Nitrogen fertilization in agriculture has serious environmental consequences, including production of the greenhouse gas nitrous oxide (N2O), pollution of groundwater with nitrate (NO3-), and river eutrophication. Nitrogen use efficiency can be increased by amending fertilizers with inhibitors to slow microbial nitrification processes, which transform ammonia to NO3-. Unfortunately, commercial inhibitors have failed to perform reliably across various agroecosystems for reasons not well understood. Using a combination of bacterial studies and soil incubations, we demonstrate here that 4-methyl-1-(prop-2-yn-1-yl)-1H-1,2,3-triazole (MPT) exhibits superior nitrification inhibitory properties. Unlike the commercial reversible inhibitors, MPT acts as a mechanistic, irreversible inhibitor of the key enzyme ammonia monooxygenase, enabling effective retention of ammonium (NH4+) and suppression of NO3- and N2O production over 21 days in several agricultural soils with pH values ranging from 4.7 to 7.5. A bacterial viability stain and a suite of freshwater and terrestrial ecotoxicity tests did not indicate any acute or chronic toxicity. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed an enhanced inhibitory effect of MPT on both ammonia-oxidizing bacteria and archaea. Thus, MPT outperforms currently available nitrification inhibitors and has great potential for broad application in various agricultural settings.

Recently, 1,4-disubstituted 1,2,3-triazoles were reported by us as a new class of nitrification inhibitors, which can outperform the commercial compound 3,4-dimethylpyrazole phosphate (DMPP) in soil incubations. In this work, the mechanism of inhibition of five 1,2,3-triazoles with different substitution patterns was explored using a bacterial assay based on the measurement of nitrite (NO2-) production by pure cell cultures of Nitrosomonas europaea and Nitrosospira multiformis. While polar functional groups, such as amines, esters, and alkoxy residues, were detrimental to inhibiting production of NO2-, triazoles carrying only aliphatic substituents showed the highest inhibition of up to 98%. The observed correlation between lipophilicity and inhibitory activity suggests that more lipophilic compounds could more easily access the membrane-bound ammonia monooxygenase (AMO), which catalyzes the first step of the nitrification process. Measurement of the Michaelis-Menten kinetics suggests that the disubstituted 1,2,3-triazoles studied in this work act as reversible, noncompetitive inhibitors. Real-time measurements of the oxygen (O-2) consumption showed that the O-2 uptake rate by AMO follows zero-order kinetics in the presence of the triazoles, confirming the nonmechanistic mode of inhibition.

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.

Nitrification inhibitors have been coformulated with nitrogen fertilizers since the 1970s to modulate the microbiological conversion of nitrogen in agricultural soils. 3,4-Dimethyl-1H-pyrazole (DMP) and dicyandiamide (DCD) are currently the most used commercial nitrification inhibitors, but their mode of action is not well understood. This work seeks to fill this void by assessing for the first time in detail their mechanism of inhibition, efficacy, and acute toxicity with pure cell cultures of Nitrosomonas europaea. Bacterial assays based on the quantification of the nitrite (NO2-) production showed that both inhibitors reversibly target ammonia monooxygenase (AMO), which catalyzes the first step of the nitrification process. Michaelis-Menten kinetics suggest that both DMP and DCD act as uncompetitive inhibitors. Real-time measurements of the oxygen (O2) consumption confirmed the nonmechanistic mode of inhibition and showed that DMP reduced the O2 uptake rate by AMO much more at considerably lower concentrations than DCD, in line with the lower inhibitory efficiency of the latter. Acute toxicity tests revealed that DCD has a 10% higher toxicity than DMP when comparing treatments at the same inhibition efficacy (i.e., DMP at 10 ppm, DCD at 100 ppm), indicating that the inhibition of the nitrification process cannot simply be achieved by increasing the inhibitor concentration. The methods presented in this study could assist the development of more reliable nitrification inhibitors in the future.

Optimizing nitrogen (N) availability to plants is crucial for achieving maximum crop yield and quality. However, ensuring the appropriate supply of N to crops is challenging due to the various pathways through which N can be lost, such as ammonia (NH3) volatilization, nitrous oxide emissions, denitrification, nitrate (NO3−) leaching, and runoff. Additionally, N can become immobilized by soil minerals when ammonium (NH4+) gets trapped in the interlayers of clay minerals. Although synchronizing N availability with plant uptake could potentially reduce N loss, this approach is hindered by the fact that N loss from crop fields is typically influenced by a combination of management practices (which can be controlled) and weather dynamics, particularly precipitation, temperature fluctuations, and wind (which are beyond our control). In recent years, the use of urease and nitrification inhibitors has emerged as a strategy to temporarily delay the microbiological transformations of N-based fertilizers, thereby synchronizing N availability with plant uptake and mitigating N loss. Urease inhibitors slow down the hydrolysis of urea to NH4+ and reduce nitrogen loss through NH3 volatilization. Nitrification inhibitors temporarily inhibit soil bacteria (Nitrosomonas spp.) that convert NH4+ to nitrite (NO2−), thereby slowing down the first and rate-determining step of the nitrification process and reducing nitrogen loss as NO3− or through denitrification. This review aims to provide a comprehensive understanding of urease and nitrification inhibitor technologies and their profound implications for plants and root nitrogen uptake. It underscores the critical need to develop design principles for inhibitors with enhanced efficiency, highlighting their potential to revolutionize agricultural practices. Furthermore, this review offers valuable insights into future directions for inhibitor usage and emphasizes the essential traits that superior inhibitors should possess, thereby paving the way for innovative advancements in optimizing nitrogen management and ensuring sustainable crop production.