BACKGROUND Lignocellulosic biomass is a promising renewable feedstock to obtain fuels and chemicals. However, a suitable pretreatment is required to separate its main components and overcome its structural resistance to enzymatic hydrolysis. In this work, a sequential pretreatment composed of autohydrolysis (AH) and organosolv delignification (ORG) is used to fractionate Pinus radiata wood and improve the enzymatic hydrolysis. RESULTS The effect of three different AH pretreatment severities on the delignification degree of organosolv solid fractions was evaluated. In addition, enzymatic hydrolysis was performed to determine the efficiency of the combination of pretreatments. The results showed that a pretreated solid with high cellulose content (88%, w/w) and high delignification degree (88%) was achieved under mild conditions of AH (150 degrees C and 30 min) and acid-catalyzed ORG (185 degrees C, 75 min, 50% (w/w) ethanol and 1% (w/w) sulfuric acid), obtaining an enzymatic hydrolysis yield of 63%. CONCLUSION This study provides the most favorable conditions to fractionate a softwood, P. radiata, employing a two-step pretreatment based on AH followed by ORG. (c) 2019 Society of Chemical Industry