AbstractAlkali-pretreated sugarcane bagasse fiber was subjected to fed-batch simultaneous saccharification and fermentation (SSF) with a pre-hydrolysis process to increase the solids loading and produce a high concentration of ethanol. The hydrolysis medium and yeast feeding modes were investigated to determine suitable conditions for high sugar yield and ethanol production. Batch addition resulted in a cumulative substrate concentration of up to 36% (w/v) and enhanced ethanol concentrations, while ethanol conversion efficiency gradually declined. Enzymatic pre-hydrolysis and fermentation with fed-batch mode contributed to the SSF process. The highest ethanol concentration was 66.915 g/L with the conversion efficiency of 72.89%, which was achieved at 30% (w/v) solids content after 96 h of fermentation. Hydrolyzed medium and yeast were added in batch mode at 24 h of enzymatic hydrolysis and fermentation, respectively. Thus, combining the fed-batch mode with pre-hydrolysis SSF produced a high yield of ethanol.