An integrated bioconversion process was developed to convert corn stover derived pentose and hexose to ethanol effectively. In this study, corn stover was pretreated by soaking in aqueous ammonia (SAA), which retained glucan (similar to 100%) and xylan (>80%) in the solids. The pretreated carbohydrates-rich corn stover was converted to ethanol via two-phase simultaneous saccharification and fermentation (TPSSF). This single-reactor process employed sequential simultaneous saccharification and fermentation (SSF), i.e. pentose conversion using recombinant Escherichia coil KO11 in the first phase, followed by hexose conversion with Saccharomyces cerevisiae D5A in the second phase. In the first phase, 88% of xylan digestibility was achieved through the synergistic action of xylanase and endo-glucanase with minimal glucan hydrolysis (10.5%). Overall, the TPSSF using 12-h SAA-treated corn stover resulted in the highest ethanol concentration (22.3 g/L), which was equivalent to 84% of the theoretical ethanol yield based on the total carbohydrates (glucan + xylan) in the untreated corn stover. (C) 2010 Elsevier Ltd. All rights reserved.