Four isolates of Clostridium genus were obtained from the activated sludge and one Escherichia coli isolated was found in sheep ruminal fluid. These isolates were identified by microscopic methods and by rRNA sequences. Growth, production of metabolic gases and production of organic acids were measured during the anaerobic cultivation of these isolates with glycerol as sole carbon source. It was found that these isolates from activated sludge were related to Clostridium botulinum, C. perfringens and C. difficile. One strain could not be assigned to any species but was similar to C. botulinum, and one ruminal bacterium was identified as Escherichia coli. All isolates grew on the medium with glycerol as sole carbon source with prolonged lag phase. The lag phase was shorter after adaptation of cells to glycerol. All these strains produced H2, and CO in concentration range 10 2 µmol L–1, and H2S in concentrations lower by one order of magnitude. Kinetics of evolution of these gases was different suggesting that they are produced by independent processes. The major organic acid produced with glycerol as the carbon source was acetic acid. Butyric, formic, and propionic acids were produced in dependence on the isolated tested. Results show that metabolic gases are produced mainly in the exponential phase of growth.