Refinement of the "Candidatus Accumulibacter" genus based on metagenomic analysis of biological nutrient removal (BNR) pilot-scale plants operated with reduced aeration.

Members of the "Candidatus Accumulibacter" genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage 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 dissolved oxygen (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 for which we propose the new species designation "Ca. Accumulibacter jenkinsii" sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, "Ca. A. necessarius" and "Ca. A. propinquus" accounted for more than 40% of the "Ca. Accumulibacter" community, whereas the newly proposed "Ca. A. jenkinsii" accounted for about 5% of the "Ca. Accumulibacter" community.IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the "Candidatus Accumulibacter" genus in pilot-scale plants operating with minimal aeration. We identified the "Ca. Accumulibacter" species enriched under these conditions, including one novel species for which we propose "Ca. Accumulibacter jenkinsii" sp. nov. as its designation. Furthermore, the MAGs obtained for five additional "Ca. Accumulibacter" species further refine the phylogeny of the "Ca. Accumulibacter" genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

Pilot-scale comparison of biological nutrient removal (BNR) using intermittent and continuous ammonia-based low dissolved oxygen aeration control systems.

Sensor driven aeration control strategies have recently been developed as a means to efficiently carry out biological nutrient removal (BNR) and reduce aeration costs in wastewater treatment plants. Under load-based aeration control, often implemented as ammonia-based aeration control (ABAC), airflow is regulated to meet desired effluent standards without specifically setting dissolved oxygen (DO) targets. Another approach to reduce aeration requirements is to constantly maintain low DO conditions and allow the microbial community to adapt to the low-DO environment. In this study, we compared the performance of two pilot-scale BNR treatment trains that simultaneously used ABAC and low-DO operation to evaluate the combination of these two strategies. One pilot plant was operated with continuous ABAC while the other one used intermittent ABAC. Both processes achieved greater than 90% total Kjehldal nitrogen (TKN) removal, 60% total nitrogen removal, and nearly 90% total phosphorus removal. Increasing the solids retention time (SRT) during the period of cold (∼12 °C) water temperatures helped maintain ammonia removal performance under low-DO conditions. However, both processes experienced poor solids settling characteristics during winter. While settling was recovered under warmer temperatures, improving settling quality remains a challenge under low-DO operation.