污水生物除磷过程中聚磷合成与分解关键酶PPK酶和PPX酶的调控机理研究

Because the environmental - biological metabolism mechanism of biological phosphorus removal is unknown clearly, the biological phosphorus removal systerm is usually unstable and inefficient in wastewater treatment practice.The removing of phosphorus during wastewater biological phosphorus removal process is essentially catalyzed by enzymes. The key enzymes which directly catalyzed the polyphosphate synthesis and degradation, such as PPK (Polyphosphate Kinase)and PPX(Exopolyphosphatases), their generation and activities regulated by the gene and the environment effects, also their controlling to the biological phosphorus removal, are not clear, and the more systematic researches should be done furthermore. The functions and the expression of the gene ppk and ppx，which encoding the PPK and PPX, also their relations with the PPK and the PPX activities and the relations with the phosphorus removal, will be investigated. The environment effects to the genes expression and enzymes activities will be also investigated. In the experiment, some molecular technologies such as homology cloning, homologous recombination, PCR and DGGE, and the pure PAO and the biological phosphorus removal activated sludge, will be used. By researching, the environments, genes, genes expression, and the enzymes activities, which mechanisms that affect the biological phosphorus removal, will be investigated. As a result, the internal regulation of the wastewater biological phosphorus removal will be elucidated, and the conclusion will provide the theoretical foundation to the wastewater biological phosphorus removal practice for stability and efficiency.

污水生物除磷系统通常存在不稳定、除磷率低等问题，究其原因是对生物除磷的环境条件-生物代谢调控机理的认识不清所致。生物除磷过程本质上是由聚磷菌胞内相应的酶催化完成的。聚磷菌胞内聚磷合成和分解的一些关键酶（如PPK和PPX）的产生及酶活性的基因调控、环境调控机理，及其对生物除磷的控制机制，目前并不清楚，尚缺乏系统的研究。本课题拟通过对聚磷菌纯菌和污水生物除磷反应器中活性污泥，采用基因同源克隆、同源重组、PCR、DGGE等技术手段，从基因和酶分子水平研究编码PPK和PPX的基因ppk和ppx的功能、基因表达及其与酶活性和生物除磷的关系，以及环境因素对基因表达和酶活性的影响，从而探讨环境因子-基因-基因表达-酶活性调控过程对除磷效果的作用机理，旨在探明生物除磷的内在调控机制，为以后工程实践中稳定高效生物除磷提供理论基础。

针对污水生物除磷过程中除磷效果不稳定，除磷机理不清楚问题。研究与除磷相关基因调控及其影响因素。得到以下主要结论：（1）采用短臂同源无痕敲除技术对聚磷微生物纯菌ppk进行敲除，发现在大肠杆菌中ppk基因的缺失会影响菌体生长但不会影响其除磷性能。(2) 采用高通量测序技术对反应器中微生物群落与除磷间的关系进行了研究。在反应器启动及运行中，随着除磷效率的提高，微生物群落发生明显规律性变化。其中占优势的为变形菌门 （Proteobacteria）其在活性污泥中的比例一直占50%以上。红环菌科的Candidatus Accumulibacter与除磷效果密切相关，Candidatus Accumulibacter的含量变化直接影响了除磷效率。（3）从高效聚磷的反应器中分离聚磷菌，分离出140余株细菌，并对其中80多株进行了聚磷能力测试，发现有三株菌有一定除磷效果，经鉴定分别为Delftia tsuruhatnsis、 Paenibacillus pasadenensisand 和Pseudomonas fluorescens。