The use of stable isotope probing to identify key iron-reducing microorganisms involved in anaerobic benzene degradation

Here, we present a detailed functional and phylogenetic characterization of an iron-reducing enrichment culture maintained in our lab with benzene as sole carbon and energy source. We used DNA-stable isotope probing to identify microbes within the enrichment most active in the assimilation of C-13-label. When C-12(6)- and C-13(6)-benzene were added as comparative substrates, marked differences in the quantitative buoyant density distribution became apparent especially for uncultured microbes within the Gram-positive Peptococcaceae, closely related to environmental clones retrieved from contaminated aquifers world wide and only distantly related to cultured representatives of the genus Thermincola. Prominent among the other constituents of the enrichment were uncultured Deltaproteobacteria, as well as members of the Actinobacteria. Although their presence within the enrichment seems to be stable they did not assimilate C-13-label as significantly as the Clostridia within the time course of our experiment. We hypothesize that benzene degradation in our enrichment involves an unusual syntrophy, where members of the Clostridia primarily oxidize benzene. Electrons from the contaminant are both directly transferred to ferric iron by the primary oxidizers, but also partially shared with the Desulfobulbaceae as syntrophic partners. Alternatively, electrons may also be quantitatively transferred to the partners, which then reduce the ferric iron. Thus our results provide evidence for the importance of a novel clade of Gram-positive iron-reducers in anaerobic benzene degradation, and a role of syntrophic interactions in this process. These findings shed a totally new light on the factors controlling benzene degradation in anaerobic contaminated environments.