This study reports the ability of Escherichia coli WDHL to metabolize pentoses commonly found in lignocellulosic hydrolysates to produce bioethanol (EtOH) and biohydrogen (H-2). A Central Composite experimental design was used to establish the effects of pH, temperature and total reducing sugars (TRS) from the wheat straw hydrolysate (WSH) on production, production rate and yield of bioethanol and biohydrogen. Optimal conditions for the simultaneous production were pH 8.2, 31 degrees C and 23.4 g TRS dm(-3). Batch culture was performed with these conditions in a 1 dm(3) bioreactors, obtaining: 8.9 g EtOH dm(-3), 0.050 g EtOH dm(-3) h(-1), 0.38 g EtOH g TRS-1, and 3277.7 cm(3) H-2, 18 cm(3) H-2 dm(-3) h(-1), 140.1 cm(3) H-2 g TRS-1. The results showed that the pH and the temperature affected the production of both biofuels. This work enriches the information of simultaneous production of bioethanol and biohydrogen by using second-generation substrates. (C) 2016 Elsevier Ltd. All rights reserved.