Grasslands are often abandoned due to lack of profitability. Extensively cultivating grassland for utilization in a biogas-based biorefinery concept could mend this problem. Efficient bioconversion of this lignocellulosic biomass requires a pretreatment step. In this study the effect of different steam explosion conditions on hay digestibility have been investigated. Increasing severity in the pretreatment induced degradation of the hemicellulose, which at the same time led to the production of inhibitors and formation of pseudo-lignin. Enzymatic hydrolysis showed that the maximum glucose yields were obtained under pretreatment at 220 degrees C for 15 min, while higher xylose yields were obtained at 175 degrees C for 10 min. Pretreatment of hay by steam explosion enhanced 15.9% the methane yield in comparison to the untreated hay. Results indicate that hay can be effectively converted to methane after steam explosion pretreatment. (C) 2014 Elsevier Ltd. All rights reserved.