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Purpose Yeasts are widely used for the production of bioethanol from biomasses rich in sugar. The present study was aimed at isolating, screening, and characterizing fermentative wild yeast recovered from bio-waste and co-products of Ethiopian sugar factories for bioethanol production using sugarcane molasses as a substrate. Method The wild yeasts were identified according to their cellular morphology and D1/D2 and ITS1-5.8S-ITS2 rDNA sequencing. Analysis of ethanol and by-product concentration was done by HPLC equipped with a UV detector. Higher alcohols, acetaldehyde, and methanol were analyzed using GC-MS equipped with a flame ionization detector (FID). Result Seven strains (Meyerozyma caribbica MJTm3, Meyerozyma caribbica MJTPm4, Meyerozyma caribbica SHJF, Saccharomyces cerevisiae TA2, Wickerhamomyces anomalus MJTPm2, Wickerhamomyces anomalus 4m10, and Wickerhamomyces anomalus HCJ2F) were found tolerant to 18% (v/v) ethanol, whereas one strain Meyerozyma caribbica MJTm3 tolerated 20%. These strains also showed tolerance to 45°C, 50% of sugar, and pH 2–10. Meyerozyma caribbica MJTm3 produced 12.7% (v/v) of alcohol with an actual ethanol concentration of 26 g L−1, an ethanol yield of 47%, 78% of theoretical yield, and a productivity of 0.54 g L−1 h−1 from 30 °Brix of molasses at 48 h incubation under laboratory scale. Based on the one variable at a time optimization (OVAT), the optimal parameters for maximum bioethanol production were at initial pH 5.5, 35 °Brix, 30°C, 15% inoculum size, 150 rpm, 4 g L−1 di-ammonium phosphate supplement, and 48 h incubation. Under these optimum conditions, 14% (v/v) alcohol, 42 g L−1 actual ethanol concentration, 69% ethanol yield, 89% of theoretical yield, and productivity of 0.88 g L−1 h−1 were obtained. Conclusion These results indicated that M. caribbica MJTm3 should further be evaluated, optimized, and improved for industrial bioethanol production due to its fermentation potential.

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