Microwave-assisted pretreatment of recalcitrant softwood in aqueous glycerol containing a series of organic and inorganic acids with different pK(a) values was examined. The pulp obtained by organosolvolysis with 0.1% hydrochloric acid (pK(a) - 6) at 180 degrees C for 6 min gave the highest sugar yield, 53.1%, based on the weight of original biomass. The pretreatment efficiency correlated linearly with the pK(a) of the acids, with the exception of malonic and phosphoric acids. Organosolvolysis with 1.0% phosphoric acid (pK(a) 2.15) gave a saccharification yield (50.6%) higher than that expected from its pK(a), while the catalytic effect of malonic acid (pK(a) 2.83) was negligible. Extensive exposure of crystalline and non-crystalline cellulose by the glycerolysis with strong inorganic acids was demonstrated by using fluorescent-labeled recombinant carbohydrate-binding modules (CBMs). Because of the low concentration of the acid catalysts and availability of glycerol as a by-product from biodiesel and fatty acid production, organosolvolysis in glycerol is an appealing process for pretreatment of recalcitrant softwood. (C) 2010 Elsevier Ltd. All rights reserved.