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Identification of intermediates formed during anaerobic benzene degradation by an iron-reducing enrichment culture

Authors

  • Umakanth Kunapuli,

    1. Institute of Groundwater Ecology, Helmholtz Center Munich, German Research Center for Environmental Health, Ingolstädter Landstraße 1, D-85764, Neuherberg, Germany.
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  • Christian Griebler,

    1. Institute of Groundwater Ecology, Helmholtz Center Munich, German Research Center for Environmental Health, Ingolstädter Landstraße 1, D-85764, Neuherberg, Germany.
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  • Harry R. Beller,

    1. Lawrence Livermore National Laboratory, PO Box 808, L-542, Livermore, CA, USA.
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    • Present address: Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.

  • Rainer U. Meckenstock

    Corresponding author
    1. Institute of Groundwater Ecology, Helmholtz Center Munich, German Research Center for Environmental Health, Ingolstädter Landstraße 1, D-85764, Neuherberg, Germany.
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*E-mail rainer.meckenstock@helmholtz-muenchen.de; Tel. (+49) 89 3187 2561; Fax (+49) 89 3187 3361.

Summary

Anaerobic benzene degradation is an important process in contaminated aquifers but is poorly understood due to the scarcity of microbial cultures for study. We have enriched a ferric iron-reducing culture that completely mineralizes benzene to CO2. With 13C6-labelled benzene as the growth substrate, ring-labelled benzoate was identified as a major intermediate by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of culture supernatants. With increasing incubation time, 13C7-labelled benzoate appeared, indicating that the carboxyl group of benzoate derived from CO2 that was produced from mineralization of labelled benzene. This was confirmed by growing the culture in 13C-bicarbonate-buffered medium with unlabelled benzene as the substrate, as the label appeared in the carboxyl group of benzoate produced. Phenol was also identified as an intermediate at high concentration. However, it was clearly shown that phenol was formed abiotically by autoxidation of benzene during the sampling and analysis procedure as a result of exposure to air. The results suggest that, in our culture, anaerobic benzene degradation proceeds via carboxylation and that caution should be exercised in interpreting hydroxylated benzene derivatives as metabolic intermediates of anaerobic benzene degradation.

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