ABSTRACT Fixed-bed reactors have been considered promise alternatives for hydrogen production due to their simple construction and increase in the biomass retention. The purpose of this study was to investigate the biological production of hydrogen in anaerobic fixed-bed reactors with cassava starch wastewater used as substrate. Different support materials and arrangements of fixed-bed were used to evaluate the biological production of hydrogen in anaerobic continuous fixed-bed reactors, with cassava starch wastewater as substrate - recycled low-density polyethylene scraps, in packed bed (R1), recycled low-density polyethylene cylinders, in ordained bed (R3) and bamboo stems, in vertical arrangement (R2 and R4). In R1 the initial pH was adjusted for 6.0, with hydraulic retention time (HRT) of 4 h and organic loading rate (OLR) of 9.5 g.L−1.d−1. In R2 the initial pH was maintained in 4.5, with HRT of 4 h and OLR of 9.5 g.L−1.d−1. R3 and R4 were operated with initial pH of 4.5, HRT of 4 h and OLR of 13.5 g.L−1.d−1. The volumetric hydrogen production (VHPR) was favored by lower OLR applied, evenin different pH ranges (6.0 and 4.5). VHPR values of 229 mL H2.L−1.d−1and 248 mL H2.L−1.d−1 were obtained in R1 and R2, respectively. Both in the bamboo stems bed as in the polyethylene cylinders bed, the increase of OLR and the reduction of the initial pH resulted in a diminishing of VHPR to 175 mL H2.d−1.L−1 (R3) and 145 m LH2 .d−1.L−1 (R4). Higher concentrations of butanol (821.32 and 1,529.22 mg.L−1) and ethanol (915.41 and 924.41 mg.L−1) were verified in the reactors with bamboo stems. In R4, the increase of OLR and the reduction of the initial pH contributed to the increase of butanol concentration in 1.8 times, diminishing the VHPR in 41.68%, yield in 63.95% and H2 in 37.6%, and indicating that the effect of pH is more pronounced with the increase of OLR, leading to the solventogenesis.