脱氮除磷系统中微丝菌表面疏水性和群体感应特性对污泥膨胀影响机制研究

The sludge bulking caused by filamentous bacteria still occurred frequently in the biological nitrogen and phosphorus removal system. In-depth study of its inducements is essential to control the sludge bulking. In this project, the physiological behaviors of Microthrix parvicella (M. parvicella) in system are investigated in terms of surface hydrophobicity and quorum sensing characteristics, and their correlation causing sludge bulking is investigated. The fluorescent probes with different hydrophobicity to identify M. parvicella are designed and prepared, Density Functional Theory is introduced to investigate the correlation between surface hydrophobicity and physical behaviors is studied combined with traditional identification method. The detection and quantification methods of AHLs, AIPs and AI-2 et al signalling molecules in sludge bulking of systems are established by biosensor, LC-MS et al techniques, and it is used to explore the correlation between quorum sensing signalling molecules and change in the property and morphology of M. parvicella. The population dynamics of M. parvicella and other filamentous bacteria in system are tested by fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) and high-throughput sequencing et al technology. The effects of signalling molecules on the changes in property and morphology of M. parvicella, messenger behavior and dynamic properties of other filamentous bacteria in system are investigated. Moreover, the mechanism on sludge bulking influenced by surface hydrophobicity and quorum sensing properties of M. parvicella in biological nitrogen and phosphorus removal system is investigated, which is great significant for further understand the essence of filamentous sludge bulking.

在生物脱氮除磷系统中，污泥膨胀现象仍频繁发生，对其诱因的深入探索是实现控制污泥膨胀的本质要求。本项目从微丝菌表面疏水性和群体感应特性两方面对系统中微丝菌的生理行为与污泥膨胀的相关性进行研究。设计合成不同疏水性识别微丝菌的荧光探针，运用密度泛函理论并结合传统识别方法研究微丝菌表面疏水性与生理行为的相关性；运用生物传感菌、LC-MS等方法建立系统内污泥膨胀发生过程中AHLs、AIPs、AI-2等信号分子的量化方法，考察系统内群体感应信号分子与微丝菌性态变化的相关性；采用FISH、PCR、高通量测序等技术，考察系统内微丝菌及伴生丝状菌等菌群演替特性；研究感应信号分子与微丝菌性态变化及与共生菌群之间的信使关系和演替规律；研究系统中微丝菌表面疏水性和群体感应特性对污泥膨胀影响机制。这对进一步加深理解丝状污泥膨胀发生的本质有重要意义。

研究过程中制备了具有不同疏水性的识别微丝菌有机荧光探针FP1、L2、L3和L4以及将 3-甲酰基-N-乙基-咔唑分别与不同碳链长度的4-甲基喹啉溴代盐和4-甲基吡啶溴代盐反应最终合成不同碳链长度的长链烷烃修饰的菁染料荧光探针。制备的探针对微丝菌均具有较好的识别效果，并采用密度泛函对探针偶极矩进行计算用以评价探针极性，结果表明探针极性大小的顺序结果和荧光探针对微丝菌的标记效果顺序一致。此外，还采用脂肪酶与不同探针进行模拟计算，模拟计算结果与平均荧光强度分析结果在染色情况上表现一致，表明通过模拟计算的方法为微丝菌找到最佳极性值范围的荧光探针从而更高效识别膨胀污泥中的微丝菌。在不同运行条件下探索生物脱氮除磷系统运行过程中群体感应信号分子的变化规律，分析系统运行、污泥膨胀特性和菌群演替与感应信号分子变化的相关性，建立了运行规律、微生物群落演替及群体感应特性调控关系。不同碳源条件下研究结果表明，吐温80为碳源时系统能够产生更多的EPS，且利于微丝菌的生长，群体感应与微生物群落之间的相互作用调控系统的运行。不同乙酸/油酸比条件下研究结果再次证明油酸利于丝状菌的增殖，特别是有利于维持微丝菌的生长，AHLs介导的群体感应不仅对污泥沉降性和污泥粒径具有显著的调控作用，同时对COD和TN的去除、EPS的产生也具有显著的调控作用。随着进水负荷的增加，系统污泥沉降性变差进而导致污泥膨胀，同时也产生更多的EPS，Nitrospira、Sphingobium和Dokdonella是不同负荷条件下主要的AHLs产生菌。波动进水负荷更容易引发污泥膨胀，群体感应对系统运行发挥更多的调控作用，基因功能预测表明波动负荷条件下氨基糖和核苷酸糖代谢和脂肪酸生物合成的代谢活性相对较高，使波动负荷条件下产生了更多的EPS和调控了更多群体感应行为。对实际污水处理厂的研究表明，微丝菌的过量生长是引发冬春季节污泥膨胀的主要原因，信号分子C6-HSL和3OC12-HSL主要调控污泥沉降性，metagenome和Ellin6067是该污水处理厂内主要的AHLs产生菌。