Corn stover is an agricultural residue consisting of lignocellulose, cellulose and hemicellulose polymers, sheeted in a lignin barrier. Corn stover can be used as feedstock for biogas production. Previous studies have shown biological pretreatment of lignocellulose materials can increase digestibility of the substrate improving hydrolysis, the rate-limiting step in biogas production. The impact of pretreating with different enzymes (laccase, manganese peroxidase and versatile peroxidase) and different incubation times, (0, 6 and 24 h) was studied. The effect on the matrix and biomethane production was determined. Pretreatments did not yield high concentrations of phenolic compounds, inhibitors of biogas production. The laccase enzyme showed an increase in biomethane production of 25% after 24 h of incubation. Pretreatment with peroxidase enzymes increased biomethane production with 17% after 6 h of incubation. As such it can be concluded that by introducing the different enzymes at different stages during pretreatment an increased biomethane production can be obtained. (C) 2014 Elsevier Ltd. All rights reserved.