Four different M2þeMgeAl hydrotalcite (HT) materials were investigated for their effect on biohydrogen enhancement, where M2þ 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 FeeZneMgeAl HTs dose (0e833 mg/L) on hydrogen production was investigated in batch tests using sucrose-fed anaerobic mixed culture at 37 C. The best catalytic activity was observed on MgeAl HT at 167 mg/L with the maximum hydrogen yield of 2.30 0.37 mol H2/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 MgeAl HT did not promote biohydrogen production, suggesting that the catalytic enhancement was related to immobilization of bacteria in the electrostatically charged HT interlayers.