This study assesses the impact of organic loading rate on biohydrogen production from glucose in an up-flow anaerobic packed bed reactor (UAnPBR). Two mesophilic UAPBRs (UAnPBR1 and 2) were tested at organic loading rates (OLRs) ranging from 6.5 to 51.4 gCOD L (1) d (1). To overcome biomass washout, design modifications were made in the UAnPBR2 to include a settling zone to capture the detached biomass. The design modifications in UAnPBR2 increased the average hydrogen yield from 0.98 to 2.0 mol-H-2 mol (1)-glucose at an OLR of 25.7 gCOD L (1) d (1). Although, a maximum hydrogen production rate of 23.4 +/- 0.9 L H-2 L (1) d (1) was achieved in the UAnPBR2 at an OLR of 51.4 gCOD L (1) d (1), the hydrogen yield dropped by 50% to around 1 mol-H-2 mol (1)-glucose. The microbiological analysis (PCR/DGGE) showed that the biohydrogen production was due to the presence of the hydrogen and volatile acid producers such as Clostridium beijerinckii, Clostridium butyricum, Megasphaera elsdenii and Propionispira arboris. (C) 2014 Elsevier Ltd. All rights reserved.