Evidence for Isotopic Exchange during Metabolism of Stable-Isotope-Labeled Formate in a Methanogenic Sediment

The disappearance of stable-isotope-labeled formate in freshwater sediment from Lake Vechten was studied by gas chromatography-mass spectrometry. Both deuterium- and (sup13)C-labeled formate were removed from pore water at a high rate (400 (plusmn) 35 [standard deviation] and 1,700 (plusmn) 600 (mu)M h(sup-1), respectively), with concomitant formation of nonlabeled formate. The removal rate of labeled and nonlabeled formate amounted to 240 (plusmn) 15 and 400 (plusmn) 40 (mu)M h(sup-1), respectively. The formation of nonlabeled formate is explained by isotopic exchange due to the activity of formate dehydrogenase rather than by the turnover of formate

Comparison of Acetate Turnover in Methanogenic and Sulfate-Reducing Sediments by Radiolabeling and Stable Isotope Labeling and by Use of Specific Inhibitors: Evidence for Isotopic Exchange

Acetate turnover in the methanogenic freshwater anoxic sediments of Lake Vechten, The Netherlands, and in anoxic sediments from the Tamar Estuary, United Kingdom, and the Grosser Jasmunder Bodden, Germany, the latter two dominated by sulfate reduction, was determined. Stable isotopes and radioisotopes, inhibitors (chloroform and fluoroacetate), and methane flux were used to provide independent estimates of acetate turnover. Pore water acetate pool sizes were determined by gas chromatography with a flame ionization detector, and stable isotope-labeled acetate was determined by gas chromatography-mass spectrometry. The appearance of acetates with a different isotope labeling pattern from that initially added demonstrated that isotopic exchange occurred during methanogenic acetate metabolism. The predominant exchange processes were (i) D-H exchange in the methyl group and (ii) (sup13)C-(sup12)C exchange at the carboxyl carbon. These exchanges are most probably caused by the activity of the enzyme complex carbon monoxide dehydrogenase and subsequent methyl group dehydrogenation by tetrahydromethanopterine or a related enzyme. The methyl carbon was not subject to exchange during transformation to methane, and hence acetate with the methyl carbon labeled will provide the most reliable estimate of acetate turnover to methane. Acetate turnover rate estimates with these labels were consistent with independent estimates of acetate turnover (acetate accumulation after inhibition and methane flux). Turnover rates from either radioisotope- or stable isotope-labeled methyl carbon isotopes are, however, dependent on accurate determination of the acetate pool size. The additions of large amounts of stable isotope-labeled acetate elevate the acetate pool size, stimulating acetate consumption and causing deviation from steady-state kinetics. This can, however, be overcome by the application of a non-steady-state model. Isotopic exchange in sediments dominated by sulfate reduction was minimal