Abstract The simultaneous ethanol production and removal during sequential cell recycle fed batch fermentation provides a complementary route to produce this biofuel from sugar mixtures, which may greatly improve yields and productivity from lignocellulosic hydrolysates. Spathaspora passalidarum is a wild-type strain able to naturally convert glucose, fructose, xylose and arabinose into ethanol. Therefore, the present work has focused on 2G bioethanol production by S. passalidarum aiming at the consumption of all sugars released after pre-treatment and enzymatic hydrolysis of sugarcane bagasse in a single fermentation step. The fermentation strategy with sequential cell recycle, fed-batch mode and ethanol removal in situ was performed on a hemicellulosic hydrolysate medium supplemented with molasses. This strategy gave improved fermentation performance and enabled the co-fermention of all sugars under microaerobic conditions. The maximum ethanol yield and productivity was 0.482 g.g−1 and 9.5 g·L-1·h−1, respectively, showing a process efficiency of 94.3%. The selective ethanol removal enables the operation of the bioreactor at low levels of ethanol (20–30 g·L-1), even with high sugar concentration inputs, accelerating the fermentation performance and avoiding inhibitory effects on yeast metabolism. Applying the cell recycle strategy, S. passalidarum was able to increase its robustness, as shown by a 10-fold increase in ethanol productivity, and it was also able to tolerate a high acetic acid concentration (4.5 g·L-1) during long-term fermentations. These results demonstrate that the bioprocess strategy has a strong potential to improve bioethanol production of rich mixed sugar from lignocellulosic hydrolysates in a single fermentation step.