Heterotrophic ammonia oxidation by <i>Alcaligenes</i> balances ROS generation and terminal electron transport

Heterotrophic nitrifiers are bacteria that aerobically oxidize ammonia in the presence of organic carbon sources, which differs from autotrophic nitrifiers that extract energy from ammonia oxidation for cell metabolism and growth. The physiological significance of heterotrophic ammonia oxidation remains unclear, even though this process has been known for decades. Here, we demonstrate that direct ammonia oxidation (Dirammox)-a heterotrophic ammonia oxidation process with dinitrogen (N₂) as the primary product-is associated with both redox balance and the electron transport chain in <i>Alcaligenes faecalis</i>. Genetic and proteomic studies indicated that disruption of Dirammox genes (<i>dnfA/dnfB/dnfC</i>) induces a transient redox imbalance and perturbation in energy metabolism, further resulting in delayed growth. In addition, we found via biochemical and physiological studies that endogenous reactive oxygen species (ROS) enhance redox fluxes to ammonia oxidation, and the genetic disruption of cytochrome <i>c</i> peroxidase results in an increased flux of electrons to ammonia oxidation, producing N₂ and N₂O. These unexpected findings provide a more thorough understanding of both the Dirammox process and the physiology of heterotrophic ammonia oxidation.