Microbial superoxide production drives biogenic nitrogen dioxide formation in soils.

Nitrogen dioxide (NO2) is a critical atmospheric pollutant and ozone precursor, yet biogenic soil sources remain poorly constrained. Current models assume soil NO2 flux is exclusively depositional. Here we demonstrate that soils can produce NO2 through microbial superoxide (O2-) production. Using controlled factorial slurry experiments, native microbial communities produced approximately 10 times more NO2 than sterile controls following nitric oxide (NO) exposure. Stimulating superoxide production with NADH increased NO2 formation 15- to 26-fold, while inhibiting NADH oxidase reduced production toward baseline levels. Superoxide dismutase decreased NO2 production by 46-71%, and O2- concentration explained 60% of variation in NO2 production rates. Addition of peroxynitrite to soil increased headspace NO2, confirming this intermediate as the mechanistic link. These findings reveal a pathway linking carbon and nitrogen cycling where heterotrophic decomposers facilitate biogenic NO-to-NO2 conversion via superoxide chemistry, potentially explaining discrepancies between satellite observations and modelled soil NOx emissions.