好氧颗粒污泥（AGS）技术是新一代变革性的废水生物处理技术，然而其关键培养技术仍不明确，限制了该技术的实际工程化应用。本质上，AGS是一种特殊形式的生物膜，是由多种微生物附着生长而形成的微生物聚集体。在AGS形成过程中，高附着性能菌株起到关键作用。理论上，细菌的高附着性能主要是由特殊的分子结构决定（比如胞外聚合物（EPS）），这些分子结构根本上是由相应附着基因的表达所控制。因此，从微生物附着生长特别是附着基因的分子水平上开展研究，可以更好地揭示颗粒生物膜的生长机制，有助于掌握AGS的关键培养技术。基于此，本研究以AGS中分离的高附着菌嗜麦芽寡养单胞菌AGS-1（研究AGS形成机制的模式菌）作为研究对象，以细菌的附着生长特性为主线，采用分子生物学技术，从附着基因的分子水平上解析颗粒污泥的生长机制以及附着基因的功能和调控机理，寻求促进AGS形成的分子调控技术和应用策略，推动AGS技术的发展。本研究所取得的主要成果如下：

（1）设计并构建了适用于高附着菌AGS-1的阿拉伯糖诱导质粒系统，实现了AGS-1中目的基因可控可预测的表达，迈出了在AGS-1中开展附着基因层面研究的第一步。选择阿拉伯糖诱导启动子（araC-P_BAD）来调节AGS-1中目的基因的表达，使用mCherry荧光蛋白来评估诱导启动子的表达水平。该系统实现了低泄漏的基础表达和高水平的最大诱导表达。基于araC-P_BAD的诱导表达可以在0.0005%-0.2% L-阿拉伯糖的宽浓度范围内进行有效调控。值得注意的是，在AGS-1中观察到“表达滞后”现象，即细菌中mCherry表达往往在细菌生长之后进行，这可能是由于碳分解代谢物抑制效应所致。此外，葡萄糖的添加会抑制该系统的诱导表达，而在细菌对数生长期加入诱导剂会使该系统的表达速率最快。使用该诱导系统，结合mCherry与AsCas12a核酸酶（源于氨基酸球菌）融合表达策略，重组AGS-1菌在0.0005%-0.1% L-阿拉伯糖浓度范围内实现了AsCas12a表达的有效诱导。这些结果表明，新构建的阿拉伯糖诱导系统可以成为研究AGS-1附着基因功能和基因编辑的重要分子生物学工具。

（2）在高附着菌AGS-1中设计并构建了一套最新的CRISPR/Cas12a基因编辑系统，通过CRISPR/Cas12a介导的附着基因rmlA的敲除，明确了该基因对于AGS-1附着生长的功能影响和调控作用。采用双质粒CRISPR/Cas12a系统，一个质粒含有由araC-P_BAD驱动的Cas12a表达盒，另一个质粒包含特异性crRNA和同源臂。采用氨基酸球菌中的AsCas12a核酸酶，并证明其对AGS-1具有轻微毒性（与Cas9相比）和强切割活性，表明对于AGS-1的基因编辑，AsCas12a是一种合适的CRISPR核酸酶。通过“突变体获取策略”，实现了CRISPR/Cas12a系统介导的rmlA敲除，编辑效率75%。rmlA敲除导致AGS-1附着性能下降了38.26%，证实rmlA是一种重要的附着基因。此外，rmlA过表达使AGS-1的附着性能增加了30%以上，这些结果表明rmlA基因的表达调控对AGS-1的附着生长具有重要作用。总之，新构建的CRISPR/Cas12a系统为研究AGS-1中附着基因的功能提供了一个有效的分子平台。

（3）基于CRISPR/Cas12a基因编辑系统，又成功实现了AGS-1中多个基因的编辑，得以验证和拓展了该系统在AGS-1基因编辑中的适用性和功能性。选择AGS-1中不同的靶基因，仅更换编辑质粒中的crRNA和相应同源臂，成功实现了CRISPR/Cas12a介导的xanB和rpfF基因敲除，证明了该系统在AGS-1基因编辑中的适用性，并确认了xanB和rpfF也是AGS-1附着生长相关的基因。值得注意的是，每一个靶基因均选择了不同的靶点并设计了相应的crRNA，但并非每一条crRNA均可实现靶基因敲除。通过切割活性测试确认，未成功实现基因敲除的crRNA无法有效引导Cas12a切割靶基因。这些靶向效果较差的crRNA往往GC含量过高或者分布不均匀，后续试验中应避免使用。为了进一步增加靶基因编辑的成功率，可以同时设计并选择两条crRNA。此外，实现了CRISPR/Cas12a系统介导的点突变，在rmlA中引入终止密码子，通过此方式确认了rmlA的功能，进一步扩展了该系统的功能性，为后续附着基因功能的研究工作提供了有力的技术支撑。

（4）通过附着基因rmlA的异源表达，验证了其提高细菌附着性能的潜力，推进了附着基因应用策略的发展。基于前期研究成果，将附着基因rmlA克隆到本研究开发的阿拉伯糖诱导质粒上，针对不同细菌更换相应抗性基因，并将新构建的rmlA表达质粒转化到大肠杆菌和铜绿假单胞菌PAO1中。rmlA的异源表达使大肠杆菌的附着性能提高了50%以上，使铜绿假单胞菌PAO1的附着性能增强了40%以上。这可能是由于rmlA的异源表达刺激了菌株中dTDP-鼠李糖（rml操纵子负责合成）的生物合成所致。这些结果表明，附着基因rmlA的异源表达可以作为一种有效改善细菌附着性能的方式。后续实验可以尝试通过此策略大量获得附着性能强化的基因工程菌，投加到絮体污泥中以促进AGS的快速形成，从而获得基于功能微生物附着生长控制的实用AGS培养技术。

    综上所述，本研究从附着基因的分子水平上解析了颗粒污泥的生长机制以及附着基因的功能和调控作用，探究了促进AGS快速形成的培养技术。该研究对深入揭示AGS形成机理并拓展其工程化应用具有重要的理论和实际意义。

Aerobic granular sludge (AGS) technology is a new generation of innovative wastewater biological treatment technology. However, the key technology of AGS cultivation is still unclear, which limits the practical engineering application of this technology. Essentially, AGS is a special form of biofilm, which is a microbial aggregate formed by the attachment of a variety of microorganisms. High-attachment bacteria are believed to play an important role in the formation of granular sludge. Theoretically, the high-attachment ability of bacteria is mainly determined by the special molecular structure, such as extracellular polymer substances (EPSs), which are subsequently controlled by the expression of relevant attachment genes. Therefore, studies on microbial attachment growth, especially on the molecular level of attachment genes, can better reveal the growth mechanism of granular biofilm and help to master the key culture technology of AGS. Based on this, this study took the high-attachment strain Stenotrophomonas maltophilia AGS-1 (the model strain for investigating the formation mechanism of AGS) isolated from AGS as the research object, focused on the attachment growth characteristics of the strain, and adopted molecular biology techniques to analyze the growth mechanism of granular sludge and the function and regulation mechanism of attachment genes at the molecular level, seeking molecular regulation techniques and application strategies to promote the AGS formation, and promoting the development of AGS technology. The main research results in this study are as follows:

(1) We designed and constructed an arabinose inducible plasmid system for the high-attachment strain AGS-1 to achieve controlled and predictable expression of target genes in AGS-1, and took the first step to conduct attachment gene research in AGS-1. The arabinose inducible promoter (araC-P_BAD) was selected to regulate the expression of target genes in AGS-1, and mCherry fluorescent protein was used to assess the expression level of the inducible promoter. The system achieved little leaky basal expression and high maximal induced expression. The araC-P_BAD-based inducible expression was modulated over a wide range of 0.0005 to 0.2% L-arabinose. Notably, a “lag expression” phenomenon was observed in which mCherry was expressed after bacterial growth, which may be caused by the carbon catabolite repression from the medium. Furthermore, the addition of glucose inhibited the induced expression of this system, whereas the addition of the inducer during the logarithmic growth phase of AGS-1 resulted in the fastest expression rate of the system. Using this induction system in combination with a fusion expression strategy of mCherry and AsCas12a nuclease (derived from Acidaminococcus sp.), the recombinant AGS-1 strain achieved effective induction of AsCas12a expression in the range of 0.0005 to 0.1% L-arabinose. These results demonstrate that the new arabinose-inducible system could be used as an important molecular tool in the gene function and genome-editing research of strain AGS-1.

(2) A state-of-the-art CRISPR/Cas12a gene editing system was designed and constructed in the high-attachment strain AGS-1, and the function and regulation role of the attachment gene rmlA was clarified by CRISPR/Cas12a-mediated knockout of the attachment gene rmlA. Using a two-plasmid CRISPR/Cas12a system, one plasmid contained a Cas12a cassette driven by an arabinose-inducible promoter, and another contained the specific crRNA and homologous arms. AsCas12a was adopted and proven to have mild toxicity (compared to Cas9) and strong cleavage activity for AGS-1, indicating that AsCas12a is a suitable CRISPR nuclease for gene editing of AGS-1. Through "the harvesting mutant strategy", CRISPR/Cas12a system-mediated rmlA knockout was achieved, with a 75% efficiency. rmlA knockout resulted in a decrease in AGS-1 attachment ability by 38.26%, demonstrating that rmlA is an important attachment gene for AGS-1. In addition, rmlA overexpression increased the attachment ability of AGS-1 by more than 30%, and these results suggest that the regulation of the rmlA gene is an important factor in the attachment growth of the AGS-1 strain. In short, the newly constructed CRISPR/Cas12a system would provide an effective tool for investigating the function of attachment genes in AGS-1.

(3) Based on the CRISPR/Cas12a gene editing system, multiple genes were successfully edited in AGS-1, which allowed us to verify and expand the applicability and functionality of the system in AGS-1. We successfully achieved CRISPR/Cas12a-mediated knockout of xanB and rpfF by selecting different target genes in AGS-1 and replacing only the crRNA and corresponding homologous arms in the editing plasmid, demonstrating the applicability of the system in AGS-1 gene editing and confirming that xanB and rpfF are also genes associated with AGS-1 attachment growth. Notably, different targets were selected and corresponding crRNAs were designed for each target gene, but not every crRNA could achieve target gene knockout. As confirmed by the cleavage activity test, the crRNAs that did not successfully achieve gene knockout could not effectively guide Cas12a to cleave the target genes. These poorly targeted crRNAs tended to have too high GC content or uneven distribution and should be avoided in subsequent experiments. To further increase the success rate of target gene editing, two crRNAs could be designed and used simultaneously. Also, CRISPR/Cas12a system-mediated point mutations were achieved, and the introduction of stop codons in rmlA confirmed the function of rmlA, further extending the functionality of the system and providing strong technical support for subsequent research work on the function of attachment genes.

(4) The heterologous expression of the attachment gene rmlA verified its potential to improve bacterial attachment ability and advanced the development of attachment gene application strategies. Based on the previous research results, the attachment gene rmlA was cloned into the arabinose inducible plasmid developed in this study, and the corresponding resistance gene was replaced for different bacteria. The newly constructed rmlA expression plasmid was transformed into Escherichia coli and Pseudomonas aeruginosa PAO1. Heterologous expression of rmlA improved the attachment ability of E. coli by more than 50% and that of P. aeruginosa PAO1 by more than 40%. This may be due to the heterologous expression of rmlA stimulating the biosynthesis of dTDP-rhamnose (the rml operon responsible for synthesis) in the strain. These results suggest that heterologous expression of the attachment gene rmlA can be used as an effective way to improve bacterial attachment ability. Follow-up experiments can try to obtain genetically engineered bacteria with enhanced attachment ability in large quantities by this strategy and inject them into flocculated sludge to promote rapid formation of AGS, thus obtaining a practical AGS culture technique based on the attachment growth control of functional microorganisms.

In summary, this study analyzed the growth mechanism of granular sludge and the functions and regulatory roles of attachment genes at the molecular level, and explored the culture techniques to promote the rapid formation of AGS. This study is of great theoretical and practical significance to reveal the mechanism of AGS formation and expand its engineering applications.