Refinement of the Candidatus Accumulibacter Genus Based on a Metagenomic Analysis of Biological Nutrient Removal (BNR) Pilot-Scale Plants Operated with Reduced Aeration Metagenome. Metagenome

Increasingly more wastewater treatment facilities are considering reduced aeration as a means to decrease operational costs and energy consumption. While successful biological nutrient removal (BNR) has been demonstrated in systems with minimal aeration, an understanding of the microbial communities that facilitate nutrient removal under these conditions is still evolving. Members of the Candidatus Accumulibacter genus are widely studied as key polyphosphate-accumulating organisms (PAO) in BNR facilities performing enhanced biological phosphorus removal (EBPR). The Ca. Accumulibacter genus is very diverse. Its current classification includes 18 Ca. Accumulibacter species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most Ca. Accumulibacter MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the Ca. Accumulibacter diversity in pilot-scale treatment trains performing BNR under low-DO conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium and high-quality MAGs for 5 of the 18 Ca. Accumulibacter species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus, and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized Ca. Accumulibacter species and for which we propose the new species designation Ca. Accumulibacter jenkinsia sp. nov. We evaluated the relative abundance of all the Ca. Accumulibacter species in the low-DO operated BNR pilot plants by using the highest quality Ca. Accumulibacter genomes assembled in this study to represent each of their corresponding species, and the reference genomes of other Ca. Accumulibacter species to represent the species for which MAGs were not recovered. Regardless of the pilot plant operational mode, Ca. A. necessarius and Ca. A. propinquus accounted for greater than 40% of the Ca. Accumulibacter community, whereas the newly proposed Ca. A. jenkinsia accounted for about 5% of the Ca. Accumulibacter community.