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 Yarrowia lipolytica remains unexplored. Here, we investigated, for the first time, the combined effects of ethanol-induced stress on lipase production and fatty acid profiles in Y. lipolytica cultivated on two hydrophobic substrates: olive mill wastewater (OMW) and Waste Frying Oil (WFO). Ethanol was applied at increasing concentrations (3%, 5%, and 7% v/v), 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 τon increasing from 30.48 ± 0.80 to 47.07 ± 3.92 min and τmax 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 Y. lipolytica 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.