One shuttle, two wins: AQDS cuts N₂O while boosting denitrification in cold, carbon-limited biofilm systems

Efficient biological nitrogen removal in biofilm systems is hampered under low-temperature and carbon-limited ("dual-low") conditions, where impaired microbial metabolism curtails denitrification and promotes nitrous oxide (N₂O). Quinone moieties of humic substances can shuttle electrons between microorganisms, yet their role in sustaining denitrification under dual-low stress remains unknown. Here we show that supplementing biofilm microcosms with the model humic analogue anthraquinone-2,6-disulfonate (AQDS) raised nitrate (NO₃^--N) removal from below 60% to 81% at 4-6 °C and C/N = 4, while curbing cumulative N₂O emissions by 12.1-26.8% and lowering net global-warming potential. Metatranscriptomics revealed coordinated upregulation of denitrification genes (narG, nirK, norB and nosZ) and extracellular electron-transfer genes (omcB, mtrC and macA), accompanied by 67.3% higher electron-transfer efficiency, elevated ATP and enhanced membrane potentials. FISH and SEM imaging captured AQDS-driven co-aggregates of electroactive microorganisms and denitrifiers bridged by nanowire-like structures, and selective enrichment of Geobacter sulfurreducens and Pseudomonas fluorescens. We identify quinone-mediated electron shuttling and direct interspecies electron transfer as the mechanistic basis of this microbial electroactive syntrophy, offering a low-energy strategy for sustainable nitrogen removal in cold, carbon-limited wastewater treatment.