High-throughput screening assay for nitrification inhibitors and the discovery of goitrin as a biological nitrification inhibitor

Nitrification inhibitors are valuable in mitigating nitrogen (N) losses from agricultural fertilizers, enhancing fertilizer use efficiency, and minimizing their environmental and climatic impacts. Currently, the portfolio of approved inhibitors is limited, creating a strong demand for new alternatives. Traditional nitrification inhibition assays rely on large soil systems, batch cultures, or, at best, microbial cultures in deep-well 96-well plates, none of which are suited for high-throughput screening. Here, we present a highly robust method that enables rapid and efficient screening of thousands of molecules on the soil-borne ammonia-oxidizing bacteria Nitrosomonas europaea and Nitrosospira multiformis. Our assay utilizes 384-well plates for both screening and read-out, requiring only 50 μL of culture per sample and low amounts of compound. The assay also allows for further characterization of nitrification inhibitors and differentiation between those targeting the ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) pathways. Finally, we applied the assay to test several oxazolidine variants and discovered goitrin as a novel biological nitrification inhibitor (BNI). Overall, this assay offers promising tools for the rapid identification of novel nitrification inhibitors, contributing to sustainable agriculture.