• Petroleum hydrocarbons;
  • Phospholipid fatty acids;
  • Isotopic labeling;
  • Whole-cell hybridization;
  • Stable carbon isotope;
  • Push-pull test;
  • Sulfate-reducing bacteria;
  • Methanogenesis


Isotopic labeling of biomarker molecules is a technique applied to link microbial community structure with activity. Previously, we successfully labeled phospholipid fatty acids (PLFA) of suspended nitrate-reducing bacteria in an aquifer. However, the application of the method to low energy-yielding processes such as sulfate reduction, and extension of the analysis to attached communities remained to be studied. To test the feasibility of the latter application, an anoxic test solution of 500 l of groundwater with addition of 0.5 mM Br as a conservative tracer, 1.1 mM SO2−4, and 2.0 mM [2-13C]acetate was injected in the transition zone of a petroleum hydrocarbon-contaminated aquifer where sulfate-reducing and methanogenic conditions prevailed. Thousand liters of test solution/groundwater mixture were extracted in a stepwise fashion after 2–46 h incubation. Computed apparent first-order rate coefficients were 0.31 ± 0.04 day−1 for acetate and 0.34 ± 0.05 day−1 for SO2−4 consumption. The δ13C increased from −71.03‰ to +3352.50‰ in CH4 and from −16.15‰ to +32.13‰ in dissolved inorganic carbon (DIC). A mass balance suggested that 43% of the acetate-derived 13C appeared in DIC and 57% appeared in CH4. Thus, acetate oxidation coupled to sulfate reduction and acetoclastic methanogenesis occurred simultaneously. The δ13C of PLFA increased on average by 27‰ in groundwater samples and 4‰ in sediment samples. Hence, both suspended and attached communities actively degraded acetate. The PLFA labeling patterns and fluorescent in situ hybridization (FISH) analyses of sediment and groundwater samples suggested that the main sulfate-reducing bacteria degrading the acetate were Desulfotomaculum acetoxidans and Desulfobacter sp. in groundwater, and D. acetoxidans in sediment.