Nitrate/nitrite-driven methane oxidation enhanced N ₂ O production in marine cold seeps

Methane oxidation coupled to sulfate reduction is the dominant process restricting methane release from cold seeps. Methane oxidation can also be coupled to nitrate/nitrite reduction, thereby linking the cycles of CH ₄ , CO ₂ , and N ₂ O. However, how this coupling shifts during seep development and its impact on N ₂ O production remains poorly understood. Here, we integrated geochemical profiling, ¹⁴ C-based labeling, and omics-based analyses to quantify the process rates, identify the key microbial mediators, and assess the net N ₂ O production across the developmental stages of cold seeps in the South China Sea. In an early-stage, polychaete-dominated seep, the depth-integrated, potential nitrate/nitrite-coupled methane oxidation rate reached 21.9 ± 3.0 mmol C/m ² /day, accounting for 36%–73% of the total methane oxidation. By contrast, in a typical epifauna-prevalent mature seep, this contribution diminished to ~1% (0.62 ± 0.16 mmol C/m ² /day), while sulfate-dependent methane oxidation became dominant. Omics data identified the predominant methane-oxidizing bacteria Methyloprofundus and QPIN01 (relative abundances of 7%–24%) as the potential mediators of nitrate/nitrite-coupled methane oxidation in the early-stage seep sediments. Consistent with the absence of N ₂ O-reduction gene nosZ in these lineages, simultaneous nitrate and methane amendments significantly stimulated net N ₂ O production by 14%–46% in the early-stage seep sediments. This increase correlated strongly with the potential nitrate/nitrite-coupled methane oxidation rate and was estimated to offset 41% ± 9% of the climate benefit gained through methane oxidation. These findings revealed the previously unrecognized successional dynamics of dominant methane oxidation pathways and necessitate the incorporation of nitrate/nitrite-coupled methane oxidation into assessments of greenhouse-gas release from these globally distributed ecosystems.