Hydrogen generation from complex substrates composed of simple sugars has the potential to mitigate future worldwide energy demand. The biohydrogen potential of a sequential microaerobic dark- and photo-fermentative system was investigated using immobilized Rhodobacter capsulatus JP91. Biological hydrogen production from glucose was carried out using a batch process and a bench-scale bioreactor. Response surface methodology with a Box-Behnken design was employed to optimize key parameters such as inoculum concentration, oxygen concentration, and glucose concentration. The maximum hydrogen production (21 +/- 0.25 mmol H-2/L) and yield (7.8 +/- 0.1 mol H-2/mol glucose) were obtained at 6 mM glucose, 4.5% oxygen and 62.5 v/v% inoculum concentration, demonstrating the feasibility of enhanced hydrogen production by immobilized R. capsulatus JP91 in a sequential system. This is the first time that a sequential process using an immobilized system has been described. This system also achieved the highest hydrogen yield obtained by an immobilized system so far.