Nitrous oxide production, truncated denitrification pathways, and omics-informed process modeling in a denitrifying phosphorus removal bioprocess

Nitrous oxide (N 2 O) is a potent greenhouse gas produced as an unintentional, undesired byproduct in nitrogen removal bioprocesses, particularly in shortcut nitrogen removal or simultaneous nitrification-denitrification configurations that produce nitrite (NO 2 − ) as a key intermediate. Similarly, denitrifying phosphorus removal (DPR) offers carbon and energy savings but can produce N 2 O in excess of conventional processes, making sustainability benefits less clear. This study assesses a DPR reactor fed with NO 2 − to understand N 2 O production and mechanisms with different carbon sources, volatile fatty acids and glucose. N 2 O production up to 50 % of influent nitrogen was observed when the reactor was fed both propionate and glucose. Through 16S rRNA amplicon and shotgun metagenomic sequencing, we found that Candidatus Accumulibacter were the dominant phosphorus accumulating organism (PAO) and encoded a complete denitrification pathway. We also found abundant populations of denitrifying glycogen accumulating organisms (GAO) and ordinary heterotrophic organisms (OHO) with truncated denitrification pathways. We developed an omics-informed process model to assess the impact of truncated denitrification pathways on N 2 O predictions. We found that modeled N 2 O profiles were the most similar to observed results when the denitrification pathways of PAO, GAO, and OHO model populations reflected denitrification gene abundances from the metagenomic sequencing analysis. Overall, our work provides insights into N 2 O production by DPAO and DGAO, provides evidence for direct glucose-driven denitrifying P uptake by Ca. Accumulibacter DPAO, and demonstrates the utility of simple omics-informed process models in predicting N 2 O trends.