Rosineide C. de Souza

Biosurfactants are amphiphilic microbial metabolites with potential to reduce surface and interfacial tension and to promote emulsification. This study investigated the biosynthetic potential, emulsifying activity, and metabolomic profile of Bacillus subtilis subsp. subtilis INPA R296 cultivated in media supplemented with residual soybean oil. Phylogenomic analyses supported the taxonomic placement of the strain close to Bacillus subtilis subsp. subtilis 168 ^T . Genome mining predicted 11 biosynthetic gene clusters (BGCs), including NRPS clusters related to surfactin and fengycin biosynthesis. Synteny analyses indicated conservation of these clusters among phylogenetically related Bacillus strains. Functional assays showed that culture-medium composition influenced cell growth, emulsification indices, and emulsifying activity. UHPLC-HRMS/MS combined with molecular networking enabled the putative annotation of metabolites belonging to several chemical classes, including cyclic lipopeptides, N -acyl amino acids, and oxylipins. The main contribution of this study is the integrated evaluation of genomic biosynthetic potential, emulsifying activity, and putative metabolite annotations in strain INPA R296 cultivated with a low-cost agro-industrial residue. Because no biosurfactant yield, surface/interfacial tension, authentic-standard validation, or structural isolation was performed, the results demonstrate emulsifying potential and the occurrence of putatively annotated amphiphilic metabolites rather than confirmed biosurfactant production. These findings identify INPA R296 as a target for further investigation of amphiphilic metabolites with potential biotechnological applications.