Urea-driven nitrification contributes to N2O production in the oligotrophic euphotic ocean

Abstract Urea is an important alternative nitrogen source to ammonium for nitrification in oligotrophic oceans, yet its role in substrate-driven nitrous oxide (N2O) production remains poorly constrained. Here, we combined N2O isotopomer profiling, 15N-tracer incubations, and metagenomics to quantify and mechanistically resolve substrate-specific archaeal nitrification in the western tropical Pacific euphotic zone. Isotopomer-based mixing and fractionation model indicated that archaeal nitrification accounted for 69.6% ± 14.1% of microbial sources of N2O in oxygenated epipelagic waters. Depth-integrated urea-driven nitrification contributed 14%–41% of total nitrification and 21%–39% of nitrification-derived N2O, with contributions regulated by substrate proportions. Acidification experiments showed that pH decline inhibited ammonium-driven nitrification (median 21.9%) and enhanced urea oxidation (median 61.9%), whereas N2O production increased for both substrates (median 35.9% and 38.0%). In addition, experimental acidification induced opposite shifts in hybrid versus double-labeled N2O, suggesting pH-driven shifts N-intermediate chemistry and intracellular partitioning. Metagenomic results support the globally widespread urea-type AOA. Together, these results indicate that urea-driven nitrification constitutes a non-negligible, substrate-dependent source of N2O in oligotrophic euphotic zones. We recommend that Earth-system N-cycle models represent urea and ammonium oxidation as distinct pathways with pH-sensitive yields to improve projections of marine nitrification and N2O fluxes under acidification.