Characterisation and antimicrobial activity of biosurfactant extracts produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa isolated from a wastewater treatment plant

Biosurfactants are unique secondary metabolites, synthesised non-ribosomally by certain bacteria, fungi and yeast, with their most promising applications as antimicrobial agents and surfactants in the medical and food industries. Naturally produced glycolipids and lipopeptides are found as a mixture of congeners, which increases their antimicrobial potency. Sensitive analysis techniques, such as liquid chromatography coupled to mass spectrometry, enable the fingerprinting of different biosurfactant congeners within a naturally produced crude extract. Bacillus amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5, isolated from wastewater, were screened for biosurfactant production. Biosurfactant compounds were solvent extracted and characterised using ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI–MS). Results indicated that B. amyloliquefaciens ST34 produced C_13–16 surfactin analogues and their identity were confirmed by high resolution ESI–MS and UPLC–MS. In the crude extract obtained from P. aeruginosa ST5, high resolution ESI–MS linked to UPLC–MS confirmed the presence of di- and monorhamnolipid congeners, specifically Rha–Rha–C₁₀–C₁₀ and Rha–C₁₀–C₁₀, Rha–Rha–C₈–C₁₀/Rha–Rha–C₁₀–C₈ and Rha–C₈–C₁₀/Rha–C₁₀–C₈, as well as Rha–Rha–C₁₂–C₁₀/Rha–Rha–C₁₀–C₁₂ and Rha–C₁₂–C₁₀/Rha–C₁₀–C₁₂. The crude surfactin and rhamnolipid extracts also retained pronounced antimicrobial activity against a broad spectrum of opportunistic and pathogenic microorganisms, including antibiotic resistant Staphylococcus aureus and Escherichia coli strains and the pathogenic yeast Candida albicans. In addition, the rapid solvent extraction combined with UPLC–MS of the crude samples is a simple and powerful technique to provide fast, sensitive and highly specific data on the characterisation of biosurfactant compounds.