Isolation and characterization of a new bacterium carboxylating phenol to benzoic acid under anaerobic conditions

A consortium of spore-forming bacteria transforming phenol to benzoic acid under anaerobic conditions was treated with antibiotics to eliminate the four Clostridium strains which were shown to be unable to accomplish this reaction in pure culture and coculture. Clostridium ghonii was inhibited by chloramphenicol (10 mu g/ml), whereas Clostridium hastiforme (strain 3) and Clostridium glycolicum were inhibited by clindamycin (20 mu g/ml), without the transformation of phenol being affected. Electron microscopic observations of resulting liquid subcultures revealed the presence of two different bacilli: a dominant C. hastiforme strain (strain 2) (width, I mu m) and an unidentified strain 6 (width, 0.6 mu m) which was not detected on solid medium. Bacitracin (0.5 U/ml) changed the ratio of the strains in favor of strain 6. C. hastiforme 2 was eliminated from this culture by dilution. The isolated strain 6 transformed phenol to benzoic acid and 4-hydroxybenzoic acid to phenol and benzoic acid in the presence of proteose peptone. Both of these activities are inducible. This strain is a gram-variable, flagellated rod with a doubling time of 10 to 11 h in the presence of phenol. It has a cellular fatty acid composition like that of C. hastiforme. However, strain 6 does not hydrolyze gelatin or produce indole. The 16S rRNA sequence of strain 6 was found to be most similar to that of some Clostridium species, with homology ranging from 80 to 86%. The evolutionary relationships of strain 6 to different groups of Clostridium and Clostridium-related species revealed that it does not emerge from any of these groups. Strain 6 most likely belongs to a new species closely related to Clostridium species.