Unprecedented N <sub>2</sub> O production by nitrate-ammonifying <i>Geobacteraceae</i> with distinctive N <sub>2</sub> O isotopocule signatures
Zhenxing Xu, Shohei Hattori, Yoko Masuda, Sakae Toyoda, Keisuke Koba, Pei Yu, Naohiro Yoshida, Zong‐Jun Du, Keishi Senoo
Abstract
ABSTRACT 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 (N 2 O), a strong greenhouse gas with ozone-depleting effects. However, it remains poorly understood how N 2 O is produced by environmental nitrate-ammonifiers and how to identify DNRA-derived N 2 O. In this study, we characterize two novel enzymatic pathways responsible for N 2 O 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 NO 2 − to NO and subsequently to N 2 O. The second pathway is observed in Nar-deficient bacteria, where the nitrite reductase (NrfA) generates NO, which is then reduced to N 2 O by Hcp–Hcr. These enzyme combinations are prevalent across the domain Bacteria. Moreover, we observe distinctive isotopocule signatures of DNRA-derived N 2 O from other established N 2 O production pathways, especially through the highest 15 N-site preference (SP) values (43.0‰–49.9‰) reported so far, indicating a robust means for N 2 O source partitioning. Our findings demonstrate two novel N 2 O production pathways in DNRA that can be isotopically distinguished from other pathways. IMPORTANCE Stimulation of DNRA is a promising strategy to improve fertilizer efficiency and reduce N 2 O emission in agriculture soils. This process converts water-leachable NO 3 − and NO 2 − into soil-adsorbable NH 4 + , thereby alleviating nitrogen loss and N 2 O emission resulting from denitrification. However, several studies have noted that DNRA can also be a source of N 2 O, contributing to global warming. This contribution is often masked by other N 2 O generation processes, leading to a limited understanding of DNRA as an N 2 O source. Our study reveals two widespread yet overlooked N 2 O 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 N 2 O production and underscore the significance of N 2 O isotopocule signatures in microbial N 2 O source tracking.