Analysis of nitrogen isotope fractionation effects is useful for tracing biogeochemical nitrogen cycle processes. Nitrification can cause large nitrogen isotope effects through the enzymatic oxidation of ammonia (NH3) via nitrite (NO2-) to nitrate (NO3-) (15εNH4+→NO2- and 15εNO2-→NO3-). The isotope effects of ammonia-oxidizing bacteria (AOB) and archaea (AOA) and of nitrite-oxidizing bacteria (NOB) have been analyzed previously. Here, we studied the nitrogen isotope effects of the complete ammonia oxidizer (comammox) Nitrospira inopinata that oxidizes NH3 to NO3-. At high ammonium (NH4+) availability (1 mM) and pH between 6.5 and 8.5, its 15εNH4+→NO2- ranged from -33.1 to -27.1‰ based on substrate consumption (residual substrate isotopic composition) and -35.5 to -31.2‰ based on product formation (cumulative product isotopic composition), while the 15εNO2-→NO3- ranged from 6.5 to 11.1‰ based on substrate consumption. These values resemble isotope effects of AOB and AOA and of NOB in the genus Nitrospira, suggesting the absence of fundamental mechanistic differences between key enzymes for ammonia and nitrite oxidation in comammox and canonical nitrifiers. However, ambient pH and initial NH4+ concentrations influenced the isotope effects in N. inopinata. The 15εNH4+→NO2- based on product formation was smaller at pH 6.5 (-31.2‰) compared to pH 7.5 (-35.5‰) and pH 8.5 (-34.9‰), while 15εNO2-→NO3- was smaller at pH 8.5 (6.5‰) compared to pH 7.5 (8.8‰) and pH 6.5 (11.1‰). Isotopic fractionation via 15εNH4+→NO2- and 15εNO2-→NO3- was smaller at 0.1 mM NH4+ compared to 0.5 to 1.0 mM NH4+. Environmental factors, such as pH and NH4+ availability, therefore need to be considered when using isotope effects in 15N isotope fractionation models of nitrification. IMPORTANCE Nitrification is an important nitrogen cycle process in terrestrial and aquatic environments. The discovery of comammox has changed the view that canonical AOA, AOB, and NOB are the only chemolithoautotrophic organisms catalyzing nitrification. However, the contribution of comammox to nitrification in environmental and technical systems is far from being completely understood. This study revealed that, despite a phylogenetically distinct enzymatic repertoire for ammonia oxidation, nitrogen isotope effects of 15εNH4+→NO2- and 15εNO2-→NO3- in comammox do not differ significantly from those of canonical nitrifiers. Thus, nitrogen isotope effects are not suitable indicators to decipher the contribution of comammox to nitrification in environmental samples. Moreover, this is the first systematic study showing that the ambient pH and NH4+ concentration influence the isotope effects of nitrifiers. Hence, these key parameters should be considered in comparative analyses of isotope effects of nitrifiers across different growth conditions and environmental samples.
Keywords: comammox; isotope fractionation; kinetic isotope effect; nitrification.