The community analysis of ammonia-oxidizing bacteria in wastewater treatment plants revealed by the combination of double labeled T-RFLP and sequencing

Abstract

The functional gene of amoA, which produces the α-subunit of ammonia monooxygenase (AMO), has been analyzed to reveal the microbial community structure of ammonia-oxidizing bacteria (AOB) by culture-independent methods. In this study, the distribution of the amoA gene in 10 wastewater treatment plants (WWTPs) was revealed by the fingerprinting method of terminal restriction fragment length polymorphism (T-RFLP) and comparative sequencing. T-RFLP showed diverse communities of AOB in the modified Ludzack-Ettinger process, in the anaerobic-anoxic-oxic processes, in the hanging biological contactor, and in the sequencing batch reactor. In all of these environments, long solid retention time (SRT) was expected to be the critical factor for maintaining the diverse AOB community structure. Because T-RFLP does not offer sufficient information to confirm the phylogenetic information of AOB, the microbial community structures were analyzed by comparative sequencing for seven samples that were selected by the statistical categorization using principal component analysis (PCA) among 14 samples. The phylogenetic tree of 21 operational taxonomic units (OTUs) among 88 clones obtained in this study revealed that AOB of Nitrosomonas oligotropha and europaea lineages were predominant in WWTPs. Double labeled T-RFLP produced group-specific terminal restriction fragments (T-RFs) representing several groups of AOB and offered advanced resolution comparing with the single labeled T-RFLP.