Co-fermentation of glucose and xylose from lignocelluloses is an efficient approach to increasing ethanol production. Simultaneous saccharification and co-fermentation (SSCF) of corn stover pretreated with aqueous ammonia was performed using engineered yeast with xylose utilization pathway. Thus far, the effect of the several key factors on SSCF was investigated, including temperature, inoculation size, pre-hydrolysis and pH. Ethanol concentration was achieved to 36.5 g/L during SSCF process with 6% glucan loading. The addition of Tween 20 reduced enzyme loading, i.e., from 15 to 7.5 FPU/g glucan with the same final ethanol concentration. The ethanol concentration was achieved to 70.1 g/L at 12% glucan loading. Yeast feeding, combined with substrate and enzyme feeding, was proved to be an efficient approach for SSCF with high solid loading. (C) 2014 Elsevier Ltd. All rights reserved.