Overliming is an effective way of conditioning to reduce the toxicity of hydrolysates generated from pretreatment of lignicellulosic biomass for ethanol production. In this work, a range of target overliming pH values from 9 to 11 was studied, and xylose fermenting Zymomonas mobilis strain 8b was used to evaluate the fermentability of overlimed corn stover hemicellulose hydrolysate. pH 11 overlimed hydrolysate was highly fermentable, but xylose losses were the greatest at this condition. Based on ethanol yield and fermentative xylose conversion, pH 10-conditioned hydrolysate produced the best results, 75% xylose utilization and 76% ethanol yield. This condition also produces the highest overall ethanol yield based on total sugars available in the unconditioned hydrolysate, 70%. Overall mass balance closures were very good averaging between 97 and 100% for all experiments. Calcium and sulfur mass balance closures ranged from 75 to 90% and indicated that approximately 50% of the calcium ends up in the gypsum cake, which is calculated to be approximately 63% gypsum. Overall result shows that the pH of the overliming process is the key factor for improving hydrolysate fermentability but too high a pH destroys some of the available sugars and reduces overall ethanol yield. Thus, it is critical to keep the overliming pH as low as possible while making the hydrolysate fermentable. (c) 2006 Elsevier Ltd. All rights reserved.