Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations
Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations
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DOI：
10.1128/aem.64.8.3042-3051.1998
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发表时间：
1998-08
影响因子：
4.4
通讯作者：
S. Juretschko;G. Timmermann;M. Schmid;K. Schleifer;A. Pommerening-Röser;H. Koops;M. Wagner
中科院分区：
文献类型：
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作者：
S. Juretschko;G. Timmermann;M. Schmid;K. Schleifer;A. Pommerening-Röser;H. Koops;M. Wagner
摘要采用多相法研究了高浓度氨氮污水处理厂硝化活性污泥中氨氧化菌和亚硝酸盐氧化菌的数量。与一组分层16 S rRNA靶向氨氧化细菌探针的原位杂交揭示了运动亚硝化球菌样细菌的优势。荧光原位杂交（FISH）提示的系统发育关系通过分离N. mobilis作为数量上占优势的氨氧化剂，随后进行比较16 S rRNA基因（rDNA）序列和DNA-DNA杂交分析。氨氧化人口的分子精细尺度分析，氨单加氧酶（amoA）的活性位点多肽编码基因的部分延伸扩增从氨氧化分离物和活性污泥提取的总DNA。然而，对活性污泥中13个amoA克隆序列的比较序列分析表明，这些序列彼此之间以及与欧洲亚硝化单胞菌Nm 50和N. mobilisisolate。结果表明，在N. mobilisisolate和N. europaea表明可能的横向基因转移事件。虽然硝化细菌菌株被分离，亚硝酸盐氧化属硝化细菌的成员在活性污泥中未检测到原位杂交。因此，我们使用rRNA方法来研究其他知名的亚硝酸盐氧化细菌属的丰度。采用三种不同的方法从活性污泥中提取DNA。对于每个DNA制备物，分别扩增编码小亚基rRNA的几乎全长基因，并用于产生三个16 S rDNA文库。通过比较序列分析，60个随机选择的克隆中有2个可以被指定为亚硝酸盐氧化细菌属Nitrospira。基于这些克隆序列，开发了特异性16 S rRNA靶向探针。用该探针对活性污泥进行FISH检测，结果表明，Nitrospira样细菌数量较多（占细菌总数的9%），并且经常出现在与N. mobilis。
ABSTRACT The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance ofNitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments ofNitrosomonas europaea Nm50 and the N. mobilisisolate. The unexpected high sequence similarity between theamoA gene fragments of the N. mobilisisolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genusNitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.