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

Accurate determinations of ammonia (R_a) and nitrite oxidation rates (R_n) are critical for understanding the nitrification process across various ecosystems. However, there is only a limited number of methods available for the quantification of R_a and R_n. Here, we report a new method coupling stable isotope tracing and acidified potassium iodide reduction (SIT-APIR) to measure R_a and R_n simultaneously. ¹⁵NH₄⁺ was added to the incubation as a tracer to quantify R_a by calculating produced ¹⁵NO₂^- and ¹⁵NO₃^-, while ¹⁵NO₂^- was employed as a tracer for R_n 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.