The impact of H2 consumption, by homoacetogens, on the overall hydrogen yield in mixed culture fermentation is the focus of this study. Batch reactors were used to test the ability of anaerobic digested sludge, heat treated to eliminate methanogens, to produce and consume molecular hydrogen. The measured average hydrogen production rate from 4.2 g glucose/L was 44 +/- 4 mmol H2/L sludge . d, while the H2 consumption rate varied much more, in the range from 4 to 62 mmol H2/L sludge . d. Hydrogen consumption rate depends on acetic acid concentration, headspace H2 partial pressure and mass transfer rates. Different but relatively long lag-phases for hydrogen consumption were observed. It is concluded that homoacetogenesis can have a significant negative effect on bio-H2 production by mixed cultures, limiting the possibilities for sustainable solutions.