Ethanol induced oxidative stress, mitochondrial dysfunction, and autophagy in Wickerhamomyces anomalus

Wickerhamomyces anomalus, a flavor-modulating non-Saccharomyces yeast used in winemaking, experiences escalating ethanol stress during fermentation, yet the physiological and structural consequences remain unclear. This study assessed responses to 9% (v/v) ethanol stress with and without exogenous antioxidants (10 mM N-acetylcysteine and 2.5 mM glutathione) by measuring: reactive oxygen species (ROS) via fluorescence staining; superoxide dismutase (SOD) and catalase (CAT) activities plus glutathione content colorimetrically; mitochondrial membrane potential using rhodamine 123; ultrastructure via transmission electron microscopy; electron transport chain complex activities and ATP levels colorimetrically; and autophagy via monodansylcadaverine staining. Results indicated ethanol exposure induced ROS overproduction and oxidative stress while concurrently activating SOD and catalase activities and increasing GSH. Furthermore, ethanol reduced mitochondrial membrane potential, inhibited electron transport chain activity, decreased ATP synthesis, and triggered autophagy. Conversely, antioxidant supplementation alleviated oxidative damage, restored mitochondrial function, suppressed autophagy, and enhanced cell viability. These findings elucidate ethanol's mechanistic toxicity in yeast and provide a foundation for developing antioxidant-enhanced, ethanol-tolerant W. anomalus strains for industrial fermentation.