Production Optimization of Lipopeptide Biosurfactant by Bacillus Subtilis Isolated from Soil Using Palm Oil Mill Effluent

Microbial biosurfactants are amphiphilic compounds with diverse industrial, biomedical, and environmental applications, yet studies on optimized lipopeptide production by Bacillus subtilis remain limited. This study aimed to isolate, identify, and characterize microbial species from soil and evaluate lipopeptide biosurfactant production by B. subtilis. Soil samples were collected aseptically, serially diluted, heat-treated to enrich spore-forming bacteria, and cultured on selective media. Pure isolates were characterized morphologically, biochemically, and molecularly using 16S rRNA gene sequencing (for bacteria) and ITS region analysis (for fungi), followed by BLAST comparison and phylogenetic analysis. Lipopeptide production was optimized by varying pH, temperature, carbon and nitrogen sources, and incubation time. Extracted biosurfactants were characterized via Thin Layer Chromatography (TLC), High-Performance Liquid Chromatography (HPLC), and Liquid Chromatography–Mass Spectrometry (LC-MS). Functional activities, including emulsification, surface tension reduction, antimicrobial activity, and minimum inhibitory concentration (MIC), were assessed. Results confirmed the identity of B. subtilis, Escherichia coli, Staphylococcus aureus, and Candida albicans. B. subtilis produced surfactin, iturin, and fengycin most efficiently at pH 7.0, 37 °C, glucose and peptone as substrates, and 48-hour incubation. The biosurfactant demonstrated strong emulsifying ability, reduced surface tension to 28–35 mN/m, and exhibited broad-spectrum antimicrobial activity, with greater effectiveness against S. aureus. In conclusion, B. subtilis-derived lipopeptide biosurfactants possess potent bioactive properties suitable for industrial, biomedical, and environmental applications. It is recommended that industrial production, therapeutic evaluation, environmental application, and advanced molecular studies be pursued to fully exploit their potential.