Structurally diverse biological nitrification inhibitors display distinct modes of inhibition in ammonia-oxidizing bacteria

Biological nitrification inhibitors (BNIs) are plant-derived compounds that suppress ammonia oxidation, representing a nature-based solution for reducing nitrogen losses and nitrous oxide emissions in agroecosystems. However, the modes of inhibition by which BNIs affect ammonia-oxidizing micro-organisms remain poorly understood. In this study, we examined the ammonia oxidation enzyme kinetics in two model species of ammonia-oxidizing bacteria (AOB), Nitrosomonas europaea and Nitrosospira multiformis, as affected by three structurally distinct BNIs: methyl 3-(4-hydroxyphenyl)propionate (MHPP), gallic acid (GA), and 6-methoxybenzoxazolinone (MBOA). GA acted as a potent, irreversible inhibitor, halting AOB activity within 1 h at 250 µM, with no recovery in cellular activity after inhibitor removal. In contrast, MHPP and MBOA exhibited a reversible mode of inhibition, with full activity restored upon removal of the inhibitor. MHPP exhibited dose-dependent, non-competitive, slow-onset inhibition of ammonia oxidation, independent of ammonium levels. By contrast, MBOA functioned as a fast-acting, uncompetitive inhibitor, with its inhibitory effect increasing with higher ammonium concentrations and MBOA doses. These results demonstrate that the efficacy of BNIs depends on compound structure, revealing distinct modes of inhibition and substrate dependencies. This study enhances our mechanistic understanding of inhibitory modes of BNI in AOB, providing a foundation for future studies with diverse nitrifiers.