This study investigated pretreatment of corn stover using solid substrate cultivation (SSC) of Phanerochaete chrysosporium (P. chrysosporium) to improve subsequent accessibility to cellulose. Thereafter, Clostridium thermocellum (C. thermocellum) was directly inoculated onto the pretreated biomass to accomplish hydrolysis, followed by solventogenesis by introducing Clostridium beijerinckii (C. beijerinckii). An enzyme suite containing laccase, lignin peroxidase and manganese peroxidase activity was detected during the cultivation of P. chrysosporium on corn stover within 288 h at an initial moisture content wH(2)O = 80% (mass of water/total mass). Incubation factors, such as substrate moisture content and cultivation temperature affected the percent of lignin removal which ranged from 14.4% to 36.4% of the original lignin. Lignin removal increased as the cultivation of P. chrysosporium continued but was accompanied by increased cellulose loss. The 7-day fungal cultivation sufficiently delignified the corn stover for the subsequent processing. Approximately 25% of the original lignin was removed; however 18% of the initial cellulose was also removed with the lignin. The investigations of the effect of fungal pretreatment were extended to miscanthus, wheat straw and switch grass. The yield of reducing sugar produced by C. thermocellum on pretreated biomass was doubled compared with non-pretreated biomass, demonstrating that pretreatment resulted in a more accessible carbon source for the solvent-producing bacterium. The final comprehensive comparison between the pretreated biomass and non-pretreated biomass on the three-stage SSC for butanol production showed pretreatment by P. chrysosporium improved microbial utilization of lignocellulosic materials for solvent production by approximate 4-7 folds. (C) 2014 Elsevier Ltd. All rights reserved.