Separation of a phenol carboxylating organism from a two-member, strict anaerobic co-culture

In a culture converting phenol to benzoic acid under anaerobic conditions a nd previously described as being constituted of only a Clostridium-like str ain 6, another bacterium (strain 7) was observed. Each organism was enriche d by centrifugation on a Percoll gradient. Strain 6 was purified by dilutio n and plating. Strain 7 did not grow on solid media, but a strain 7 culture , cleared of strain 6, was obtained by subculturing in the presence of ampi cillin and by dilution. In fresh medium, phenol was transformed by the reco nstituted co-culture but not by each strain alone. In a supernatant from a co-culture or from a strain 6 culture, strain 7 alone transformed phenol bu t not strain 6. Maintenance of an active strain 7 in fresh medium instead o f co-culture supernatant became possible when phenol was replaced by 4-hydr oxybenzoate (4-OHB), which is decarboxylated to phenol before being transfo rmed to benzoate. Even with 4-OHB, the use of co-culture (or strain 6 cultu re) supernatant resulted in faster transformation activity and growth rate. A phylogenetic analysis placed strain 7 in a cluster of uncultivated or no nisolated bacteria (92-96% homology). Strain 7 is also related to Desulfoto maculum, Desulfitobacterium, Desulfosporosinus, Moorella, and Sporotomaculu m genera (87-92% homology).