Microbial community analysis of enhanced biological phosphorus removal process and identification of polyphosphate accumulating organisms by molecular approach

强化生物除磷工艺的微生物群落分析及聚磷积累微生物的分子鉴定

基本信息

批准号：
14350283
负责人：
MINO Takashi
金额：
$ 9.6万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Fundamental research about enhanced biological phosphorus removal (EBPR) process is now making great progress because of the new microbial community analysis methods such as molecular techniques. This research was carried to reveal the relationship between microbial community and function of EBPR sludge using 16S-rDNA approach.In this research, at first, laboratory scale reactors were operated using acetate, glutamate, propionate, glucose or peptone as a main carbon source respectively. The expression of phosphorus removal activity was monitored by batch experiments and the microbial community change was monitored by Denaturing gradient gel electrophoresis (DGGE) using 16S-rDNA fragments. By comparing phosphorus removal activity and microbial community change, polyphosphate accumulating organisms were screened. In addition, by sequencing the whole length of 16S-rDNA of target bacteria by using cloning method and developing gene probes which can specifically detect the target bacteria and combining with the polyphosphate staining method and fluorescent in situ hybridization, capability of accumulating polyphosphate by screened bacteria was investigated.As a result, it was demonstratedd that gram-positive bacteria with high GC content accumulated polyphosphate in the reactor fed with mainly peptone and this gram positive bacteria was considered as the second putative polyphosphate accumulating organisms (PAOs) after Rhodocyclus related bacteria, which has been already considered as PAOs candidates. In addition, as another bacteria which accumulated polyphosphate but was not Rhodocyclus related bacteria nor gram positive bacteria was detected, it was clearly shown that still unknown PAOs should exist. In this way, it was shown that the EBPR is achieved by the combination of several PAOs

由于分子技术等新的微生物群落分析方法的出现，强化生物除磷（EBPR）工艺的基础研究目前正在取得长足的进展。本研究旨在利用 16S-rDNA 方法揭示微生物群落与 EBPR 污泥功能之间的关系。在本研究中，首先分别使用乙酸盐、谷氨酸盐、丙酸盐、葡萄糖或蛋白胨作为主要碳源运行实验室规模的反应器。通过批量实验监测除磷活性的表达，并通过使用16S-rDNA片段的变性梯度凝胶电泳（DGGE）监测微生物群落变化。通过比较除磷活性和微生物群落变化，筛选聚磷积累微生物。另外，通过克隆方法对目标细菌的16S-rDNA全长进行测序，开发能够特异性检测目标细菌的基因探针，结合多磷酸染色法和荧光原位杂交，筛选出具有积累多磷酸盐能力的细菌。结果表明，具有高GC含量的革兰氏阳性菌在主要添加蛋白胨的反应器中积累多磷酸盐，并且该革兰氏阳性菌被认为是第二假定的多磷酸盐积累物。继Rhodocyclus相关细菌之后的生物体（PAO），该细菌已被认为是PAO的候选者。此外，由于检测到另一种积累多磷酸盐的细菌，但既不是红环属相关细菌，也不是革兰氏阳性细菌，这清楚地表明仍然存在未知的PAO。这样，表明EBPR是通过多种PAO的组合来实现的