Dark fermentative biohydrogen production mainly relies on strains that follow the butyric acid fermentation pathway and substrate that could be easily fermented, i.e., hexose. In this paper, biohydrogen production was performed by using xylose, a typical pentose which is not easily fermented, as the substrate and Klebsiella strain, which follows the formate cleavage pathway, as the fermenter. The full reaction stoichiometry of xylose fermentation was constructed based on electron equivalent (e(-) equiv) balance. The (Delta G degrees) values of the full reactions in the batch experiments were all negative and showed no significant difference in the hydrogen generation performance. However, thermodynamics analysis on each step identified ferment xylose to acetyl-CoA as the thermodynamic bottlenecks (i.e., (Delta G degrees)' similar to-1.6 KJ/e(-)) for all batch experiments. Moreover, the formate cleavage and ethanol-producing reactions became the thermodynamic bottlenecks in the presence of carboxyl azo dye-MR due to the inhibitions on enzymes activity. The results of this work provide a clear metabolic reaction mechanism on fermentative biohydrogen production from xylose by formate cleavage pathway.