AbstractPretreatment is a prerequisite step for increasing the enzymatic digestibility of agricultural residues for conversion to fuels and chemicals in biorefineries. In this study, a sequential acid and alkaline process was developed for pretreatment of rice straw for ethanol fermentation. Effects of key parameters in acid pretreatment were studied using a full factorial design model, which showed the higher influence of time compared to acid concentration and temperature on reducing sugar yields. The combined sequential process involved an initial hemicellulose solubilization by dilute acid using 1% (w/v) H2SO4 at 125 °C for 10 min, followed by alkaline delignification using 1.25% NaOH at 90 °C for 10 min. Under these conditions, a glucose recovery yield of 70.9% from saccharification of the cellulose enriched fraction was obtained with 2- to 4-fold savings in chemical usage as compared with single-step processes. Scanning electron microscopy revealed modification of biomass micro-structure and increases in reactive surface area. Simultaneous saccharification and fermentation of the solid residues by Saccharomyces cerevisiae, using 25 FPU/g Accellerase® 1500, led to a final ethanol concentration of 21.0 g/L with the productivity of 0.27 g/L/h, equivalent to 84.6% theoretical yield. The results indicate the potential of the sequential process for increasing pretreatment efficiency and allowing stepwise separation of lignocellulose components for multi-product biorefineries.