New high-throughput screening assays for nitrification inhibitors identify an oxazolidine-thione as biological nitrification inhibitor

Nitrification is a microbial two-step process whereby ammonia (NH3) is first oxidized into nitrite (NO2-), by ammonia-oxidizing microorganisms, and then into nitrate (NO3-) by nitrite-oxidizing bacteria (NOB) in a mutualistic symbiosis. Due to high nitrogen (N) input in agriculture, this process needs to be slowed down as NO2- and NO3- easily leach, becoming unavailable for plants and leading to eutrophication. Moreover, they may be further processed into the very strong greenhouse gas nitrous oxide (N2O). Nitrification thus results in inefficient use of fertilizer and leads to detrimental environmental impact. Inhibitors can be used to mitigate these negative effects and are shown to positively affect plant growth and crop yield and strongly reduce nitrogen (N) pollution. However, currently, only a limited portfolio of nitrification inhibitors is commonly available. These show a variable efficiency and come with logistic complexities. As such, there is a high demand for a broader portfolio of novel molecules. To contribute to the discovery phase of new inhibitors and enable the evaluation of a large number of molecules, we developed miniaturized high-throughput screening assays on the soil-borne ammonia-oxidizing bacteria (AOB) Nitrosomonas europaea and Nitrosospira multiformis. We give a detailed overview of the procedure and illustrate its use by testing structural variants of oxazolidine-thiones that were previously discovered as nitrification inhibitors. This led to the discovery of the natural compound goitrin as a new biological nitrification inhibitor (BNI). Overall, the presented assays are promising discovery tools, as demonstrated by the newly discovered BNI, and may contribute to the advancement of a more sustainable agriculture.