Abstract: The aim of this study was to investigate the influence of some environmental factors on bacterial metabolism. Fermentative hydrogen production by C. acetobutylicum, using glucose as the substrate. The effect of initial pH (4-8), inoculum size (1-20% (v/v)) and glucose concentration (1-30 g L-1) on hydrogen production were studied. The optimum cultivation temperature for hydrogen production was at 30 °C. The results show that substrate concentration and inoculum size resulted in hydrogen yield (YP/S) of 391 mL g-1 glucose utilized with maximum hydrogen productivity of 77.5 mL/L/h. Higher substrate concentration or inoculum size adversely affects hydrogen production, which decreases hydrogen yield by 15% to 334 mL g-1 glucose utilized when 30% (v/v) inoculum size was used. The use of 30 g L-1 substrate concentration resulted in a 75% decrease to 97 mL g-1 glucose supplied. Concluded that proper Xo/So enhanced the hydrogen production.