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Abstract Purpose Co-culturing is a widely used method to improve bioethanol production from biomass enriched in fermentable sugars. This study aims to produce bioethanol from sugarcane molasses by simultaneous co-fermentation of S. cerevisiae isolate TA2 and W. anomalus isolate HCJ2F-19. Methods Response surface methodology (RSM) based on the central composite design (CCD) was employed to optimize fermentation conditions, including mixing rate (110–150 rpm), temperature (25–35 °C), molasses concentration (25–35 obrix), and incubation time (36–72 h). The ethanol concentration was analyzed using HPLC equipped with a UV detector. Results The monocultureS. cerevisiae isolate TA2 produced 17.2 g.L−1 of ethanol, 0.33 g.g−1 of ethanol yield, and 0.36 g.L−1.h−1 of productivity compared to W. anomalus isolate HCJ2F that produced 14.5 g.L−1, 0.30 g.g−1 and 0.28 g.L−1.h−1 ethanol, ethanol yield, and productivity under laboratory conditions, respectively. In comparison to single cultures of S. cerevisiae TA2 and W. anomalus HCJ2F, the co-fermentation using both isolates showed an increased ethanol yield of 29% and 53% compared to the single species fermentations, respectively. The results showed that the growth of W. anomalus HCJ2F-19 and S. cerevisiae TA2 was not influenced by each other during the co-fermentation process. The one variable at a time optimization (OVAT) analysis resulted in an ethanol concentration of 26.5 g.L−1 with a specific yield and productivity of 0.46 g.g−1, 0.55 g.L−1.h−1, respectively, at pH 5.5, 25 obrix, 48 h, 150 rpm, 30 °C, 60:40 inoculum ratio, and 10% overall inoculum size. The maximum ethanol concentration of 35.5 g.L−1 was obtained by co-fermentation using the RSM-CCD tool at 30 obrix, 30 °C, 54 h, and 130 rpm. Conclusion The results suggested that the co-fermentation of S. cerevisiae isolate TA2 and W. anomalus isolate HCJ2F improves bioethanol production from sugar cane molasses under optimum fermentation conditions. |