Sorghum grain (Sorghum bicolor (L.) Moench) grown in a temperate climate was used as a feedstock for bioethanol production. Mixed cultures of Zymomonas mobilis and Saccharomyces cerevisiae were used for fermentation under the conditions and parameters established through the design of experiments (DOE, 2(K-p) standard design). The most effective parameters for the simultaneous saccharification and fermentation of the liquefied and granular starch fermentation methods were selected. Analyses revealed that the granular starch fermentation method (with Stargen 002) combined with mixed cultures yielded higher bioethanol by about an average of 5% compared to single cultures. Distillates obtained in fermentations with mixed cultures were investigated qualitatively and quantitatively for particular volatile byproducts. Distillates from fermentation with mixed cultures contained higher amounts of volatile byproducts (9.04-11.38 g/L) than single bacterial cultures (4.10-6.68 g/L) but less than single yeast cultures (9.27-13.14 g/L). These findings indicate the potential of mixed cultures in granular starch fermentation, at the same time suggesting that challenges such as selecting microbial consortia, accelerating the process, and ensuring consistent yields must be solved for widespread industrial application.