A two-stage process using aqueous ammonia and hot-water has been investigated to fractionate corn stover. To maximize hemicelluloses recovery and purity in the liquid hydrolyzate by optimizing the fractionation process, the experiments were carried out employing response surface methodology (RSM). A central composite design (CCD) was used to evaluate and confirm the effectiveness and interactions of factors. The optimal fractionation conditions were determined to be as follow: (1) First-stage reactor operated in batch mode using a 15% NH4OH solution (W-NH3 = 15%) at 1:10 solid:liquid ratio, 60 degrees C, and 24 h; (2) second stage percolation reactor operated using hot-water at 20 cm(3) min(-1), 200 degrees C, and 10 mm. The model predicted 51.5% xylan recovery yield and 82.4% xylan purity under these conditions. Experiments confirmed the maximum xylan recovery yield and purity were 54.7% and 83.9% respectively under the optimal reaction conditions. With the solids resulting from the two-stage treatment, 87%-98% glucan digestibilities were obtained with 15 FPU of GC 220 per g-glucan and 30 CBU of Novo 188 per g-glucan enzyme loadings. Xylan digestibility of xylooligomer hydrolysates reached 76% with 8000 GXU per g-xylan of Multifect-Xylanase loading. In the simultaneous saccharification and fermentation (SSF) test using treated solids and Saccharomyces cerevisiae (D(5)A), 86 % to 98% of ethanol yield was obtained on the basis of the glucan content in the treated solids. (C) 2011 Elsevier Ltd. All rights reserved.