Environmental and kinetic controls on aerobic heterotrophic denitrification and N2O emission in acidic aerated biofilms.

Acidic, nitrite-rich aerobic systems are emerging hotspots for substantial N2O and NO emissions. Aerobic denitrification has been implicated as a major driver, but the controls governing its occurrence and persistence remain poorly understood. We provide direct experimental evidence that aerobic heterotrophic denitrification predominates in these systems and drives N2O-associated nitrogen loss. Long-term acid selection enriched a community maintaining denitrification across pH 4.5-7.0 and dissolved oxygen up to 8 mg O2l-1, with external organic carbon essential for activity. Raising nitrite from 1.81 to 18.84 mg N l-1 increased the N2O share of aqueous total nitrogen loss from 39.8% to 70.6%, so nitrite accumulation intensifies N2O emission. Raising dissolved oxygen from 0 to 4 mg O2l-1 increased the apparent Km for nitrite nearly fourfold to 13.60 ± 2.44 mg N l-1, while Vmax fell 25% to 17.79 ± 1.55 mg N l-1 h-1. Aeration therefore constrained nitrite affinity more than reduction capacity, allowing nitrogen loss and N2O production to persist under oxygenation and intensify as nitrite accumulates. The findings provide a mechanistic basis for understanding N2O production in acidic, oxygenated, nitrite-available systems and have broader implications for predicting and managing nitrogen transformations in engineered and natural environments.