In this study, individual pretreatments (acid and ultrasonic) and a combination of these pretreatments were optimized to enhance the efficiency of dark fermentative hydrogen production (DFHP) from microalgal biomass. The experimental results show that the maximum H-2 production performance of 42.1 mL H-2/g dry cell weight (dcw) was predicted at 0.79% (v/w) HCl and at a specific energy input (SEI) of 49,600 kJ/kg dcw in the combined pretreatment, while it was limited in both individual pretreatments. Repeated batch testing of the predicted optimal conditions revealed that the combined pretreatment conditions for DFHP from microalgal biomass were successfully optimized by increasing the solubilization of the feedstock and by reducing the formation of the toxic 5-hydroxymethylfurfural (HMF). (C) 2013 Elsevier Ltd. All rights reserved.