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Carbon and Hydrogen Isotope Fractionation during Anaerobic Toluene Oxidation by Geobacter metallireducens with Different Fe(III) Phases as Terminal Electron Acceptors

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Microbial oxidation of BTEX compounds under iron-reducing conditions is an important attenuation process for fuel-contaminated sites. We evaluated the use of compound-specific isotope analysis for the identification and quantification of anaerobic toluene oxidation by Geobacter metallireducens. 13C and 2H enrichment of toluene was measured in laboratory batch systems and varied significantly for a solid vs a dissolved Fe(III) phase provided as terminal electron acceptor. 13C enrichment factors (ϵC) in suspensions of a solid Fe(III) phase were between −1.0 and −1.3‰, whereas ϵC-values were significantly higher in solutions of Fe(III) citrate (−2.9 to −3.6‰). The same trend was observed for 2H fractionation. Solid phase reduction resulted in an ϵH-value of −34.6 ± 0.9‰, compared to −98.4 ± 3.0‰ for the reduction of dissolved Fe(III). The linear correlation of δ2H vs δ13C during toluene oxidation resulted in nearly identical slopes for both systems, confirming that the reaction mechanism, that is enzymatic methyl-group oxidation, was the same. We hypothesize that smaller 2H and 13C fractionation in suspensions is due to toluene transport limitations to cells of G. metallireducens at surfaces of solid Fe(III) phases. Enrichment factors determined in Fe(III) mineral suspensions should be more representative for anaerobic toluene degradation owing to the abundance of solid Fe(III) in soils and aquifers. Toluene isotope fractionation is larger if dissimilatory Fe(III) reduction occurs in solution instead of Fe(III) mineral suspensions owing to toluene transport limitations.

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