基于丝化诱导产物消减及群体感应调控的酵母菌处理废水稳定性研究

The transformation of yeast cell from yeast to hypha is a crucial limitation to the application of yeasts in wastewater treatment. Enhancement of quorum sensing and elimination of hypha-induced metabolites in wastewater degradation will be adopted to inhibit the formation of hypha so as to solve the system bulking caused by yeast hypha. First, hypha-induced metabolites and quorum sensing molecules (QSMs) will be screened in wastewater treatment system. Secondly, reduction of hypha-induced metabolites will be fulfilled by optimizing the nutrient factors in wastewater and operation parameters as well as synergistic reaction of yeast strains. The linkage of external factors (nutrition and environment), yeast cell morphology and metabolites will be given and the external factors resulted in hypha formation and inherent mechanism will also be revealed. On the other hand, the external factors will be optimized to enhance the secretion of QSMs. The response of yeast strains to endogenous/exogenous QSMs will be investigated by which the optimized combination of yeast strains will be obtained. The threshold concentration and optimum addition time will be investigated to inhibit the hypha formation and the responding genes related with hypha induction or inhibition and the transduction pathways for these factors will be elucidated for the further gene regulation of the transformation of cell morphology. Thirdly, the optimized nutrient factors and operation parameters will be validated in a sequencing bioreactor (SBR) to augment the secretion and mediation of QSMs and reduce the hypha-induced metabolites. Meanwhile, the control and recovery of unstable SBR with hypha transformation will be explored by supplement of QSMs. Finally, an efficient and stable yeast-wastewater treatment system will be established.

酵母细胞由酵母型向菌丝型转化是限制酵母菌废水处理技术进一步应用的技术瓶颈。本研究拟从消减废水降解过程中的丝化诱导代谢产物和强化酵母细胞群体感应（QS）调控两个方面控制细胞的形态转化，从而解决因细胞丝化而导致的系统膨胀问题。首先筛查出系统中的丝化诱导产物及群体感应信号物质（QSMs）；接着优化水质营养因子和运行参数并利用菌株间协同作用消减丝化诱导产物；探索物质因子、环境因子与酵母细胞形态、丝化诱导产物之间的相关性，查明引起细胞丝化的外部因素及内在机制；另一方面，优化外部因素强化QSMs的分泌，揭示各菌株对内、外源QSMs的响应并优化菌群体系；确定QSMs调控细胞丝化的浓度阈值和调控时间点，判明丝化诱导和丝化抑制的调控基因和相应的信号传导途径；随后在序批式反应器（SBR）中验证水质营养因子及运行参数，评价QSMs对丝化失稳系统的调控和恢复能力；最终构建高效、稳定的酵母菌废水处理系统。

酵母菌处理高浓度有机废水能同时实现高负荷、无剩余污泥且可将污染物转化为单细胞蛋白。然而，酵母细胞由酵母型向菌丝型转化是限制该技术进一步应用的技术瓶颈。本研究从消减丝化诱导代谢产物和强化酵母细胞群体感应（QS）两个方面调控细胞的形态转化，基本解决了因酵母细胞丝化而导致的系统膨胀问题。首先利用代谢组学方法动态监测了细胞丝化前后系统出水中的代谢产物，证实了长链脂肪酸和酪氨酸等氨基酸是细胞丝化诱导物；氮源缺乏、低溶解氧及短污泥龄会导致丝化诱导物浓度增加。随后建立了针对酵母群体感应信号分子（QSMs）的质谱联用分析方法，在连续运行的SBR反应器中检测出了两种QSMs,反式-法尼醇和2-苯乙醇（2-PE）；在丝化诱导培养基（YCBP）中只检测出2-PE，说明体系中酵母菌株能够产QSMs且与培养基质有关；此外检测到了一种细胞丝化抑制物（CFIs）-2,4-二叔丁基苯酚（2,4-Di-TBP）。添加一定浓度的外源QSMs或者CFIs可有效抑制酵母细胞丝化，丝化初期的0-3 h是最佳调控时间点。氮源、碳源影响3株功能酵母分泌QSMs或者CFIs的种类且浓度因菌株而易，以油脂废水为碳源均可分泌法尼醇；W1产生较高浓度的2-PE且受葡萄糖刺激明显。相比之下，菌株O2分泌的QSMs量较低。细胞密度对QSMs分泌有重要影响，菌株O2在高细胞密度的丝化程度远低于低细胞密度，其本质为QS对细胞形态的调控作用，高密度下降低了细胞对丝化诱导产物的吸收以及小分子醇、酸等代谢产物浓度。5 mM的Zn离子能完全抑制苯丙氨酸对酵母细胞丝化的诱导作用，cAMP/PKA通路有关基因Ras1、TPK1和TPK2发生差异表达，酵母菌胞外代谢产物中的中、长链脂肪酸含量下降，对苯丙氨酸的利用率降低，而2-PE以及2,4-Di-TBP的分泌增强。接着筛选出一株能高产2-PE的酵母菌株（Sa），碳源是影响Sa产QSMs的主要因素，氮源影响相对较小。最后通过向系统中添加Sa进行菌株复配，显著提升了系统运行的稳定性， COD去除率均大于85%， SVI低于82 mL/g，且有利于功能菌株丰度的保持；抑制酵母丝化的基因TPK1、TPK2表达活跃，激发丝化的基因表达量显著降低。本研究对最终构建高效、稳定的酵母菌废水处理系统具有重要意义。