MBR中多糖降解细菌的关键酶识别与分子改造及其对膜污染的控制机制

Polysaccharide is the primary membrane foulant in membrane bioreactors (MBRs) for wastewater treatment. Hence, inhibiting the production of polysaccharide in sludge mixed liquor or enhancing its biodegradation is very crucial for efficient control of MBR fouling. In this project, the bacteria for degradation of alginate, a typical fouling-causing polysaccharide, are obtained by screening from MBRs. Their degradation behaviors to polysaccharide and the related impact factors, as well as their potential to mitigate membrane fouling, will be comprehensively evaluated. At a molecular level, first of all, the properties of the key lyase in alginate degradation pathway will be analyzed to clarify the enzymatic basis for efficient degradation of alginate. Afterwards, carbon metabolic repression (CCR), a potential important influence factor restraining activities of polysaccharide-degrading bacteria, and its regulation pattern towards the expression of the key alginate lyases will be investigated in detail. Meanwhile, the exopolysaccharide synthesis and secretion pathway in polysaccharide-degrading bacteria will be analyzed to identify the key enzymes accounting for the accumulation of exopolysaccharides. Finally, the engineering strains, with the characteristics of highly expressing the key alginate lyase, eliminating CCR, knockouting the key genes for the synthesis/secretion of exopolysaccharides, will be constructed by using molecular biology techniques. Based on all the results mentioned above, the mechanism of MBR fouling control by the engineering strains will be further clarified. Overall, the findings of this project will be aid in understanding of polysaccharide-degrading bacteria and provide an important alterative for MBR fouling control.

多糖是膜生物反应器（MBR）污水处理工艺的优势膜污染物。因此，抑制污泥混合液中多糖的产生或强化其生物降解是实现MBR膜污染有效控制的关键。本项目拟从MBR中筛选出高效降解海藻酸钠（典型的膜污染多糖）的细菌，以此为基础，深入分析降解菌对多糖的降解特性及其影响因素与膜污染控制效果。在分子生物学水平上，首先，分析海藻酸降解途径中关键裂解酶的酶学性质，阐述海藻酸被高效降解的酶学基础。其次，系统识别影响菌株降解活力的关键因子，确定碳源代谢阻遏（CCR）行为及其对关键海藻酸裂解酶的表达调控方式。同时，分析菌株胞外多糖的合成及分泌途径，鉴定影响胞外多糖积累的关键酶。最后，运用分子生物学手段构建高表达关键裂解酶、消除CCR、敲除胞外多糖合成/分泌关键基因的工程菌株，并结合关键酶的研究结果，阐明其对膜污染控制的机制。研究结果将有助于拓展对多糖降解菌的认识，为MBR膜污染的有效控制提供重要理论依据和技术支持。

膜生物反应器（MBR）中膜污染的识别与控制研究是近几年的研究热点，而MBR 中多糖的有效控制是解决膜污染的关键。本项目从MBR中筛选获得了两株高效多糖降解菌Strain Q-1和Strain Y-2，16S rDNA测序结合理化性质分析表明，其分别为Klebsiella属和Pseudonocardia属的新种。进一步系统分析了它们在单一多糖和混合多糖中的生长规律与表型、以及其多糖降解特性和降解产物类型。结果显示，两者对单一多糖海藻酸钠、葡聚糖和混合多糖EPS-多糖均具有明显降解作用，而Strain Y-2表现出比Strain Q-1更高效的多糖降解速率。相较于Strain Q-1，Strain Y-2展示出了一系列更有利于MBR膜污染控制的特性，即其生长速率更慢，剩余污泥产量少；自身多糖和无机盐胶体合成较少，新引入的膜污染物量少；粒径更大，膜污染潜力小。同时，LC-OCD对降解产物的分析结果显示，它们除了能够将多糖大分子大幅降解为小尺寸分子及矿化外，也对另一重要膜污染物EPS-蛋白有一定的降解作用。此外，将两株菌分别向MBR活性污泥中添加后，系统的SOUR、SNR和SPRR均明显提升，SPUR无影响，仅SDNR受到小幅抑制，表明多糖降解菌投加对MBR原有活性污泥的脱氮除磷功能有促进作用。最后，对性能更优越的Strain Y-2进行全基因组测序分析，识别出了25种EPS-多糖合成关键酶基因，可为后续分子改造奠定基础，但始终未识别出疑似的多糖裂合酶基因，而该菌优越的多糖降解特性提示其至少含有一种新型多糖裂合酶。我们既而对垃圾渗滤液处理厂的活性污泥进行宏基因组测序分析发现，其中含有大量多糖降解相关酶类，可为后续多糖降解酶筛选和研究提供酶源。本项目的研究成果可为应用多糖降解菌控制MBR膜污染提供前期理论基础，鉴定出的两个新种及预测的新型多糖裂合酶，具有一定的科学价值。