Investigations were carried out to determine the effect of the pH, the nitrogen source, iron and the dilution rate (h−1) on fermentative hydrogen production from glucose by the newly isolated strain Citrobacter freundii CWBI952. The hydrogen production rate (HPR), hydrogen yield, biomass and soluble metabolites were monitored at 30 °C in 100 mL serum bottles and in a 2.3 L bioreactor operated in batch, sequenced-batch and semicontinuous mode. The results indicate that hydrogen production activity, formate biosynthesis and glucose intake rates are very sensitive to the culture pH, and that additional formate bioconversion and production of hydrogen with lower biomass yields can be obtained at pH 5.9. In a further series of cultures casein peptone was replaced by (NH4)2SO4, a low cost alternative nitrogen source. The ammonia-based substitute was found to be suitable for H2 production when a concentration of 0.045 g/L FeSO4 was provided. Optimal overall performances (ca. an HPR of 33.2 mL H2/L h and a yield of ) were obtained in the semicontinuous culture applying the previously optimized parameters for pH, nitrogen, and iron with a dilution rate of 0.012 h−1 and degassing of biogas by N2 at a 28 mL/min flow rate.