Physiological Influence of Fe and Cu Availability on Nitrogen Isotope Fractionation during Ammonia Oxidation

Microbially mediated cycling processes play central roles in regulating the speciation and availability of nitrogen, a vital nutrient with wide implications for agriculture, water quality, wastewater treatment, ecosystem health, and climate change. Ammonia oxidation, the first and rate-limiting step of nitrification, is carried out by bacteria (AOB) and archaea (AOA) that require the trace metal micronutrients copper (Cu) and iron (Fe) for growth and metabolic catalysis. While stable isotope analyses for constraining nitrogen cycling are commonly used, it is unclear whether metal availability may modulate expression of stable isotope fractionation during ammonia oxidation, by varying growth or through regulation of metabolic metalloenzymes. We present the first study examining the influence of Fe and Cu availability on the kinetic nitrogen isotope effect in ammonia oxidation (¹⁵ε_AO). We report a general independence of ¹⁵ε_AO from the growth rate in AOB, except at a low temperature (10 °C). With AOA Nitrosopumilus maritimus SCM1, however, ¹⁵ε_AO decreases nonlinearly at lower oxidation rates. We examine assumptions involved in the interpretation of ¹⁵ε_AO values and suggest these dynamics may arise from physiological constraints that push the system toward isotopic equilibrium. These results suggest important links between isotope fractionation and environmental constraints on physiology in these key N cycling microorganisms.