To enhance the enzymatic conversion of cotton stalks to glucose at high substrate loadings and subsequently to produce ethanol, biomass was subjected to ionic liquid and alkaline pretreatments. Ionic liquid, 1-ethyl-3-methylimidazolium acetate (EMIMAc), which reduced biomass crystallinity, was effective in improving the conversion of cotton stalks to glucose (82-85%) regardless of the substrate loading used during enzymatic hydrolysis. On the other hand, the glucose yield decreased from 67% to 55% with an increase in substrate loading from 3% to 15% (w/v) for the alkaline pretreated biomass. Fermentation of the glucose obtained upon enzymatic hydrolysis of the pretreated cotton stalks at the highest substrate loading (15%) resulted in ethanol yields of 74% and 46% for EMIMAc and alkaline pretreated biomass, respectively. These results highlight the advantage of EMIMAc pretreatment to maintain increased substrate loadings during enzymatic hydrolysis of lignocellulosic biomass for achieving high concentrations of products with improved yields. (C) 2013 Elsevier B.V. All rights reserved.