Biosurfactants produced by halophilic bacteria are gaining attention as eco-friendly and biocompatible alternatives to synthetic surfactants due to their high surface activity, stability under extreme conditions, and intrinsic antimicrobial properties. These amphiphilic biomolecules hold great promise for bioremediation, biomedical, and pharmaceutical applications. In this study, moderately halophilic bacteria capable of biosurfactant production were isolated from saline mud collected at the Burgas solar salterns (Bulgaria). The halophilic microbiota was enriched in Bushnell–Haas (BH) medium containing 10% NaCl amended with different carbon sources. Primary screening in BH liquid medium evaluated the isolates’ ability to degrade n-hexadecane while at the same time producing biosurfactants. Thirty halophilic bacterial strains were isolated on BH agar plates supplemented with 2% n-hexadecane, 2% olive oil, or 2% glycerol. Four isolates—BS7OL, BS8OL, BS9GL, and BS10HD—with strong emulsifying activity (E₂₄ = 56%) and reduced surface tension in the range of 27.3–45 mN/m were derived after 7 days of batch fermentation. Strain BS10HD was chosen as the most potent biosurfactant producer. Its phylogenetic affiliation was determined by 16S rRNA gene sequence analysis; according to the nucleotide sequence, it was assigned to Halomonas ventosae. The extract material was analysed by thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR). Upon spraying the TLC plate with ninhydrin reagent, the appearance of a pink spot indicated the presence of amine functional groups. FTIR analysis showed characteristic peaks for both lipid and peptide functional groups. Based on the observed physicochemical properties and analytical data, it can be suggested that the biosurfactant produced by Halomonas ventosae BS10HD is a lipopeptide compound.

Surfactants are widely used in industrial applications, creating a growing demand for sustainable and cost-effective production processes. Biosurfactants, on the other hand, are environmentally friendly alternatives to synthetic surfactants because of their biodegradability, low toxicity, and diverse functional properties. Among biosurfactant-producing microorganisms, filamentous fungi remain comparatively underexplored despite their considerable biotechnological potential. This study evaluated the biosurfactant-producing ability of filamentous fungi isolated from petrochemical waste-contaminated soil collected near a fuel station in Sofia, Bulgaria. Three isolates grown on n-hexadecane and olive oil were selected as promising fungi for biosurfactant production, following surface tension reduction and enhancement of emulsification. Based on ITS region sequencing, the isolates were identified as Penicillium cavernicola LL-1 (LC899784.1), Trichoderma longibrachiatum LL-2 (LC899785.1), and Aspergillus niger LL-3 (LC899786.1). Culture conditions were optimized, and the use of mineral–organic media supplemented with 2% sunflower seed oil at pH 7 was found most appropriate. The optimal cultivation temperature was 28 °C for P. cavernicola LL-1 and 35 °C for both T. longibrachiatum LL-2 and A. niger LL-3. Maximum biosurfactant activity occurred during the stationary growth phase. Among the isolates, T. longibrachiatum LL-2 reduced the surface tension to 31.8 mN/m and achieved an emulsification index (E₂₄) of 58% with motor oil, whereas P. cavernicola LL-1 reduced the surface tension to 34.8 mN/m and E₂₄ = 56% for gasoline, and A. niger LL-3 reduced the surface tension to 37.4 mN/m and E₂₄ was 57% for triolein. E₂₄ assay was performed with the same hydrophobic substrates (gasoline, diesel fuel, motor oil, olive oil or trioleins) for all of the studied strains. Based on the primary screening experiments (E₂₄ assay and surface tension measurement), T. longibrachiatum LL-2 was chosen as the most promising biosurfactant producer. For this reason, subsequent experiments were conducted with this strain for a deeper investigation, as it is the least researched. The combined chromatographic, spectroscopic, and mass-spectrometric analyses suggest that the bioproduct obtained from T. longibrachiatum LL-2 exhibits features consistent with a glycolipid compound and certain characteristics reported for sophorolipid-like compounds. However, these preliminary findings do not allow the carbohydrate moiety to be identified conclusively. This novel bioproduct exhibited pronounced surface-active properties, indicating its potential as a fungal-derived biosurfactant for sustainable biotechnological applications.