Electron shuttling promotes denitrification and mitigates nitrous oxide emissions in lakes

Abstract Eutrophication is an emerging global issue that is becoming increasingly severe due to the rising nutrient inputs and limited availability of electron donors for nitrogen removal. In sediments where redox conditions fluctuate dramatically, extracellular electron transfer (EET) critically supports microbial metabolism. However, the biogeochemical significance of EET-coupled denitrification and its EET mechanisms remain unclear. Here, through field investigations and laboratory 15 N isotope experiments, we reveal that humic substance (HS)-mediated electron shuttling significantly enhances denitrification primarily by stimulating bacterial outer membrane c -type cytochrome. Specifically, EET mitigates the emission of greenhouse gas nitrous oxide by enriching nosZII -type reducers. Notably, the efficacy of exogenous HS amendment attenuates in sediment with high native HS concentration. Metagenomic binning further reveals multiple cytochromes forming a complete EET-coupled denitrification electron transport chain. Our findings elucidate the microbial mechanisms underlying electron shuttling-driven denitrification in lakes, thereby expanding the understanding of biogeochemical cycles.