The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H-2) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H-2 production at various growth temperatures, (2) investigate the effect of oxygen (O-2) on H-2 production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85 degrees C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H-2 production reached maximum levels in both the 77 degrees C and 85 degrees C experiments. The fermentation stoichiometry for T. neapolitana at 85 degrees C was 3.8 mol H-2, 2 mol CO2, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H-2 production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H-2 through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1035-1042, 2009