含挥发性脂肪酸废水中活性污泥混合培养合成PHA机制研究

The treatment of residual activated sludge is one of the global problems. In our previous research, the activated sludge which could treat propylene oxide (abbreviated as PO) saponification wastewater, could utilize wastewater containing volatile fatty acids (abbreviated as VFAs) to accumulate PHAs after acclimation. There are about 57.8% of PHAs-accumulating organisms (abbreviated as PHAAOs) in this activated sludge, of which proportion is highest among all kinds of activated sludge. The study of PO activated sludge can not only solve the utilization of waste resource, but also offer materials for the research of microbiome in the activated sludge. In this project, PHAAOs in the PO saponification wastewater activated sludge will be firstly acclimated and enriched to reveal the inherent relationship between the supplement of different VFAs and the bacterial community succession. In addition, the bacterial community structure and the richness of PHAAOs in the sludge were explored. Genes related to PHAs metabolism in different acclimated activated sludge metagenomics were investigated, and the expression levels of these genes were also detected. As a result, the possible metabolic pathway of PHAs can be verified, and the roles of PHAs metabolism associated genes from different microbes were clarified. This project may be propitious to elucidate the PHAs biosynthesis mechanism of mixed microorganisms in VFAs containing wastewater, and provide theory base for the utilization of PO wastewater residual sludge in large scale.

剩余活性污泥处理是世界性难题。我们在前期研究发现处理环氧丙烷(PO)皂化废水的活性污泥经驯化可利用含挥发性脂肪酸（VFAs）废水大量积累PHA。同时发现该污泥菌群是已知的具有合成PHA能力的微生物占比最高的污泥菌群，比例达57.8%。以PO皂化废水剩余污泥为材料开展研究，不仅可以解决废弃物资源化的问题，还可为活性污泥中微生物组研究提供丰富的资料，具有潜在应用价值。本项目拟利用不同VFAs条件驯化PO皂化废水污泥富集PHA合成菌群，研究驯化过程中菌群结构与具有合成PHA能力菌群丰富度变化规律，揭示不同VFAs驯化条件与PHA合成菌群演替间的内在关联；同时挖掘驯化后污泥菌群PHA代谢相关基因，分析菌群PHA代谢通路，明确不同菌种来源PHA代谢相关基因在积累PHA过程中的作用，初步阐明含VFAs废水中污泥菌群混合培养合成PHA机制，为实现规模化利用剩余污泥生产PHA奠定理论基础。

本项目采用两个序批式反应器（SBR），分别以乙酸钠、乙酸钠与丙酸钠混合液（1:1）为唯一碳源，采用微好氧-好氧模式将环氧丙烷（PO）皂化废水剩余污泥驯化35天后发酵积累PHA。使用乙酸钠为唯一碳源驯化后，污泥的最大PHA积累量占细胞干重的34.37%，PHA单体以3-羟基丁酸（3-HB）为主，占95%以上；利用乙酸钠与丙酸钠混合液驯化后剩余污泥的最大PHA积累量为细胞干重的51.23%，3-羟基戊酸（3-HV）的比例增加，3-HB和3-HV的质量比约为2:1。本项目采用了三代测序中的单分子实时测序技术分析剩余污泥驯化前后微生物群落结构变化。在门水平，变形菌门（Proteobacteria）在驯化前后都是污泥群落中的优势微生物，利用乙酸钠驯化后的污泥（AccAS）和利用乙酸钠和丙酸钠驯化后的污泥（PrAccAS）该种群分别比驯化前（OriAS）提高了52.28%、55.23%，并且该种群中的多数细菌已被鉴定为能够合成PHA。在属水平，显著减少的是Tessaracoccus，由驯化前的31.87%在AccAS和PrAccAS分别减少至3.63%、1.50%；显著提高的是假单胞菌属（Pseudomonas）由驯化前的0.0025%，在AccAS和PrAccAS分别增加至24.35%、3.22%，另外，在PrAccAS中，优势属还有Phycisphaera和固氮弧菌属（Azoarcus），由驯化前的0.06%、未检测到分别增加至9.32%、47.68%。增加的这些菌属具有合成PHA的能力，而那些不能适应驯化环境的菌属逐渐被淘汰。对从污泥中筛选到的潜在新种lm1T、lm2 T、L72 T分别进行了基于遗传学、表型特征及细胞结构组成的分类鉴定，分别鉴定为Rhodoligotrophos defluvii sp.nov、Chryseobacterium binzhouense sp. nov.、Propylenella binzhouense sp. nov。基于本项目研究内容，共发表研究论文5篇，获授权发明专利2项，培养硕士研究生2人，获山东省高等学校科学技术三等奖1项，受邀作会议报告3次。