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Summary: Enzyme determinations in bacteria-free extracts and dual-wavelength experiments with membrane suspensions established that Propionibacterium freudenreichii converted glycerol into triose phosphate via glycerol kinase and NAD-independent glycerol 1-phosphate dehydrogenase which is closely linked to cytochrome b. Glycerol 1-phosphate dehydrogenase uses fumarate as a final hydrogen acceptor. The enzyme system catalysing fumarate reduction with glycerol 1-phosphate as a hydrogen donor, is membrane bound and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO). Fumarate reduction with reduced benzyl-viologen is not inhibited by HOQNO. Cytochrome b is therefore probably involved in the anaerobic electron transport from glycerol 1-phosphate to fumarate. Molar growth yields and fermentation balances were determined for P. freudenreichii and P. pentosaceum growing on glucose, fructose, glycerol and lactate and ATP yields (mol of ATP formed/mol of substrate fermented) were calculated assuming that 1 mol ATP is formed in the electron transport from glycerol 1-phosphate and lactate to fumarate, and that 2 mol ATP are formed in the electron transport from NADH to fumarate. Mean Y ATT values (g dry wt bacteria/mol ATP) were 15.2 and 12.9 for P. freudenreichii, and 16.4 and 11.8 for P. pentosaceum each growing on complex or synthetic medium respectively. The observation that for each strain YATP values were constant for the same medium, supported our assumptions on energy generation in propionic acid bacteria.