Identification of Desulfosporosinus as toluene-assimilating microorganisms from a methanogenic consortium

To understand the potential for toluene removal under electron acceptor depleted conditions, stable isotope probing (SIP) was applied to a methanogenic toluene degrading culture to identify the microorganisms responsible for toluene assimilation. Both bacterial and archaeal communities were investigated. The approach involved addition of labeled and unlabeled toluene to microcosms, DNA extraction, ultracentrifugation, and analysis of the generated fractions, as well as the total genomic DNA. Three genes were investigated in the fractions, including the 16S rRNA gene, bssA (encoding for benzylsuccinate synthase α-subunit) and bamA (encoding for 6-oxocylcohex-1-ene-1-carbonyl-CoA hydrolase). Analysis of the total genomic 16S rRNA gene clone library indicated the microbial community was reasonably diverse, containing microorganisms from six phyla (Proteobacteria, Firmicutes, Acidobacteria, Actinobacteria, Deferribacteres, Bacteroidetes). In contrast, only four phylotypes were found in the heavy fraction 16S rRNA gene clone library (from three phyla: Firmicutes, Acidobacteria, Actinobacteria). When these data were correlated with the TRFLP fragments enriched in the heavy fractions, three phylotypes were identified. Specifically, a Desulfosporosinus phylotype was highly enriched in the heavy fractions and was therefore the key consumer of the labeled carbon from toluene. Two other phylotypes, Peptostreptococcaceae and Pseudonocardia were presumed to consume daughter products and produce methane precursors, which in turn were likely utilized by Methanomicrobia to produce methane. Further, the SIP results suggested that the enzymes encoding by functional genes (bssA and bamA) were likely to be harbored by the Desulfosporosinus phylotype.