Integrated Stable Isotope Tracing and Acidified Potassium Iodide Reduction (SIT-APIR) Method to Determine Microbial Nitrification Rates across Environments

Accurate determinations of ammonia (Ra) and nitrite oxidation rates (Rn) are critical for understanding the nitrification process across various ecosystems. However, there is only a limited number of methods available for the quantification of Ra and Rn. Here, we report a new method coupling stable isotope tracing and acidified potassium iodide reduction (SIT-APIR) to measure Ra and Rn simultaneously. 15NH4+ was added to the incubation as a tracer to quantify Ra by calculating produced 15NO2- and 15NO3-, while 15NO2- was employed as a tracer for Rn by determining produced 15NO3-. 15NO2- and 15NO3- were chemically reduced to 15NO with acidified potassium iodide and the produced 15NO was subsequently measured by a membrane inlet mass spectrometer (MIMS). Under the optimized experimental conditions, the detection limit of this method is 0.1 μmol L-1. This method is accurate (relative error: 4.45% for 15NO2-, 0.65% for 15NO3-) and stable (relative standard deviation: 5.56% for 15NO2-, 8.97% for 15NO3-) to quantify the concentrations of 15NO2- (0.1-200 μmol L-1) and 15NO3- (0.1-80 μmol L-1) with excellent calibration curves (R2 ≥ 0.994). Furthermore, the optimal incubation time for nitrification rate measurements was determined by simulating the kinetics of 15NO2- and 15NO3- during incubation with the Gompertz model. In addition to its application to water and sediment samples, this method can also be extended to soil samples. Overall, this newly developed method significantly improved the accuracy and efficiency for determining the nitrification rate in diverse ecosystems with low sample volume and cost.