Bioprospecting spore-forming bacteria: bioactivity-guided fractionation and profiling of metabolite-enriched fractions from Lysinibacillus fusiformis IWSA

Soil-derived spore-forming bacteria remain an important but underexplored source of biologically active microbial metabolites. In this study, bacterial isolates belonging to the family Bacillaceae were selectively recovered from soil samples and characterized using morphological and 16S rRNA gene analyses. Crude extracts obtained from the isolates were evaluated for antioxidant, antimicrobial, and cytotoxic activities. Phylogenetic analysis clustered the isolates within the genera Lysinibacillus and Bacillus. Among them, Lysinibacillus fusiformis IWSA (S2b) exhibited consistent antioxidant activity and broad-spectrum antimicrobial effects against Staphylococcus aureus and Escherichia coli. Bioactivity-guided fractionation of S2b resolved the metabolites into two major pooled fractions (F2-3 and F4-5) exhibiting differential spatial bioactivity patterns. Fraction F4 -5 demonstrated enhanced antimicrobial and cytotoxic activity, with an IC₅₀ value of 4.8 µg/mL in Caco-2 cells, compared to the moderate activity observed in F2-3. Morphological assessment further revealed variation in cellular responses between fractions. GC-MS profiling of pooled active fractions tentatively indicated a chemically complex mixture predominantly consisting of fatty acids and fatty acid methyl ester derivatives. Overall, the study demonstrates the utility of bioactivity-guided fractionation for resolving biologically active metabolite-enriched fractions from L. fusiformis IWSA and supports the potential of soil-derived spore-forming bacteria as sources of bioactive microbial metabolites.