Denitrifying communities enriched with mixed nitrogen oxides preferentially reduce N₂O under conditions of electron competition in wastewater

The reduction rate of nitrous oxide (N₂O) is affected by the electron competition among the four denitrifying steps, limiting the mitigation of N₂O emissions during wastewater treatment. We foresee essential to understand how combinations of electron acceptors (EAs) affect the microbial composition and reduction mechanisms of denitrifying communities. We enriched three denitrifying communities from activated sludge biomass with equivalent loads of different EAs: NO₃^– (R1), N₂O (R2), and NO₃^– + N₂O (R3). The resulting enrichments were compared in terms of (1) reduction of nitrogen oxides in absence/presence of other EAs (NO₃^–, NO₂^–, N₂O), (2) their denitrification gene composition and (3) their microbial community composition. Batch results showed the presence of NO₃^– and NO₂^– suppressed N₂O reduction rates in in all three reactors. The effect was lower in the mixed-substrate feed community than in the single-substrate feed under infinite sludge retention time and chemostat operation modes. N₂O-reducers of type nosZ II were enriched when N₂O serves as the sole EA, whereas nosZ I type N₂O-reducers were more prone to enrichment with NO₃^– as EA. The EA composition rather than the sludge retention mode differentiated the microbial communities. The genus Flavobacterium seems to play a significant role in alleviating the suppression of the N₂O reduction rate caused by electron competition. Limited conditions of electron supply are the norm independently of high C/N levels, and a community co-enriched with NO₃^– and N₂O alleviates more the competition for electrons in the nitrous oxide reductase enzyme than communities enriched with NO₃^– or N₂O individually.