芽孢杆菌AHLs降解酶介导群体感应调控生物膜脱氮的作用机制

Biofilm is regarded as the key link in the wastewater treatment system. Our previous experiments found that adding Bacillus subtilis to biofilm system could significantly improve the denitrification efficiency of aquaculture wastewater, but the regulation mechanism was not clear. Quorum sensing system in biofilm was mainly mediated by N-acyl homoserine lactones (AHLs). In addition, Bacillus had the ability to produce AHLs-degrading enzyme. Therefore, excluding the influence of denitrification of bacillus self, we concluded that B. subtilis promoted biofilm denitrification via quorum sensing mediated by AHLs-degrading enzyme. To verify the inference, on the experiment basis of building the B. subtilis aiiA gene knockout strain and the Escherichia coli aiiA gene insertion strain, We will add five treatment strains (B. subtilis aiiA gene knockout strain, E. coli aiiA gene insertion strain, B. subtilis, Supernatant liquid of B. subtilis and E. coli) to the mature biofilm system. We will investigate the denitrification efficiency of biofilm system using water quality analysis, and confirm that AHLs-degrading enzyme can regulate biofilm denitrification. Using high performance liquid chromatography and gas chromatography, quantitative real-time PCR technologies investigate the concentrations of extracellular polymeric substances, the concentrations of AHLs, and the expression levels of quorum sensing gene (luxI and luxR), respectively. We will get the reliable evidence that AHLs-degrading enzyme have a direct correlation with biofilm crowd behavior. This study will not only reveal the molecular mechanism that AHLs-degrading enzyme regulated biofilm denitrification, but also provide a theoretical basis for the regulation mechanism of biofilm.

生物膜是水处理的核心环节，申请人前期实验发现生物膜系统中添加芽孢杆菌能显著提高养殖废水的脱氮效率，但作用机制不清楚。生物膜整个生命活动的群体感应调节系统主要由N-酰基高丝氨酸内酯（AHLs）介导，而芽孢杆菌具有分泌AHLs降解酶的能力。因此，在排除芽孢杆菌自身代谢脱氮的影响下，我们推断芽孢杆菌促进生物膜脱氮是经由其分泌的AHLs降解酶调控AHLs介导的群体感应完成的。为验证该推断，在构建枯草芽孢杆菌aiiA敲除株和大肠杆菌aiiA转入株的基础上，项目拟将不同菌株添加到成熟生物膜系统中，通过系统脱氮效率测定，验证AHLs降解酶是否调控生物膜脱氮，通过高效气相、液相色谱及RT-PCR等技术测定胞外多聚物浓度、AHLs浓度及群体感应系统基因luxI、luxR表达情况，获得AHLs降解酶与生物膜群体行为直接作用的可靠证据，进而揭示芽孢杆菌调控生物膜快速脱氮的分子机制，为生物膜脱氮调控提供理论指导。

生物膜是水处理的核心环节，前期实验发现生物膜系统中添加芽孢杆菌能显著提高养殖废水的脱氮效率，但作用机制不清楚。生物膜整个生命活动的群体感应调节系统主要由N-酰基高丝氨酸内酯（AHLs）介导，而芽孢杆菌具有分泌AHLs降解酶的能力。因此，推断芽孢杆菌促进生物膜脱氮是经由其分泌的AHLs降解酶调控AHLs介导的群体感应完成的。为此，项目以生物膜群体感应为研究切入点，通过构建重组大肠杆菌aiiA、aiiO基因转入改造株，开展了芽孢杆菌AHLs降解酶（aiiA、aiiO基因）降解信号分子、脱氮能力以及应用效果等实验，研究结果如下：1) 从苏云金芽孢杆菌等中获得aiiA、aiiO基因，验证了aiiA、aiiO基因具有分泌AHLs降解酶能力，并证实对AHLs介导的群体感应有调节作用；2）通过信号分子AHL指示菌Agrobacterium tumefaciens A136实验，验证了芽孢杆菌及分泌的AHLs降解酶对促进养殖处理系统生物膜脱氮的作用；3）开展了水产养殖处理系统生物膜群体行为特征及影响研究，通过测定脱氮效率、群体感应信号分子AHLs浓度、脱氮基因等指标，证实了AHLs降解酶与生物膜群体行为直接作用的可靠证据，并探讨了其作用机制，揭示了关键控制基因调控生物膜快速脱氮的分子机制，为生物膜脱氮调控提供理论基础；4）在应用领域，将生物膜应用于池塘原位和尾水处理系统中，形成了强化型生物膜技术，获得良好的脱氮效果。本研究生物膜群体感应结果为水产养殖尾水处理系统脱氮强化提供了新的技术手段，未来将信号分子调控基因应用到生物膜脱氮调控过程中，可实现高效脱氮，为水产养殖尾水综合治理及池塘工程学科提供了新的工艺模式方案和理论指导。