Four different M2+-Mg-Al hydrotalcite (HT) materials were investigated for their effect on biohydrogen enhancement, where M2+ is Fe and/or Zn. HTs were synthesized by the coprecipitation method and characterized by infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD). The effect of Fe-Zn-Mg-Al HTs dose (0-833 mg/L) on hydrogen production was investigated in batch tests using sucrose-fed anaerobic mixed culture at 37 degrees C. The best catalytic activity was observed on Mg-Al HT at 167 mg/L with the maximum hydrogen yield of 2.30 +/- 0.37 mol H-2/mol sucrose, which was 44% higher than the control. The major metabolites detected in the test were acetic acid (3.6 g/L), butyric acid (4.1 g/L), and lactic acid (0.5 g/L). The basic properties of the different catalysts played an important role in stimulating or inhibiting the activity of hydrogen producing bacteria. Calcined Mg-Al HT did not promote biohydrogen production, suggesting that the catalytic enhancement was related to immobilization of bacteria in the electrostatically charged HT interlayers. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.