The growth kinetics of hydrogen producing bacteria using three different substrates, namely sucrose, non-fat dry milk (NFDM), and food waste were investigated in dark fermentation through a series of batch experiments. The results showed that hydrogen production potential and hydrogen production rate increased with an increasing substrate concentration. The maximum hydrogen yields from sucrose, NFDM, and food waste were 234, 119, and 101 mL/g COD, respectively. The low pH (pH < 4) inhibited hydrogen production and resulted in lower carbohydrate fermentation at high substrate concentration. Michaelis-Menten equation was employed to model the hydrogen production rate at different substrate concentrations. The equation gave a good approximation of the maximum hydrogen production rate and the half saturation constant (K-S) with correlation coefficient (R-2) over 0.85. The K-S values of sucrose, NFDM, and food waste were 1.4, 6.6, and 8.7 g COD/L, respectively. Based on K-S values, the substrate affinity of the enriched hydrogen producing culture was found to depend on carbohydrate content of the substrate. The substrate containing high carbohydrate showed a lower K-S value. The maximum hydrogen production rate was governed by the complexity of carbohydrates in the substrate. (c) 2006 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.