Isotopic fingerprint of heterotrophic nitrification by Alcaligenes faecalis

The discovery of heterotrophic nitrification has expanded our view of nitrification beyond the canonical chemolithoautotrophs. Yet, the role of heterotrophic bacteria in nitrification across environmental and engineered systems remains unclear, partly due to limited physiological characterization and the absence of robust diagnostic tools. The analysis of nitrogen (N) isotope fractionation effects has been used for tracing biogeochemical N cycle processes and offers the potential to resolve underlying biochemical pathways. While autotrophic nitrification is known to generate substantial N isotope effects during ammonia (NH₃) oxidation to nitrite (NO₂⁻), comparable constraints for heterotrophic nitrifiers are lacking. Here, we report for the first time the N isotope effects associated with heterotrophic nitrification by Alcaligenes faecalis, an organism capable of converting NH₃ into several nitrogenous products. In batch incubations with 2.2 mM ammonium (NH₄⁺) as the sole N source, A. faecalis produced up to 0.67 ± 0.04 mM NH₂OH, 0.11 ± 0.01 mM NO₂⁻, and 12 ± 1.2 µM N₂O, while the remaining NH₄⁺ was assimilated into biomass. Therefore, the main NH₄⁺consumption pathway of A. faecalis is, in fact, best described by ammonium assimilation, which supports previous findings. Total NH₄⁺ consumption showed an isotope effect of 13.8 ± 0.4‰, exceeding that of biomass formation (4.8 ± 0.2‰). Both values fall within the known range for bacterial NH₄⁺ assimilation, but the disparity suggests additional fractionating steps beyond assimilation alone. NH₂OH, NO₂⁻, and N₂O were initially strongly ¹⁵N-depleted relative to the NH₄⁺ source, and became progressively enriched as NH₄⁺ was consumed. N₂O exhibited a variable site preference (24–38‰), indicating contributions from at least two production pathways. Overall, our findings show that heterotrophic nitrification produces N-isotopic signatures fundamentally distinct from canonical ammonia oxidation. These characteristic patterns in both NH₄⁺ and NO₂⁻ pools highlight the diagnostic potential of stable isotopes for identifying heterotrophic nitrification in complex systems.