Metatranscriptomics Reveals Potential Hybrid Electron Routing in Comammox Nitrospira and NO-Supplemented Cooperation in a Partial Nitritation-Anammox Bioreactor under Dynamic Aeration

Intermittent aeration drives partial nitritation-anammox (PN-A) systems, yet how microbiomes regulate aerobic and anaerobic respiration during rapid, oxygen-limited redox shifts remains poorly understood. Here, we applied genome-resolved metatranscriptomics to evaluate transcriptional dynamics in a long-term PN-A bioreactor treating low concentrations of ammonium. Under intermittent aeration, active populations did not follow strict metabolic switching, but instead exhibited complementary regulation by coordinately transcribing aerobic and anaerobic respiratory pathways. Notably, comammox Nitrospira exhibited a severe transcriptional bottleneck, with terminal oxidase expression 2–3 orders of magnitude lower than that of amo and nirK. Gene coexpression and pathway mapping support a novel hybrid electron transport chain where electrons from ammonia oxidation are directly routed to nirK. This alternative electron-transfer route functionally couples energy dissipation with obligatory nitrite detoxification. Concurrently, a severe transcriptional deficit of nirS relative to hdh in anammox Ca. Kuenenia indicates a partial bypass of intracellular nitrite reduction. Collectively, these patterns support a novel nitric oxide-supplemented cooperation: anammox bacteria scavenge extracellular nitric oxide generated by comammox Nitrospira, conserving their own electrons while acting as a vital sink to relieve comammox nitric oxide toxicity. These findings provide a mechanistic blueprint for optimizing advanced PN-A microbiomes through engineered aeration control.