Detection of anaerobic toluene and hydrocarbon degraders in contaminated aquifers using benzylsuccinate synthase (bssA) genes as a functional marker

Abstract

Benzylsuccinate synthase (Bss) is the key enzyme of anaerobic toluene degradation and has been found in all anaerobic toluene degrading bacterial isolates tested. However, only a few pure cultures capable of anaerobic toluene oxidation are available to date, and it is important to understand the relevance of these model organisms for in situ bioremediation of hydrocarbon-contaminated aquifers. Due to their phylogenetic dispersal, it is not possible to specifically target anaerobic toluene degraders using marker rRNA genes. We therefore established an assay targeting a approximately 794 bp fragment within the Bss alpha-subunit (bssA) gene, which allows for the specific detection and affiliation of both known and unknown anaerobic degraders. Three distinct tar-oil-contaminated aquifer sites were screened for intrinsic bssA gene pools in order to identify and compare the diversity of hydrocarbon degraders present at these selected sites. We were able to show that local diversity patterns of degraders were entirely distinct, apparently highly specialized and well-adapted to local biogeochemical settings. Discovered at one of the sites were bssA genes closely related to that of Geobacter spp., which provides evidence for an importance of iron reduction for toluene degradation in these sediments. Retrieved from the other two sites, dominated by sulfate reduction, were previously unidentified bssA genes and also deeply branching putative bssA homologues. We provide evidence for a previously unrecognized diversity of anaerobic toluene degraders and also of other hydrocarbon degraders using fumarate-adding key reactions in contaminated aquifers. These findings enhance our current understanding of intrinsic hydrocarbon-degrading microbial communities in perturbed aquifers and may have potential for the future assessment and prediction of natural attenuation based on degradation genes.

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

“…Certain species were also reported to reduce ferric iron (Knoblauch et al, 1999). Moreover, a single sulfate-reducing bacterium related to Desulfocapsa was reported to anaerobically utilize toluene with sulfate as the electron acceptor (Meckenstock, 1999;Winderl et al, 2007). Whether the observed Desulfobulbaceae in our enrichment were directly reducing ferric iron, or shuttling electrons for example via a sulfide/sulfur shuttle (Straub and Schink, 2004) cannot be discerned with the present results.…”

“…The resulting PCR products were cloned and sequenced, and the sequence data were assembled to contigs as described previously (Winderl et al, 2007). Closely related 16S rRNA sequences were identified using BlastN search program (http://ncbi.nlm.nih.gov/ BLAST).…”

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment

“…Accordingly, microbes potentially involved in on-site toluene degradation can be detected using the genes of benzylsuccinate synthase (Bss), the key enzyme of anaerobic toluene degradation (Boll et al, 2002). At the investigated former coal gasification site, we have previously detected unidentified and deeply branching environmental homologs of benzylsuccinate synthase alpha-subunit (bssA), only distantly related to the known proteobacterial bssA sequences (Winderl et al, 2007). However, mere PCR detection of bssA or related catabolic genes in situ did not allow identifying the microbes carrying these genes, and it also remained unanswered under which conditions they are active.…”

“…We wanted to identify involved bssA genes, and to elucidate whether an active role can be verified for unidentified bssA homologs previously detected at the site (Winderl et al, 2007). Therefore, we exposed freshly sampled aquifer sediments containing a complex in situ microbial community to fully labeled 13 C 7 -toluene under close-to-in situ conditions.…”

Transcriptome of Geobacter uraniireducens growing in uranium-contaminated subsurface sediments

“…Molecular and culturing studies have demonstrated that Geobacter species are the predominant microorganisms in a wide diversity of subsurface environments in which dissimilatory Fe(III) reduction is an important process for the degradation of both natural organic matter and organic contaminants or for the stimulation of in situ bioremediation of metal-contaminated subsurface environments (Rooney-Varga et al, 1999;Anderson et al, 2003;Lovley et al, 2004;North et al, 2004;Coates et al, 2005;Lin et al, 2005;Sleep et al, 2006;Holmes et al, 2007;Winderl et al, 2007). By designing an isolation medium that mimics subsurface conditions, it has become possible to isolate Geobacter species that have 16S rRNA gene sequences that are identical or highly similar to the 16S rRNA sequences that predominate in Fe(III)-reducing subsurface environments Shelobolina et al, 2008;Holmes et al, 2008a).…”