOA, AOB and their metabolic functions in wastewater treatment will be investigated using DNA/RNA-SIP technique. On the basis of previous studies (NSFC. 51078007), typical WWTPs will be chosen. Then through in field label or microcosm incubation, 13C-labeled substrate DNA/RNA-SIP will be employed to reveal whether AOA in wastewater treatment is autotrophic or heterotrophic, whether nitrification was driven by AOA or AOB, the relative contributions of AOA and AOB to ammonia oxidation, AOA's metabolic pathway, activity of AOA and AOB during nitrificaiton, and the temporal and spatial heterogeneity of AOA and AOB. And time-course DNA-SIP will be used to systematically investigate the effects of environmental factors such as ammonium levels, inhibitors, pH value, alkalinity, organic carbon, temperature, salinity, DO levels and sludge retention time on the growth of AOA and AOB, changes in AOA and AOB abundance and composition, activities of AOA and AOB, the linking between phylogenies of AOA, AOB and their metabolic functions. A combined approach of 13C-labeled substrate DNA/RNA-SIP, catalyzed reporter deposition-FISH (CARD-FISH) and stable isotope tracer technique of 15N and 18O will be employed to detect the potential metabolic coupling of AOA and AOB in nitrification/denitrification, short-cut nitrification/denitrification, simultaneous nitrification and denitrification and anaerobic ammonium oxidization process. On the basis of this project, the linking between phylogenies of AOA, AOB and their metabolic functions in wastewater treatment will be better understood. Furthermore this project will provide microbial foundation for regulating and developing novel biological nitrogen removal process.