A methanogenic consortium transforming phenol to benzoic acid was submitted to different treatments to characterize the carboxylating microorganisms and eventually to facilitate their isolation. Under aerobic conditions, phenol was not transformed by the consortium and no growth was observed on solid medium. The consortium from an inoculum that was treated with heat, or heat and ethanol, retained the ability to carboxylate phenol under strictly anaerobic conditions. Electron microscopic observations of the consortium from an inoculum that was heated for 15 min at 80 °C revealed only Gram-positive bacilli. In this culture, methane production was not detected and benzoic acid accumulated. Five colonies with distinct morphologies were isolated from this culture on solid medium. Four of these strains were identified as Clostridium spp. In contrast to the untreated culture, none of the strains isolated were able to carboxylate phenol in pure culture or in coculture, nor could they decarboxylate or dehydroxylate 4-hydroxybenzoic acid, or oxidize 2-hydroxybenzyl alcohol, or O-demethylate anisole or 2-methoxyphenol. Also, the consortium from a treated inoculum retained its ability to decarboxylate and dehydroxylate 4-hydroxybenzoic acid forming phenol and benzoic acid, respectively, but could not accomplish the other reactions. These results suggest that spore-forming microorganisms are involved in the carboxylation of phenol and in the decarboxylation and dehydroxylation of 4-hydroxybenzoic acid.Key words: spore-forming bacteria, phenol, benzoic acid, methanogenic conditions, carboxylation.
