Project description:We demonstrate the feasibility of total RNA-SIP in experiments where microbes from a hydrocarbon-contaminated aquifer were studied in microcosms with 13C-labelled-toluene to understand their adaptation to the simultaneous availability of low levels of different electron acceptors. SIP successfully resolved the involvement of microaerobic vs. aerobic and anaerobic populations. Under microoxic, nitrate-amended conditions hydrocarbon degradation was actually stimulated, but transcripts of denitrification showed no signs of 13C-labelling. The expression of distinct oxygenase-based catabolic pathways for toluene degradation was clearly apparent in 13C-labelled mRNA. We discuss how these direct insights into the gene expression and adaptation mechanisms within complex degrader communities can guide more integrated approaches in monitoring and restoration of contaminated sites.
Project description:Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil contaminated aquifer in Germany, where a specialized community of contaminant degraders co-dominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase alpha-subunit, bssA) genes detected in situ appeared more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with 13C7-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae for sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This, and the absence of 13C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy amongst anaerobic toluene degraders on site. 2 samples examined from the different electron-acceptors (sulphate or ferric iron) incubates at the time point of maximal toluene degradation.
Project description:Bioavailability of electron acceptors is probably the most limiting factor in the restoration of anoxic, contaminated environments. The oxidation of contaminants such as aromatic hydrocarbons, particularly in aquifers, often depends on the reduction of ferric iron or sulphate. We have previously detected a highly active fringe zone beneath a toluene plume at a tar-oil contaminated aquifer in Germany, where a specialized community of contaminant degraders co-dominated by Desulfobulbaceae and Geobacteraceae had established. Although on-site geochemistry links degradation to sulphidogenic processes, dominating catabolic (benzylsuccinate synthase alpha-subunit, bssA) genes detected in situ appeared more related to those of Geobacter spp. Therefore, a stable isotope probing (SIP) incubation of sediment samples with 13C7-toluene and comparative electron acceptor amendment was performed. We introduce pyrosequencing of templates from SIP microcosms as a powerful new strategy in SIP gradient interpretation (Pyro-SIP). Our results reveal the central role of Desulfobulbaceae for sulphidogenic toluene degradation in situ, and affiliate the detected bssA genes to this lineage. This, and the absence of 13C-labelled DNA of Geobacter spp. in SIP gradients preclude their relevance as toluene degraders in situ. In contrast, Betaproteobacteria related to Georgfuchsia spp. became labelled under iron-reducing conditions. Furthermore, secondary toluene degraders belonging to the Peptococcaceae detected in both treatments suggest the possibility of functional redundancy amongst anaerobic toluene degraders on site.
Project description:Benzene is abundant air pollutant that has been associated with several diseases in particularly cardiovascular disease. Despite of known health risks, effects of benzene on cardiovascular health is not well studied. In this study, in a well-controlled animal model, we are characterizing how benzene influences cardiovascular system.
Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms.
Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms.
Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms.
Project description:au12-01_detox; Improving the knowledge of the metabolic pathways of benzene in higher plants Cabbages (Brassica oleracea Var. Prover)are directly submitted to a one dose of benzene (3ppm) during 1 hour or 3 hours. The comparison is realized between samples treated and untreated to the benzene for both exhibitions.
Project description:au11_04_benzene - benzene effects on brassica leaves - Improving the knowledge of the metabolic pathways of benzene in higher plants. - Cabbages (Brassica oleracea variety Prover), placed in a 300L exposure chamber, are directly subjected to a single dose of benzene (3ppm) for 24 hours.