Unprecedented N2O production by nitrate-ammonifying Geobacteraceae with distinctive N2O isotopocule signatures.

Dissimilatory nitrate reduction to ammonium (DNRA), driven by nitrate-ammonifying bacteria, is an increasingly appreciated nitrogen-cycling pathway in terrestrial ecosystems. This process reportedly generates nitrous oxide (N₂O), a strong greenhouse gas with ozone-depleting effects. However, it remains poorly understood how N₂O is produced by environmental nitrate-ammonifiers and how to identify DNRA-derived N₂O. In this study, we characterize two novel enzymatic pathways responsible for N₂O production in Geobacteraceae strains, which are predominant nitrate-ammonifying bacteria in paddy soils. The first pathway involves a membrane-bound nitrate reductase (Nar) and a hybrid cluster protein complex (Hcp-Hcr) that catalyzes the conversion of NO₂^- to NO and subsequently to N₂O. The second pathway is observed in Nar-deficient bacteria, where the nitrite reductase (NrfA) generates NO, which is then reduced to N₂O by Hcp-Hcr. These enzyme combinations are prevalent across the domain Bacteria. Moreover, we observe distinctive isotopocule signatures of DNRA-derived N₂O from other established N₂O production pathways, especially through the highest ¹⁵N-site preference (SP) values (43.0‰-49.9‰) reported so far, indicating a robust means for N₂O source partitioning. Our findings demonstrate two novel N₂O production pathways in DNRA that can be isotopically distinguished from other pathways.IMPORTANCEStimulation of DNRA is a promising strategy to improve fertilizer efficiency and reduce N₂O emission in agriculture soils. This process converts water-leachable NO₃^- and NO₂^- into soil-adsorbable NH₄⁺, thereby alleviating nitrogen loss and N₂O emission resulting from denitrification. However, several studies have noted that DNRA can also be a source of N₂O, contributing to global warming. This contribution is often masked by other N₂O generation processes, leading to a limited understanding of DNRA as an N₂O source. Our study reveals two widespread yet overlooked N₂O production pathways in Geobacteraceae, the predominant DNRA bacteria in paddy soils, along with their distinctive isotopocule signatures. These findings offer novel insights into the role of the DNRA bacteria in N₂O production and underscore the significance of N₂O isotopocule signatures in microbial N₂O source tracking.