Simultaneous saccharification and fermentation (SSF) of lignocellulosic substrate is limited by low glucose availability in the early stages of SSF, different temperature optima for enzymatic hydrolysis and fermentation, and low ethanol productivity. These were eliminated in this work by carrying out the pre-saccharification step at the optimum temperature for cellulolytic enzymes. When inoculation was delayed for 12 h (dSSF process), ethanol productivity was enhanced and the process was shortened by 60% compared to SSF, while the final ethanol concentration remained the same. Increased loading of acid-pretreated wheat straw in batch mode did not improve the ethanol concentration; although pre-concentration of substrate from 36% to 66% of wet solid enabled a 48% increase in glucose concentration it negatively influenced fermentation. The fed-batch dSSF process, with gradual feeding of pre-hydrolysed medium, carried out in two bioreactors, enabled us to increase the final ethanol concentration and to eliminate problems associated with high concentrations of inhibitors in batch mode and high viscosity of the medium. (C) 2014 Elsevier Ltd. All rights reserved.