Lignocellulosic biomass is a promising alternative energy source, which after pretreatment can be efficiently used as feedstock for biological production of second generation bioftels. Hydrogen is considered an ideal biofuel and its production through dark fermentation has been recognized as a sustainable process. However, more studies with continuous systems are needed for its application at industrial Scale. In this study, enzymatic hydrolysate of Agave tequilana bagasse was used for long-term continuous hydrogen production in both, a continuous stirred tank reactor (CSTR) and a trickling bed reactor (TBR), which were operated up to 87 days under different organic loading rates (OLR) ranging from 17 to 60 g COD/L-d. Volumetric hydrogen production rate (VHPR) and hydrogen molar yield (HMY) in CSTR displayed an inverse correlation with maximum values of 2.53 L H-2/L-d and 1.35 mol H-2/mol substrate, attained at OLR 52.2 and 40.2 g COD/L-d, respectively. In contrast, increasing OLR up to 52.9 g COD/L-d simultaneously enhanced VHPR and HMY in TBR, attaining values of 3.45 L H-2/L-d and 1.53 mol H-2/mol substrate, respectively. Acetate and butyrate were the main metabolites in both reactors, while lactate and propionate were detected in minor concentrations. Metabolites distribution, electron balances and hydrogen production trends obtained from both reactors suggest that CSTR may be more susceptible to inhibition by hydrogen accumulation than TBR. Apparent hydrogen consumption and susceptibility to high solids load were found to limit further OLR increments in CSTR and TBR, respectively. (C) 2016 Elsevier B.V. All rights reserved.