Isolation, identification and characterization of fungal lipids from biorefinery waste sites

Oleaginous fungi represent a sustainable platform for biodiesel production; however, most studies rely on conventional wild strains, while fungi adapted to harsh, waste-rich environments remain scarcely explored. In this study, filamentous fungi were isolated from a petroleum-contaminated biorefinery waste site and screened for lipid-producing potential. Six isolates (PET1–PET6) belonging to Aspergillus and Trichoderma genera were identified, among which Aspergillus sydowii PET2 exhibited the highest preliminary lipid yield (3.53% w /w) and was selected for further investigation. Lipid production by PET2 was optimized using Response Surface Methodology (RSM) based on a Central Composite Design (CCD), evaluating the effects of temperature, glucose concentration, agitation, and initial pH. The model was statistically significant ( p < 0.05), with temperature identified as the dominant factor influencing lipid accumulation. Under optimized conditions (pH 6.0, glucose 57.26 g/L, agitation 150 rpm, 20 °C), validation experiments showed that PET2 achieved a biomass concentration of 5.37 g/L and a lipid content of 5.21% ( w /w) in shake-flask cultivation. Scale-up in a 3-L air L-shaped bioreactor further enhanced performance, yielding 5.64 g/L biomass and a markedly higher lipid content of 18.40% ( w /w), corresponding to a 3.53-fold increase in lipid accumulation compared to shake-flask culture. The extracted lipids were efficiently converted into FAME with a balanced fatty acid composition (40.06% saturated, 31.91% monounsaturated, and 28.03% polyunsaturated), supporting favourable fuel stability, cold-flow properties, and combustion performance. The biodiesel complied with ASTM D6751 and EN 14214 standards, except for density and oxidation stability, which can be readily improved through antioxidant supplementation or blending. Future research should prioritise genetic engineering to enhance lipid biosynthetic pathways, incorporate additional RSM parameters (e.g., nitrogen availability, dissolved oxygen, and trace element supplementation) to further maximize productivity, and pilot-scale studies to validate process scalability. • sydowii PET2 from biorefinery waste yielded the highest lipids • Biomass and lipid yield optimized using response surface methodology • Optimum pH 6, glucose 57.26 g/L, agitation 150 rpm, temperature 20 °C • Bioreactor lipid yield 3.53-fold higher than shake flask cultivation • Lipids converted to FAME rich in SFA, meeting ASTM & EN biodiesel standards.