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The potential of using a two-stage hydrogen and methane fermentation of sugarcane juice was studied. The effects of pure and mixed culture as inocula on hydrogen production were compared. Additionally, the effects of pure culture inocula form, i.e., free cells or immobilized cells of Clostridium butyricum TISTR1032 was compared to different forms of mixed cultures, i.e., granules or suspended cells of heat-treated upflow anaerobic sludge blanket (UASB) granules. The hydrogenogenic effluents from all treatments were used as substrates to evaluate the potential of methane production by non-pretreated UASB granules. Results showed that a pure culture gave a higher hydrogen production potential and a shorter lag time in comparison to mixed cultures. Immobilized cells of C. butyricum TISTR1032 gave a hydrogen production potential that was 1.2 times higher than that of free cells. However, there was no significant difference in