Minute-Scale Oxic-Anoxic Cycling Enables Nitrous Oxide Reductase Reactivation under Anoxic Conditions to Reduce N ₂ O Emissions in Wastewater Treatment

Nitrous oxide (N₂O) emissions from biological wastewater treatment pose a disproportionate challenge to carbon neutrality, as small releases substantially elevate the greenhouse gas footprint. Conventional treatment systems operate with aerobic phases lasting hours, during which oxygen suppresses nitrous oxide reductase (NosZ) activity and allows dissolved N₂O to accumulate. Shortening these phases to the minute scale through dynamic feedback-controlled aeration could couple N₂O production and reduction, but whether frequent oxic-anoxic cycling enables NosZ reactivation during postoxic anoxia remains unclear. In 1 L lab-scale suspended-growth reactors, we compared oxygen-only pulsing without imposed anoxia (continuous perturbation, CP) and paired oxic-anoxic cycling (intermittent perturbation, IP). At an oxygen ceiling of 2 mg/L, CP increased the N₂O fraction of gaseous nitrogen (GN) to 0.25-0.74, whereas IP maintained a low N₂O-N/GN ratio of 0.02-0.24 while releasing 79% of nitrified ammonium-N as GN. After adaptation, dissolved N₂O consumption rates were 4-6.5 times higher under IP than CP. Metaproteomic evidence confirmed NosZ enrichment under IP, with <i>Hyphomicrobium</i> as a key contributor. These results establish minute-scale oxic-anoxic cycling as a key operational prerequisite for NosZ reactivation during postoxic anoxia, recoupling N₂O production and reduction, and providing a practical framework for reducing N₂O emissions through PLC-based aeration control without retrofitting.