Integrated omics analyses reveal differential gene expression and potential for cooperation between denitrifying polyphosphate and glycogen accumulating organisms

Unusually high accumulation of the potent greenhouse gas nitrous oxide (N₂O) has previously been documented in denitrifying biological phosphorus (P) removal bioprocesses, but the roles of differential denitrification gene expression patterns and ecological interactions between key functional groups in driving these emissions are not well understood. To address these knowledge gaps, we applied genome-resolved metagenomics and metatranscriptomics to a denitrifying bioprocess enriched in as-yet-uncultivated denitrifying polyphosphate accumulating organisms (PAOs) affiliated with Candidatus Accumulibacter. The six transcriptionally most active populations in the community included three co-occurring Accumulibacter strains affiliated with clades IF (a novel clade identified in this study), IA and IC, a competing glycogen accumulating organism (GAO) affiliated with Competibacteraceae (GAO1), a Gammaproteobacteria PR6 and an Anaerolineae CH7. Strongly elevated expression of nitrite reductase genes compared to nitrous oxide reductase genes was observed in the overall community and in Accumulibacter populations, suggesting a strong role for differential gene expression in driving N₂O accumulation. Surprisingly, while ~90% of the nirS gene transcripts were expressed by the three co-occurring PAO populations, ~93% of the norB gene transcripts were expressed by GAO1 and ~75% of the norZ gene transcripts were mapped to PR6 and several other non-PAO flanking populations. This suggests the potential for cooperation between flanking populations and PAOs in reducing denitrification intermediates. Such cooperation may benefit the community by reducing the accumulation of toxic nitric oxide.