Fermentable sugar obtained from lignocellulosic material exhibits great potential as a renewable feedstock for the production of bio-ethanol. One potentially viable source of fermentable sugars is pyrolysis oil, commonly called bio-oil. Depending on the type of lignocellulosic material and the operating conditions used for pyrolysis, bio-oil can contain upwards of 10 wt% of 1,6-anhydro-beta-D-glucopyranose (levoglucosan, LG), an anhydrosugar that can be hydrolyzed to glucose. This research investigated the extraction of levoglucosan from pyrolysis oil via phase separation, the acid-hydrolysis of the levoglucosan into glucose, and the subsequent fermentation of this hydrolysate into ethanol. Optimal selection of water-to-oil ratio, temperature and contact time yielded an aqueous phase containing a levoglucosan concentration of up to 87 g/L, a yield of 7.8 wt% of the bio-oil. Hydrolysis conditions of 125 degrees C, 44 min and 0.5 M H2SO4 resulted in a maximum glucose yield of 216% (when based on original levoglucosan), inferring other precursors of glucose were present in the aqueous phase. The aqueous phase contained solutes which inhibited fermentation, however, up to 20% hydrolysate solutions were efficiently fermented (yield = 0.46 g EtOH/g glucose; productivity = 0.55 g/L, h) using high yeast inoculums (1 g/L in flask) and micro-aerophilic conditions. (C) 2009 Elsevier Ltd. All rights reserved.