短程反硝化除磷菌代谢过程中关键酶的交互作用机制及应用研究

Up to now, nitrogen and phosphorus removal with high efficiency and low cost has become the environmental issue both at home and abroad. The shortcut denitrification and phosphorus removal technology with nitrite as electron acceptor is the hot topic today. The present research mostly included its technology respect, but the research on the characterization and key enzymes of shortcut simultaneous denitrification and phosphorus removal bacteria were very poor. Therefore, this research starts with the screening of shortcut denitrification and phosphorus removal bacteria with nitrite as electron acceptor, and chooses typical key enzymes for denitrification and phosphorus removal. The functional genes encoding key enzymes can be acquired using modern molecular biology techniques, and the interaction mechanism among them can be studied by adopting DNA shuffling and gene knockout. Furthermore, its synergistic effect is optimized. Then the functional genes are shuffling, protoplasts and recombination to the screened host strain, and construct shortcut denitrification and phosphorus removal bacterium for the strengthen expression of multifunctional genes. The denitrification and phosphorus removal enhanced system is constructed by adding engineering strain. The best growth environment and removal law of nitrogen and phosphorus in enhanced system are also studied. Through the studies in the project, it could be very helpful understand the biological simultaneous denitrification and phosphorus removal mechanism, and provide the theory basis of the reasonable construction of new multi-genes recombinant engineering bacteria and the engineering application.

高效低耗去除水中氮磷污染物是国内外广泛关注的环境问题，而以亚硝酸盐为电子受体的短程反硝化除磷技术则是当前的研究热点。目前对短程反硝化除磷的研究多集中在工艺方面，但对短程反硝化除磷菌株的特性及代谢过程中关键酶等方面的研究较少。本课题拟从短程反硝化除磷菌的筛选入手，选择菌株代谢过程中的典型关键酶，运用现代分子生物学技术和手段，获得编码各个关键酶的功能基因片段，并利用DNA Shuffling和基因敲除等技术，研究关键酶之间的交互作用机制，优化其协同脱氮除磷效果。然后将其进行多功能基因体外拼接，融合重组到筛选出的宿主菌中，完成多功能基因增强表达的短程反硝化除磷工程菌的重组构建。投加工程菌构建反硝化除磷生物强化系统，对系统内工程菌的最佳生长环境及氮磷污染物去除规律进行研究。通过本课题的研究，可进一步丰富生物反硝化脱氮和同步除磷的机理内涵，同时为多基因重组工程菌的合理构建及工程化应用提供理论依据。

本研究以提高微生物的反硝化脱氮和除磷性能为目标，从短程反硝化除磷菌的筛选入手，以实验室保存的六株反硝化除磷菌为对象，进行复壮。经过3～5代的传代培养后，各菌株的功能均有所恢复。挑选经复壮后，代时较短、生长良好的菌株，以短程反硝化和除磷的性能作为主要考察因素对其进行复筛，得到七株高效短程反硝化除磷菌株，并对经过复壮的短程反硝化除磷菌进行了复配研究。考察环境因素对短程反硝化除磷菌的生长和反硝化脱氮除磷性能的影响，得到最佳环境条件。探讨单一碳源和混合碳源对短程反硝化除磷菌性能的影响。对菌株代谢过程中短程反硝化脱氮的关键酶——亚硝酸盐还原酶Nir的基因研究表明，短程反硝化除磷菌1K1为nirS+和nirK-型。选择菌株代谢过程中与除磷相关的典型关键酶ppk、ppx和phoU，运用现代分子生物学技术和手段，获得编码各个关键酶的功能基因片段，研究关键酶之间的交互作用机制，优化脱氮除磷效果。根据生物信息学的研究，设计PCR引物，经PCR扩增后得到这三类目的基因条带，将其经克隆后测序，分别得到了phoU，ppx和ppk的基因序列，在生物脱氮除磷过程中起着关键性的作用。用复筛后的短程反硝化除磷菌作为种源，构建生物强化反应器，对其研究表明，生物强化能在一定程度上改善活性污泥的性能，提高系统的处理效果，尤其是对磷的去除和反硝化功能方面，同时探讨了反应机理。