Isotopic signature of <scp>N₂O</scp> produced during sulfur‐ and thiosulfate‐driven chemoautotrophic denitrification in freshwaters

Abstract Chemoautotrophic denitrification plays an important role in nitrogen removal and the formation of a greenhouse gas (N 2 O) in aquatic environments. Natural stable isotopes support the tracing of nitrogen sources and the identification of biogeochemical processes in field research. However, the isotopic characteristics of N 2 O produced during chemoautotrophic denitrification have not been investigated so far. In this study, we analyzed isotopic signatures of nitrate and N 2 O in sulfur‐ and thiosulfate‐dependent denitrifying enrichments obtained from freshwater lakes. Chemoautotrophic denitrification exhibited a nitrate isotope pattern similar to heterotrophic denitrification: the 18 ε/ 15 ε‐nitrate followed a ratio close to 1. However, chemoautotrophic denitrification produced N 2 O with lower δ 15 N, δ 18 O, and higher site preference (SP = δ 15 N α ‐δ 15 N β ) values, compared to heterotrophic denitrification. The SP value, approximately 5.1‰, was characteristic in detecting chemoautotrophic denitrification driven by different sulfur forms. δ 18 O varied with specific electron donors, around 20‰ and 40‰ during sulfur‐ and thiosulfate‐dependent denitrification, respectively. The unique N 2 O isotope characteristics were likely regulated by nitric oxide reductases of Burkholderiaceae populations during sulfur‐dependent denitrification and Sulfurovum during thiosulfate‐dependent denitrification. These findings improve our understanding of N 2 O production processes and have important implications for predicting N 2 O emissions at a greater spatial and temporal resolution.