好氧颗粒污泥（Aerobic granular activated sludge，AGAS）工艺作为一种高效的废水处理技术，相较于絮体活性污泥（Floc activated sludge，FAS）技术，具有无可比拟的优势。AGAS在本质上是一种颗粒状的生物自聚集体，因此，从微生物角度来研究AGAS中微生物的生长及其相互作用与联系是十分有必要的。微生物自身的群体感应（Quorum sensing，QS）系统会通过产生的自体诱导物（即信号分子）对微生物的生长和代谢等生理行为进行调控。本研究即是以微生物的QS系统作为切入点，针对N-酰化高丝氨酸内酯类分子（N-Acyl-homoserine lactones，AHLs）介导的种内QS系统与呋喃酰硼酸二酯类分子（Furanosyl borate diester，Autoinducter-2，AI-2）介导的种间QS系统，通过构建AGAS中微生物QS相关基因缺失重组突变菌株，阐明QS系统对于微生物的附着生长与胞外多聚物（Extracellular polymeric substances，EPS）的调控与分子作用机制，明晰QS系统对于微生物生长与相互作用的影响，进而探究其在AGAS形成过程中的贡献，为阐明AGAS形成的内在机理提供新的理论依据。

本研究得到的主要研究成果如下：

（1） 实际污泥样品（FAS、AGAS）以及AGAS分离菌株（AGAS-1,2,3,4,5,6）都能产生AHLs与AI-2。其中，AGAS中的AHLs、AI-2与EPS均比FAS高，且微生物产生的AHLs、AI-2、EPS中的胞外多糖物质（Extracellular polysaccharides，PS）以及胞外蛋白物质（Extracellular proteins，PN）与其自身附着性能呈现一定的正相关性。

（2） 根据三维荧光光谱（The three dimensional excitation-emission-matrix fluorescence spectra，3D-EEM）分析结果，AGAS与FAS产生的EPS组分较为相似，但含量有一定的差异；而在AGAS分离菌株中，高附着菌与低附着菌的EPS在组分与含量上均存在差异；并且在实际污泥样品与AGAS分离菌株的EPS中存在一种可能对微生物的附着性能具有促进作用的物质。

（3） 不同实际污泥样品与AGAS分离菌株中的QS相关基因具有差异性。根据PCR（Polymerase Chain Reaction，聚合酶链式反应）扩增结果，以AGAS中的高附着分离菌株AGAS-1作为研究主体，利用自杀质粒同源重组基因编辑技术成功构建了基因lasR（AHLs介导的QS系统相关）与基因tqsA（AI-2介导的QS系统相关）缺失重组突变菌株。通过荧光定量PCR研究发现，以AGAS-1的野生型菌株（AGAS-1-Wild Type，AGAS-1-WT）作为参比，基因lasR缺失重组突变菌株（AGAS-1-ΔlasR）中基因lasR的表达、基因tqsA缺失重组突变菌株（AGAS-1-ΔtqsA）中基因tqsA的表达明显下降。而利用转录组学分析——RNA测序（Ribonucleic acid sequencing，RNA-seq，核糖核酸测序）分析发现，在转录组水平上，AGAS-1-ΔlasR中基因lasR的相对表达含量显著下降11.92倍；AGAS-1-ΔtqsA中基因tqsA的相对表达含量下降2.33倍，表明这些QS基因的表达与微生物的信号分子、EPS产生以及附着生长等性能密切相关。

（4） 相较于野生菌株AGAS-1-WT，基因lasR缺失重组突变菌株AGAS-1-ΔlasR的AHLs、EPS与附着性能相对含量均呈下降趋势。而根据RNA-seq分析结果，基因lasR及其相关基因调控的AHLs介导的QS系统在AGAS-1中会通过影响其产生的D-丙氨酸、脂肪酸与Psl（Polysaccharide synthesis locus，一种通过多糖合成位点合成的PS）等物质，从而影响了菌体的附着性能。

（5） 相较于野生菌株AGAS-1-WT，基因tqsA缺失重组突变菌株AGAS-1-ΔtqsA的AI-2与EPS相对含量下降，而附着性能上升。研究发现，基因tqsA及其相关基因调控的AI-2介导的QS系统在AGAS-1中会对AI-2的受体分子LuxQ/ArcB以及信号传导分子BarA产生影响，并共同调控菌体中PS、粘附素、脂肪酸等物质的产生以及与运动相关、菌毛相关等基因的表达，进而影响菌体的附着性能。

综上，本研究探索了QS相关基因lasR与基因tqsA在AGAS-1中的分子代谢通路与调控作用，揭示了由AHLs/AI-2介导的QS系统对微生物附着性能的调控机制。即基于两种信号分子AHLs/AI-2介导的QS系统会通过调控AGAS-1中的多种物质而共同影响着其附着性能，从而可能在AGAS的形成过程中发挥作用。这些研究结果对更好地建立和开发AGAS的关键培养技术，实现其快速稳定培养，进而推广AGAS技术在实际废水处理中的大规模工程应用，具有重要的理论意义和实际价值。

Aerobic granular activated sludge (AGAS), as an efficient wastewater treatment technology, had incomparable advantages compared to floc activated sludge (FAS). AGAS was a granular biological aggregation formed by self-immobilization microorganisms, thus, it was necessary to study the growth, interactions and associations of microorganisms in AGAS. Quorum sensing (QS) system could regulate the physiological behavior (e.g., growth and metabolism etc.) of microorganisms through the autoinducters (i.e., signal molecules) which were produced by themselves. Therefore, in this study, the QS was selected as the observation point and for the N-Acyl-homoserine lactones (AHLs) and the Furanosyl borate diester (Autoinducter-2, AI-2), the molecular and regulatory mechanism of the QS system on the adhesive growth and extracellular polymeric substances (EPS) of the microorganisms in AGAS would be investigated by constructing mutated strains with non-QS-associated genes. On the basis of it, the effect of QS on the growth and function of microorganisms in AGAS would be elucidated, which would clarify the contribution of QS to the formation of AGAS, so as to provide a new theoretical basis for understanding the mechanism of AGAS formation. The main research results obtained in this study were as follows:

(1) FAS, AGAS and the strains-AGAS-1,2,3,4,5,6 isolated from AGAS could all produce AHLs and AI-2. The AHLs, the AI-2 and the EPS in AGAS were higher than FAS, and it had a positive correlation between the attachment ability and the AHLs, the AI-2, the extracellular polysaccharides (PS) and the extracellular proteins (PN) in EPS produced by microorganisms.

(2) According to the 3 dimensional excitation-emission-matrix fluorescence spectra(3D-EEM), it was found that the components of the EPS in AGAS were similar with FAS, while the concentrations were different between them. Both the concentration and component of the EPS were different between high attachment strains and low attachment strains which were isolated from AGAS. Furthermore, a component of the EPS was found in the different sludges and all strains isolated from AGAS, which might promote the adhesion of microorganisms.

(3) There were the variations for the QS genes in different sludge samples and the strains isolated from AGAS. According to the polymerase chain reaction(PCR), the gene lasR (AHLs-mediated QS system) and the gene tqsA (AI-2-mediated QS system) mutants (knockout) of AGAS-1 were constructed by suicide plasmid. The expression of the gene lasR in AGAS-1-ΔlasR (AGAS-1 without gene lasR) and the gene tqsA in AGAS-1-ΔtqsA (AGAS-1 without gene tqsA) which was analyzed by the fluorescent quantitative PCR, were significantly different with AGAS-1-Wild Type (AGAS-1-WT). And it was found that the relative expression of the gene lasR, analyzed by Ribonucleic acid sequencing (RNA-seq) in AGAS-1, was decreased by 11.92 times after knocked out, and the gene tqsA was fallen down by 2.33 times. This showed that the expression of QS genes was significantly related to the signal molecules, the EPS and the attachment ability of microorganisms.

(4) The attachment ability, the AHLs and the EPS of AGAS-1-ΔlasR was shown a down trend compared with AGAS-1-WT. According to the results of RNA-seq analysis, the QS system mediated by AHLs with gene lasR and its related genes would affect the attachment ability of AGAS-1 by affecting the D-alanine, fatty acids and Psl (Polysaccharide synthesis locus, a kind of PS) in it.

(5) The AI-2 and the EPS of AGAS-1-ΔtqsA showed a down trend as well, while the attachment ability was increased when compared with AGAS-1-WT. It was found that the QS system mediated by AI-2 with gene tqsA and its related genes would affect the receptor molecule LuxQ/ArcB of AI-2 and the signal transduction molecule BarA, and then affect PS, adhesin, fatty acids, cell motility and fimbria, thereby affect the attachment ability of AGAS-1.

In summary, this study explored the molecular metabolic pathways of QS-related genes (lasR and tqsA) which revealed the regulation mechanism of QS system mediated by AHLs and AI-2 on microbial attachment ability. That was the QS system which would regulate the production of multifarious substances in AGAS-1, jointly affect the adhesion, consequently the role in the formation of AGAS. It had an important theoretical significance and practical application value for developing the key cultivation technology for AGAS, achieving a rapid and stable formation, and then finally promoting the full-scale application.