Pelleting increases the efficiency of handling and transportation of biomass for conversion into biofuels either through biochemical or thermochemical process. Pretreatment of biomass makes lignin fragments accessible by disrupting the lignocellulosic structure, and ensures the production of high-quality pellets. In this study, biological pretreatment using solid-state fermentation (SSF) was investigated as a means to improve the quality of pellets produced from wheat straw. SSF of wheat straw using Trametes versi color 52J (TV52J), T. versicolor m4D (TVm4D) and Phanerochaete chrysosporium (PC) were conducted. Response surface methodology was employed by using a four-factor, three-level Box-Behnken design with moisture content (% mass fraction of water), hammer mill screen size used for chopping wheat straw (mm), fermentation time (days) and fermentation temperature (degrees C) as process parameters. Pellet density, dimensional stability and tensile strength were the response variables. The fermentation temperature significantly affected all the responses. The optimization options of moisture content (70% mass fraction of water), hammer mill screen size (50-52.8 mm) and fermentation temperature (22-22.1 degrees C) of wheat straw with pretreatment using the three fungal strains were very similar. The optimized fermentation time of wheat straw pretreated with PC was the longest at 35 days; the shortest at 21 days was for wheat straw pretreated with TV52J. The microscopic structural changes induced by microbial pretreatment were examined by scanning electron microscopy (SEM). Results showed that the combination between single fibers became relatively loose, and the connection was never tight which was advantageous to improve the physical quality of the compressed pellets. Crown Copyright (C) 2016 Published by Elsevier Ltd. All rights reserved.