Marine ammonia-oxidizing archaea (AOA) are pivotal to in the global climate system by dominating nitrous oxide (N₂O) production and contributing significantly to dark carbon fixation in the ocean. Although dissolved oxygen (DO) has long been considered as the primary regular of ammonia oxidation and N₂O production, emerging evidence indicates that N₂O production pathways in AOA are also governed by the ammonia to nitrite ratio. However, the combined effects of DO and substrate ratio on AOA-mediated N₂O production and carbon fixation remain unexplored. In this study, a comprehensive series of isotope-labeling experiments were conducted to investigate the responses of the model marine AOA strain Nitrosopumilus maritimus under varying DO levels and substrate ratios. Our results demonstrate that DO not only directly controls ammonia oxidation and N₂O production rates but also determines the partitioning of N₂O production pathways. The substrate ratio primarily reshapes N₂O source structure without significantly altering overall ammonia oxidation or N₂O production rates. Elevated DO generally suppresses N₂O yield, with the extent of suppression modulated by the substrate ratio, revealing a co-regulatory role of DO and ammonia-to-nitrite ratio in both the magnitude and mechanism of N₂O production. Furthermore, elevated DO consistently enhance carbon fixation across all substrate treatments. Collectively, our findings suggest that ongoing ocean deoxygenation may amplify climate feedbacks by simultaneously increasing marine N₂O emissions while weakening AOA-driven dark carbon fixation, thereby reinforcing climate change.
 

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