Display omitted •H2 production was investigated in anaerobic packed-bed reactor at acidic conditions.•Produced hydrogen yields (mol H2/mol glucose) were 1.80 (R1)>1.74 (R2)>1.46 (R3).•Main metabolic products were found to be acetic acid, butyric acid and ethanol.•Higher hydrogen yield correlates with low acetate-to-butyrate (HAc/HBu) ratio.•Quantity of attached biomass (gTVS/gsupport) were 0.06 (R1), 0.04 (R2) and 0.035 (R3). This study assesses the impact of different support materials (Mutag BioChip™, expanded clay and activated carbon) on microbial hydrogen production in an anaerobic packed-bed reactor (APBR) treating synthetic waste water containing glucose as the main carbon source at low pH value. The APBRs were inoculated with acid pretreated anaerobic sludge and operated at pH value of 4±0.2 and hydraulic retention time (HRT) of 3h. The maximum hydrogen yield of 1.80mol H2/mol glucose was achieved for the APBR packed with Mutag BioChip™ (R1), followed by expanded clay (R2, 1.74mol H2/mol glucose) and activated carbon (R3, 1.46mol H2/mol glucose). It was observed that the investigated support materials influenced the immobilization of hydrogen producing bacteria and consequently hydrogen production performance as well as composition of soluble metabolites. The main metabolic products were acetic acid and butyric acid accompanied with a smaller content of ethanol. The data indicated that in reactors with higher hydrogen yield (R1 and R2), acetate/butyrate (HAc/HBu) ratios were 1.7 and 1.6, respectively, while in the reactor with the lowest hydrogen yield (R3) the obtained HAc/HBu ratio was 4.8. Finally, stable hydrogen and organic acids production throughout the steady-state operation period at low pH values was achieved in all reactors.