Identification of key nitrous oxide production pathways in aerobic partial nitrifying granules

Summary: The identification of the key nitrous oxide (N₂O) production pathways is important to establish a strategy to mitigate N₂O emission. In this study, we combined real-time gas-monitoring analysis, ¹⁵N stable isotope analysis, denitrification functional gene transcriptome analysis and microscale N₂O concentration measurements to identify the main N₂O producers in a partial nitrification (PN) aerobic granule reactor, which was fed with ammonium and acetate. Our results suggest that heterotrophic denitrification was the main contributor to N₂O production in our PN aerobic granule reactor. The heterotrophic denitrifiers were probably related to Rhodocyclales bacteria, although different types of bacteria were active in the initial and latter stages of the PN reaction cycles, most likely in response to the presence of acetate. Hydroxylamine oxidation and nitrifier denitrification occurred, but their contribution to N₂O emission was relatively small (20-30%) compared with heterotrophic denitrification. Our approach can be useful to quantitatively examine the relative contributions of the three pathways (hydroxylamine oxidation, nitrifier denitrification and heterotrophic denitrification) to N₂O emission in mixed microbial populations.