Genome-centric metagenomics resolves microbial diversity and prevalent truncated denitrification pathways in a...

Denitrification is the stepwise microbial reduction of nitrate or nitrite (NO₂^-) to nitrogen gas via the obligate intermediates nitric oxide (NO) and nitrous oxide (N₂O). Substantial N₂O accumulation has been reported in denitrifying enhanced biological phosphorus removal (EBPR) bioreactors enriched in denitrifying polyphosphate accumulating organisms (DPAOs), but little is known about underlying mechanisms for N₂O generation, prevalence of complete versus truncated denitrification pathways, or the impact of NO₂^- feed on DPAO-enriched consortia. To address this knowledge gap, we employed genome-resolved metagenomics to investigate nitrogen transformation potential in a NO₂^- fed denitrifying EBPR bioreactor enriched in Candidatus Accumulibacter and prone to N₂O accumulation. Our analysis yielded 41 near-complete metagenome-assembled genomes (MAGs), including two co-occurring Accumulibacter strains affiliated with clades IA and IC (the first published genome from this clade) and 39 non-PAO flanking bacterial genomes. The dominant Accumulibacter clade IA encoded genes for complete denitrification, while the lower abundance Accumulibacter clade IC harbored all denitrification genes except for a canonical respiratory NO reductase. Analysis of the 39 non-PAO MAGs revealed a high prevalence of taxa harboring an incomplete denitrification pathway. Of the 27 MAGs harboring capacity for at least one step in the denitrification pathway, 10 were putative N₂O producers lacking N₂O reductase, 16 were putative N₂O reducers that lacked at least one upstream denitrification gene, and only one harbored a complete denitrification pathway. We also documented increasing abundance over the course of reactor operation of putative N₂O producers. Our results suggest that the unusually high levels of N₂O production observed in this Accumulibacter-enriched consortium are linked in part to the selection for non-PAO flanking microorganisms with truncated denitrification pathways.