Isolation, screening, and molecular characterization of indigenous yeast strains from fruits for bioethanol production using molasses

The increasing global demand for fossil fuels, driven by rapid population growth, has led to resource depletion and rising energy costs, prompting the search for renewable alternatives such as bioethanol. This study aimed to isolate, screen, and characterize potent yeast strains from selected fruits and evaluate their bioethanol production using molasses as a substrate. Eighteen fruit samples were randomly collected from Gondar City, Tikil Dingay, and Kola Diba in the Amhara Regional State, Ethiopia. Yeasts were isolated using the serial dilution technique and screened based on gas production and medium color change. Identification was conducted through morphological, physiological, biochemical, and molecular analyses, including sequencing of the ITS1, ITS2, and 5.8 S rRNA regions. A total of 22 yeast isolates were obtained, of which four (ASMS2, A1IS3, AS2M2, and AS3M10) demonstrated strong fermentation potential. Optimal growth was observed at 37 °C and pH 5. All selected isolates tolerated up to 5% ethanol and 30% glucose concentrations and fermented glucose, sucrose, and maltose, but not galactose or lactose. Ethanol concentrations ranged from 0.780 to 1.218%, with fermentation efficiencies between 36.0% and 47.0%, and ethanol productivity from 0.16 to 0.23 g/L/h. Molecular identification revealed A1IS3 as Pichia kudriavzevii and AS2M2 as Pichia species. The findings indicate that mango and avocado fruits are promising sources of ethanol-producing yeasts, and further optimization of fermentation conditions is recommended.