This study investigated the effect of granules formation, hematite nanoparticles and bio-film carriers on biohydrogen production from sucrose wastewater in continuous stirring tank reactors operated at 12 h HRT, pH of 5.5 and 35 degrees C. Granular-based bioreactor was subjected to acid incubation period for 24 h by shifting the pH from 5.5 to 3. Before application of the acid incubation, hydrogen-producing granules (HPGs) diameter and hydrogen production rate (HPR) of 0.5 mm and 4.3 L/L.d, respectively were measured at 10 g-sucrose/L. Application of acid incubation enhanced the granulation process, where the particle size increased to 2.8 mm and'higher HPR of 7.8 L/L.d was obtained. Higher sucrose concentration (15-30 g\L) enhanced HPGs diameter and increased the HPR. At 10 g-sucrose/L, addition of hematite nanoparticles increased the HPR to 5.9 L/L.d higher than 3.87 L/L.d measured in control reactor. Biofilm-based reactor showed HPR of 2.48 L/L.d lower than the control reactor. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.