Bacillus subtilis CECT30572 for biological fertilizer stabilization: an alternative to synthetic nitrification inhibitors

Introduction Modern intensive agriculture faces the critical challenge of reduced nitrogen use efficiency and severe environmental impacts associated with nitrate (NO 3 − ) leaching. Although chemical nitrification inhibitors such as 3,4-dimethylpyrazole phosphate (DMPP) are widely used to reduce nitrogen losses, there is growing interest in sustainable, bio-based alternatives. This study investigates Bacillus subtilis CECT30572, the active biological component of the commercial nitrogen stabilizer NSAFE ® , as a microbial strategy to reduce nitrate leaching. Methods Hybrid whole-genome sequencing was performed to characterize genes associated with nitrogen metabolism. In vitro assays evaluated nitrate depletion, and soil column leaching experiments were conducted under contrasting soil types using different fertilizer formulations. Results Hybrid whole-genome sequencing revealed a conserved nasABCDEF cluster and regulatory elements ( tnrA, glnR, codY, gltC ) associated with nitrogen assimilation. The genome also encodes the respiratory narGHJI operon for nitrate reduction to nitrite (NO 2 > − ), while lacking canonical genes for complete denitrification ( nirK/S, norBC, nosZ ), indicating a non-denitrifying nitrogen metabolism. In vitro assays showed substantial nitrate removal, with a net depletion of 455 mg L −1 NO 3 −1 . The nitrate removed from the medium was not recovered predominantly as extracellular or intracellular-associated NH 4 + . Although its specific fate cannot be determined from the present measurements, we hypothesize that it could have been assimilated with the bacteria in the form of amino acids. Soil column leaching experiments under contrasting soil types showed that coating or mixed fertilizers (granular NP, granular N, UAN) with CECT30572 significantly reduced cumulative NO 3 − leaching, with reductions of a magnitude similar to those observed with DMPP under several experimental conditions. Discussion Overall, these results indicate that the nitrate removal observed for B. subtilis CECT30572 under in vitro conditions may contribute to the reduction in nitrate losses observed in soil columns, supporting its potential as a bio-based strategy for reducing nitrate leaching.