Impact of Ethanol Stress on Yarrowia lipolytica for Sustainable Bioconversion of Agro-Food Oil Wastes into Lipases and Lipids

Ethanol stress profoundly affects yeast metabolism, yet its integrated impact on lipase activity and lipid remodeling in <i>Yarrowia lipolytica</i> remains unexplored. Here, we investigated, for the first time, the combined effects of ethanol-induced stress on lipase production and fatty acid profiles in <i>Y. lipolytica</i> cultivated on two hydrophobic substrates: olive mill wastewater (OMW) and Waste Frying Oil (WFO). Ethanol was applied at increasing concentrations (3%, 5%, and 7% <i>v</i>/<i>v</i>), and the physiological responses were monitored over time (48, 72, and 96 h). Our results reveal a substrate-dependent and dose-dependent response to ethanol. Lipase activity was significantly enhanced at 5% ethanol, reaching 0.55 ± 0.11 U/mL in the OMW medium after 48 h. In comparison, mild stress (3%) induced the de novo synthesis of C20:1 (eicosenoic acid) and C20:2 (eicosadienoic acid), indicating reprogramming of lipid biosynthetic pathways. Oxidative stability, assessed by pressurized differential scanning calorimetry (PDSC), markedly improved in OMW-derived lipids, with τ_on increasing from 30.48 ± 0.80 to 47.07 ± 3.92 min and τ_max from 35.73 ± 0.62 to 54.04 ± 1.99 min under 3% ethanol. Conversely, WFO-derived samples exhibited lower oxidative stability and less pronounced changes in lipid composition. These findings demonstrate that <i>Y. lipolytica</i> adapts its lipid metabolism differently depending on the substrate, and that controlled ethanol exposure can enhance both lipase secretion and lipid oxidative resistance, underscoring its potential as a robust biocatalyst for sustainable biorefineries and the valorization of agro-food oil wastes.