Increased Nitrogen Loading Facilitates Nitrous Oxide Production through Fungal and Chemodenitrification in Estuarine and Coastal Sediments

Estuarine and coastal environments are assumed to contribute to nitrous oxide (N₂O) emissions under increasing nitrogen loading. However, isotopic and molecular mechanisms underlying N₂O production pathways under elevated nitrogen concentration remain poorly understood. Here we used microbial inhibition, isotope mass balance, and molecular approaches to investigate N₂O production mechanisms in estuarine and coastal sediments through a series of anoxic incubations. Site preference of the N₂O molecule increased due to increasing nitrate concentration, suggesting the changes in N₂O production pathways. Enhanced N₂O production under high nitrate concentration was not mediated by bacterial denitrification, but instead was mainly regulated by fungal denitrification. Elevated nitrate concentration increased the contribution of fungal denitrification to N₂O production by 11-25%, whereas it decreased bacterial N₂O production by 16-33%. Chemodenitrification was also enhanced by high nitrate concentration, contributing to 13-28% of N₂O production. Elevated nitrate concentration significantly mediated nirK-type denitrifiers structure and abundance, which are the keystone taxa driving N₂O production. Collectively, these results suggest that increasing nitrate concentration can shift N₂O production pathways from bacterial to fungal and chemodenitrification, which are mainly responsible for the enhanced N₂O production and have widespread implications for N₂O projections under ongoing nitrogen pollution in estuarine and coastal ecosystems.