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

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)工艺的基础研究取得了很大进展。本研究采用16S-rDNA方法，分别以乙酸盐、谷氨酸、丙酸、葡萄糖和蛋白酵素为主要碳源，在实验室规模的反应器中运行，研究了微生物群落与EBPR污泥功能之间的关系。通过间歇实验监测除磷活性的表现，利用16S-rDNA片段的变性梯度凝胶电泳(DGGE)监测微生物群落的变化。通过比较除磷活性和微生物群落变化，筛选出聚磷菌。此外，通过克隆的方法对目标菌的16S-rDNA全长进行测序，研制出能特异性检测目标菌的基因探针，并结合聚磷酸盐染色方法和荧光原位杂交，对筛选出的细菌积累聚磷酸盐的能力进行了研究。结果表明，GC含量较高的革兰氏阳性菌在以蛋白调为主的反应器中积累聚磷酸盐，该革兰氏阳性菌被认为是仅次于红环菌属的第二类聚磷菌(PAOS)，已被认为是聚磷酸盐候选菌。此外，由于检测到另一种聚磷菌，但不是红环菌相关细菌，也不是革兰氏阳性菌，因此清楚地表明仍然存在未知的聚磷菌。结果表明，EBPR是由多个PAO组合而成的

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