Investigations were carried out to determine the effect of various concentrations of organic and ammonium nitrogen sources on fermentative hydrogen production by the strain Clostridium butyricum CWBI1009. The results indicated that the H-2-producing metabolism of the strain is favoured within the range (0.56-0.062 g(N)/L) of peptone and (NH4)(2)SO4. Optimal overall performance (i.e. 1.43 +/- 0.08 mol H-2/mol glucose and 1.08 +/- 0.03 mL(H2)/h, respectively) was achieved with 0.062 g(N)/L of casein peptone. The study of the amino acid uptake and the gene expression pattern for four [FeFe]-hydrogenases and the nitrogenase showed that nitrogen was in excess in all the experiments with a nitrogen concentration above 0.062 g(N)/L and, at that optimal concentration, the expression of the HydB2 gene would be responsible for the much higher H-2 yield. (C) 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.