This study used the fungus, Phanerochaete chrysosporium, to pretreat cotton stalks with two methods, shallow stationary and agitated cultivation, at three supplemental salt concentrations. Pretreatment efficiencies were compared by evaluating lignin degradation, solid recovery and carbohydrate availability over a 14-day period. Shallow stationary cultivation with no salts gave 20.7% lignin degradation along with 76.3% solid recovery and 29.0% carbohydrate availability. The highest lignin degradation of 33.9% at a corresponding solid recovery and carbohydrate availability of 67.8% and 18.4%, respectively, was obtained through agitated cultivation with Modified NREL salts. Cultivation beyond 10 days did not significantly increase lignin degradation during 14 days of pretreatment. Manganese addition during shallow stationary and agitated cultivation resulted in higher solid recoveries of over 80% but lower lignin degradation. Although agitated cultivation resulted in better delignification, results indicate that pretreatment under submerged shallow stationary conditions provides a better balance between lignin degradation and carbohydrate availability. (C) 2009 Elsevier Ltd. All rights reserved.