Isolation and Screening of Wild Indigenous Yeasts from Molasses and Sugary Substrates in Bioethanol Production

The growing global demand for energy has primarily encountered by fossil fuels, particularly petroleum. However, due to its rising cost, environmental impact, and non-renewable nature, there is an urgent need to develop sustainable and eco-friendly alternatives such as bioethanol. In this study, 35 samples of sugary substrates were collected from Wonji Shoa Sugar Factory for wild yeasts isolation following the standard protocols. A total 305 yeast isolates were retrieved and screened using physiological and osmotic stress tolerance tests. Fermentative and potent wild yeasts were identified using morphological and biolog-based biochemical methods. A total 305 wild yeast isolates obtained from various substrates at Wonji Shoa Sugar Factory. These isolates exhibited diverse colony morphologies and predominantly had oval-shaped cells. Out of the total, 20 isolates (6.56%) tolerated 18% ethanol, and seven isolates (2.29%) survived at 20% ethanol concentration. Among those ethanol-tolerant strains, 17 showed thermotolerance at 45°C temperature for 48 hours. Selected isolates also demonstrated significant gas production from glucose and tolerance to high sugar concentrations (up to 35% glucose), low pH (as low as pH 2), and 7% NaCl. Biolog system confirmed the species as Kluyveromyces lodderae, Saccharomyces cerevisiae, Saccharomyces boulardii, Zygosaccharomyces rouxii, and Torulaspora globosa. In batch fermentation using molasses, these species produced bioethanol concentrations of 12.62%, 11.61%, 10.58%, 10.82%, and 10.44% (v/v) at 48 hours, with corresponding fermentation efficiencies of 98.0%, 97.8%, 93.1%, 90.8%, and 88.1%, respectively. Based on their super ethanol yield, fermentation efficiency, and tolerance to industrial stressors, K. lodderae and S. cerevisiae emerged as the most promising candidates for bioethanol production. Keywords: Bioethanol, fermentation efficiency, indigenous yeast, stress tolerance, and thermotolerant yeast