The application of pyrolytic sugars for biofuel production through fermentation is challenged by inhibitory contaminant compounds. Inhibition is so severe that only 0.25% sugar syrup can be used. In this study, overliming was tested as a simple detoxification method, using the Escherichia coli KO11+ lgk to directly convert levoglucosan into ethanol. After treatment with at least 14.8 g/L of Ca(OH)(2), fermentation with 2% (w/v) pyrolytic sugar syrup was observed with no inhibition of ethanol production. Further investigation of treatment time and temperature showed that 8-16 h of treatment at 20 degrees C, and 1-4 h of treatment at 60 degrees C are necessary to obtain consistent ethanol production. The samples treated with 18.5 g/L Ca(OH)(2) at 60 degrees C for 4 h showed no inhibition at 2.5%. Multiple contaminants removed by the overliming treatment were identified. This study demonstrates that overliming is a promising method for detoxification of pyrolytic sugars for fermentation. (C) 2013 Elsevier Ltd. All rights reserved.