Functional characterization of an anaerobic benzene‐degrading enrichment culture by DNA stable isotope probing

Abstract

The flow of carbon under sulfate-reducing conditions within a benzene-mineralizing enrichment culture was analysed using fully labelled [13C6]-benzene. Over 180 days of incubation, 95% of added 13C-benzene was released as 13C-carbon dioxide. DNA extracted from cultures that had degraded different amounts of unlabelled or 13C-labelled benzene was centrifuged in CsCl density gradients to identify 13C-benzene-assimilating organisms by density-resolved terminal restriction fragment length polymorphism analysis and cloning of 16S rRNA gene fragments. Two phylotypes showed significantly increased relative abundance of their terminal restriction fragments in 'heavy' fractions of 13C-benzene-incubated microcosms compared with a 12C-benzene-incubated control: a member of the Cryptanaerobacter/Pelotomaculum group within the Peptococcaceae, and a phylotype belonging to the Epsilonproteobacteria. The Cryptanaerobacter/Pelotomaculum phylotype was the most frequent sequence type. A small amount of 13C-methane was aceticlastically produced, as concluded from the linear relationship between methane production and benzene degradation and the detection of Methanosaetaceae as the only methanogens present. Other phylotypes detected but not 13C-labelled belong to several genera of sulfate-reducing bacteria, that may act as hydrogen scavengers for benzene oxidation. Our results strongly support the hypothesis that benzene is mineralized by a consortium consisting of syntrophs, hydrogenotrophic sulfate reducers and to a minor extent of aceticlastic methanogens.

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

“…Most relevantly, we used freshly sampled aquifer sediment material containing a complex in situ microbial community for our experiment. This contrasts the present study from related recent studies, in which SIP has been used as a tool to functionally dissect previously enriched, less complex laboratory cultures of anaerobic BTEX degraders (Kunapuli et al, 2007;Oka et al, 2008;Bombach et al, 2010;Herrmann et al, 2010). …”

“…Thus, degrader anabolism must have substantially diluted the incorporation of 13 C from the contaminant with an unlabeled carbon source. Again, this low labeling efficiency coincides with labeling ratios observed in recent SIP studies of BTEX-degrading, iron-or sulfate-reducing enrichments (Kunapuli et al, 2007;Bombach et al, 2010;Herrmann et al, 2010). Besides medium carbonate, no other C source was added to the microcosms.…”

“…This electron recovery was similarly low as in a recent SIP study of a benzene-degrading, sulfate-reducing enrichment culture (Herrmann et al, 2010), irrespective of the fact that we were working with fresh sediment samples containing a complex aquifer microbiota. Part of the produced sulfide could have been missed due to precipitation with sedimentary ferrous iron; however, a delayed appearance of sulfide with respect to 13 CO 2 production was not observed.…”

“…Members of the Peptococcaceae have been previously described as important constituents of microbial communities in contaminated aquifers (Robertson et al, 2001;Franzmann et al, 2002), and two respective isolates have been described to oxidize toluene (Liu et al, 2004;Morasch et al, 2004). More recently, related microbes have also been identified to be involved in the anaerobic degradation of benzene (Kunapuli et al, 2007;Abu Laban et al, 2009;Herrmann et al, 2010). Taken together, this evidence strengthens the hypothesis that members of the Peptococcaceae do not only represent important microbial community constituents in deep, oligotrophic subsurface environments (Chivian et al, 2008), but also in shallow, contaminated aquifers (Franzmann et al, 2002;Kunapuli et al, 2007).…”

“…In recent years, especially the use of stable isotopes in combination with molecular tools has enabled researchers to identify such key anaerobic contaminant degraders, not only in enrichments stemming from aquifers (Kasai et al, 2006;Kunapuli et al, 2007;Bombach et al, 2010;Herrmann et al, 2010), but also from a wide variety of other sediments and soils (Gallagher et al, 2005;Kittelmann and Friedrich, 2008;Liou et al, 2008;Oka et al, 2008). Stable isotope probing (SIP) of nucleic acids allows for the specific identification of microorganisms catabolizing and assimilating carbon from a particular 13 C-labeled substrate, and if performed for DNA, also to identify and affiliate involved catabolic genes on labeled genomes (Jeon et al, 2003;Leigh et al, 2007).…”