To enhance enzymatic saccharification of pine biomass, the pretreatment reagents peracetic acid (PAA) and ionic liquid (IL) were validated in single reagent pretreatments or combination pretreatments with different sequences. In a 1 h saccharification, 5-25% cellulose conversion was obtained from the single pretreatment of PAA or IL In contrast, a marked enhancement in conversion rates was achieved by PAA-IL combination pretreatments (45-70%). The PAA followed by IL (PAA + IL) pretreatment sequence was the most effective for preparing an enzymatic digestible regenerated biomass with 250-fold higher glucose formation rates than untreated biomass and 2- to 12-fold higher than single pretreatments with PAA or IL alone. Structural analysis confirmed that this pretreatment resulted in biomass with highly porous structural fibers associated with the reduction of lignin content and acetyl groups. Using the PAA + IL sequence, biomass loading in the pretreatment step can be increased from 5% to 15% without significant decrease in cellulose conversion. (C) 2013 Elsevier Ltd. All rights reserved.