Non-canonical reductive nitrous oxide production pathways in a seasonally stratified lake basin

Nitrous oxide (N ₂ O), a potent greenhouse gas and ozone-depleting agent, was detected at high concentrations in the anoxic bottom-waters of a monomictic eutrophic lake basin in Switzerland (Lake Lugano). The observed high site-specific nitrogen (N) isotope preference (SP), was inconsistent with bacterial denitrification, which typically exhibits low SP, thereby challenging its role as the primary N ₂ O source. This pointed to chemo-denitrification and/or fungal denitrification, both characterized by high SP, as possible alternative pathways. We conducted incubation experiments with sediment and bottom-water samples to assess N ₂ O production and reduction dynamics and associated natural-abundance stable isotope signatures. We demonstrate that N ₂ O accumulation predominantly originated from sedimentary production, and that elevated SP values in the bottom water reflected fractional bacterial N ₂ O reduction. Using an isotope mass balance mixing model, we identified bacterial denitrification as the dominant sedimentary process (∼75%), followed by chemo-denitrification (∼20%), and fungal denitrification (∼5%). Additional ¹⁵ N tracer incubation experiments, combined with selective inhibitors to quantify isolated pathways, confirmed model-estimated contributions. These findings validate the use of literature-based SP values in mixing models, and provide evidence for non-canonical N ₂ O production via chemo- and fungal denitrification, highlighting the need to broaden our understanding of N ₂ O cycling in lakes beyond classical bacterial pathways.