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The greenhouse gas nitrous oxide (N₂O) has strong potential to drive climate change. Soils are a major source of N₂O, with microbial nitrification and denitrification being the primary processes involved in such emissions. The soybean endosymbiont <i>Bradyrhizobium diazoefficiens</i> is a model microorganism to study denitrification, a process that depends on a set of reductases, encoded by the <i>napEDABC</i>, <i>nirK</i>, <i>norCBQD</i>, and <i>nosRZDYFLX</i> genes, which sequentially reduce nitrate (NO₃⁻) to nitrite (NO₂⁻), nitric oxide (NO), N₂O, and dinitrogen (N₂). In this bacterium, the regulatory network and environmental cues governing the expression of denitrification genes rely on the FixK₂ and NnrR transcriptional regulators. To understand the role of FixK₂ and NnrR proteins in N₂O turnover, we monitored real-time kinetics of NO₃⁻, NO₂⁻, NO, N₂O, N₂, and oxygen (O₂) in a <i>fixK</i>₂ and <i>nnrR</i> mutant using a robotized incubation system. We confirmed that FixK₂ and NnrR are regulatory determinants essential for NO₃⁻ respiration and N₂O reduction. Furthermore, we demonstrated that N₂O reduction by <i>B. diazoefficiens</i> is independent of canonical inducers of denitrification, such as the nitrogen oxide NO₃⁻, and it is negatively affected by acidic and alkaline conditions. These findings advance the understanding of how specific environmental conditions and two single regulators modulate N₂O turnover in <i>B. diazoefficiens</i>.