Rapid and Inexpensive Assay for Testing the Efficiency of Potential New Synthetic Nitrification Inhibitors

The microbial conversion of ammonia to nitrite in soils involves three enzymatic steps. Nitrification inhibitors (NIs) are designed to inhibit ammonia monooxygenase (AMO), the enzyme performing the initial oxidation of ammonia to hydroxylamine, to mitigate excessive nitrogen fertilizer losses in agricultural systems. Because the efficiency of the current commercial NIs is highly unreliable, novel, better performing compounds need to be developed. Previously, time-consuming soil incubation studies were required as the first step to test new potential NIs. We present here a simple and cost-efficient colorimetric assay that has been developed for the rapid assessment of the efficiency of new synthetic NIs to identify the most promising compounds for subsequent soil studies. This protocol enables screening of the inhibitor activity of multiple compounds at the same time with high reproducibility and can be manipulated to determine pH and temperature-dependent effects on NIs.