| Summary: | Glucose, alcohol stillage and glycerol were used as substrates for bio-hydrogen production by the newly isolated strain <i>Clostridium beijerinckii</i> 6A1 under batch conditions. High molar yields of hydrogen from the studied organic substrates were observed. There was a neat difference in the metabolic pathways of substrate digestion when hexose-based substrate or glycerol were used. The products of glycerol digestion showed that a pathway with no formic acid formation as intermediate was probable. In this case, considerable concentrations of acetic and propionic acid (up to 6 g dm<sup>−3</sup>) and small amounts of butanol were observed after 48 h. When glucose or hexose-based substrates were used, considerable amounts of formic acid (up to 6 g dm<sup>−3</sup>), i.e., the pathway proposed for <i>Clostridia</i> mixed cultures, were appropriate for the observed process of hydrogen release. For these substrates, considerable amounts of propionic acid in concentrations up to 1 g dm<sup>−3</sup> were observed. That is why the pathway proposed for mixed cultures seemed more appropriate for our experiments carried out with hexose-based substrates. When hexoses were used, substrate digestion stopped the formation of acetic acid, propionic acid and ethanol. Probably, these intermediates are inhibitors to the further digestion to other products.
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