Hydrogen production by dark fermentation is one of the most promising methods for obtaining clean energy. Inoculum pretreatments allow the selection of bacteria that have better performance in hydrogen production, because the selection of pretreatment limits the presence of some species while favoring others. In order to elucidate the inoculum pretreatment influence during the operation of two EGSB reactors, two pretreatments were assayed: heat shock and cell wash-out. Different organic loading rates (24-60 g glucose/L d) and hydraulic retention times (10-4 h) were applied to both reactors to determine population dynamics along 100 days of operation. Reactors exhibited differences in both volumetric hydrogen production rate and molar yield but with cell wash-out pretreatment showing better performance than heat shock pretreatment. Maximum molar yield (0.92 mol H-2/mol hexose) and volumetric hydrogen production rate (4.23 L H-2/L d) were obtained with organic loading rates of 36 g glucose/L d at HRT of 10 h in EGSB reactor inoculated with cell wash-out pretreated sludge. The microbial community of the reactors samples was analyzed by 16S rRNA genes profiles and the predominant bands were excised and their DNA sequence determined. Clostridium and representatives of Enterobacteriaceae were dominant, with a strong presence of Lactobacillus genus. The whole result indicates that the inoculum pretreatment has a strong initial effect during early stages of fermentation, after which the operating conditions have a greater impact on reactor performance. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.