H-2 production by Petrotoga miotherma, Thermosipho africanus, Thermotoga elfii, Fervidobacterium pennavorans, and Thermotoga neapolitana was compared under microaerobic conditions. Contrary to these previously reported strains being strict anaerobes, all tested strains grew and produced H-2 in the presence of micromolar levels of O-2. T. neapolitana showed the highest H-2 production under these conditions. Microscopic counting techniques were used to determine growth curves and doubling times, which were subsequently correlated with optical density measurements. The Biolog anaerobic microtiter plate system was used to analyze the carbon source utilization spectrum of T. neapolitana and to select non-metabolized or poorly metabolized carbohydrates as physiological buffers. Itaconic acid was successfully used as a buffer to overcome pH-induced limitations of cell growth and to facilitate enhanced production of CO-free H-2.