Phenotypic diversity and hybridization of wild Saccharomyces for improving bioethanol production

Background The transition toward sustainable energy sources requires alternatives to fossil fuels that are both efficient and environmentally friendly. Bioethanol has emerged as a promising substitute for gasoline; however, its production is limited by substrate complexity, fermentation inhibitors, and microbial stress tolerance. Conventional bioethanol relies largely on Saccharomyces cerevisiae , which has a restricted capacity to metabolize pentose sugars and withstand industrial stresses such as high ethanol and osmotic pressure. Expanding the diversity of yeasts used in bioethanol processes may help overcome these limitations. Saccharomyces eubayanus , a wild yeast species from Patagonia, exhibits exceptional tolerance to extreme environments, particularly low temperatures, and shows extensive population genetic and phenotypic diversity. Its adaptability and reproductive compatibility make it a strong candidate for industrial biotechnology applications, including the generation of intraspecific hybrids with enhanced stress resistance and improved fermentative performance. Results In this study, we evaluated the phenotypic diversity of S. eubayanus isolates under conditions relevant to bioethanol fermentation and applied mass-mating approaches to generate hybrids with improved fermentative traits. The resulting strains were assessed for their performance under stressors that mimic second-generation bioethanol production, including high ethanol concentrations, osmotic stress, and inhibitory compounds derived from lignocellulosic biomass pretreatments. Our analysis demonstrated substantial variation among isolates and identified hybrid strains with enhanced tolerance and fermentative potential. Conclusions Our findings highlight the untapped potential of S. eubayanus diversity for bioethanol research and demonstrate the value of mass-mating as a strategy to generate robust, high-performing strains. This work provides a framework for harnessing natural genetic resources to advance efficient, resilient, and sustainable biofuel production.