Stable hydrogen and carbon isotope fractionation during microbial toluene degradation: Mechanistic and environmental aspects

Primary features of hydrogen and carbon isotope fractionation during toluen e degradation were studied to evaluate if analysis of isotope signatures ca n be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d(8) and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic i sotope fractionation was calculated from the shift of the molar ratios of t oluene-d8 and nondeuterated toluene. The D/H isotope fractionation varied s lightly for sulfate-reducing strain TRM1 (slope of curve [b] = -1.219), Des ulfobacterium cetonicum (b = -1.196), Thauera aromatica (b = -0.816), and G eobacter metallireducens (b = -1.004) and was greater for the aerobic bacte rium Pseudomonas putida mt-2 (b = -2.667). The D/H isotope fractionation wa s 3 orders of magnitude greater than the C-13/C-12 carbon isotope fractiona tion reported previously. Hydrogen isotope fractionation with nonlabeled to luene was 1.7 and 6 times less than isotope fractionation with per-deuterat ed toluene-d(8) and nonlabeled toluene for sulfate-reducing strain TRM1 (b = -0.728) and D. cetonicum (b = -0.198), respectively. Carbon and hydrogen isotope fractionation during toluene degradation by D. cetonicum remained c onstant over a growth temperature range of 15 to 37 degreesC but varied sli ghtly during degradation by P. putida mt-2, which showed maximum hydrogen i sotope fractionation at 20 degreesC (b = -4.086) and minimum fractionation at 35 degreesC (b = -2.138). D/H isotope fractionation was observed only if the deuterium label was located at the methyl group of the toluene molecul e which is the site of the initial enzymatic attack on the substrate by the bacterial strains investigated in this study. Use of ring-labeled toluene- d(5) in combination with nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobi c toluene degradation pathway, benzylsuccinate synthase, was measured in ce ll extracts of D. cetonicum with an initial activity of 3.63 mU (mg of prot ein)(-1). The D/H isotope fractionation (b = -1.580) was 30% greater than t hat in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluen e molecules by the enzyme were retained in the same molecules after the pro duct was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fraction ation. Similar D/H fractionation factors for toluene degradation by anaerob ic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the firs t enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.