Sequential electron beam-steam explosion (EB-SE) pretreatment was applied to hardwood (Birch) and softwood (Pine) substrates in an effort to enhance their enzymatic saccharification. The effect of these two pretreatments on the structure and composition of the individual cell wall components was examined. The combination of these treatments showed a synergistic effect on the conversion of hemicelluloses into water soluble oligomers, and enhanced the overall enzymatic saccharification of wood substrates. Even after the combined pretreatment Pine was more recalcitrant than Birch, which was attributed to differences in the lignin. Model systems created from cellulose and isolated high molecular weight (HMW) lignin fractions were found to inhibit enzymatic conversion of cellulose by 20% over a control. This inhibition is likely related to the unproductive binding of the cellulose enzymes to the HMW lignin. Additionally, the presence of the HMW lignin reduces the swelling capacity of the wood substrate, and thus its accessibility to enzymes. Conversely, low molecular weight lignin fragments were found to be slightly beneficial for enzymatic hydrolysis of cellulose substrates. These results provide insight to the complex interactions between lignin and cellulase enzymes, and highlight the need for pretreatment processes that can effectively cleave lignin into oligomeric fragments. (C) 2017 Elsevier Ltd. All rights reserved.