A continuous mixed immobilized sludge reactor (CMISR) using activated carbon as support carrier for dark fermentative hydrogen production from enzymatic hydrolyzed food waste was developed. The effects of immobilized sludge packing ratio (10-20%, v/v) and substrate loading rate (OLR) (8-40 kg/m(3)/d) on biohydrogen production were examined, respectively. The hydrogen production rates (HPRs) with packing ratio of 15% were significantly higher than the results obtained from packing ratio of 10% and 20%. The best HPR of 353.9 ml/h/L was obtained at the condition of packing ratio = 15% and OLR = 40 kg/m(3)/d. The Minitab was used to elicit the effects of OLR and packing ratio on HPR (Y) which could be expressed as Y = 5.31 OLR + 296 packing ratio + 40.3 (p = 0.003). However, the highest hydrogen yield (85.6 ml/g food waste) was happened at OLR of 16 kg/m(3)/d because of H-2 partial pressure and oxidization/reduction of NADH. (C) 2014 Elsevier Ltd. All rights reserved.