ACTIVITY AND DIVERSITY OF METHANOGENS IN A PETROLEUM-HYDROCARBON CONTAMINATED AQUIFER

Methanogenic microorganisms may contribute considerably to the mineralization of petroleum hydrocarbons in contaminated environments. However, quantification of methanogenesis in the subsurface is problematic since the product of this process, methane, is soluble in water only to a limited extent and readily volatilizes into the unsaturated zone. The objective of this study was to quantify methanogenesis in situ in the methanogenic zone of a petroleum hydrocarbon-contaminated aquifer using single-well push-pull tests. These tests consist of three phases, injection, incubation and extraction. Furthermore, we wanted to compare activity data from push-pull tests with the actual community composition determined using molecular methods (denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH)). In the push-pull tests, we used different substrates known to be consumed by different populations of methanogenic Archaea (acetate, methanol, formate, and CO2 + H2). The highest degradation rate was obtained for formate (1.85 mM per day), followed by H2 (0.90 mM per day), acetate (0.38 mM per day), and methanol (0.11 mM per day). These data suggested the presence of metabolically different types of, but mainly formate- and H2-degrading methanogenic Archaea. In agreement with these results, DGGE indicated a high diversity of Archaea with at least 3 dominant species. However, FISH data suggested that acetate-consuming methanogens were dominant (Methanosaeta spp.), accounting for approximately 55% of the archaeal population both in water and sediment samples. In conclusion, methanogenesis was successfully quantified in a petroleum hydrocarbon-contaminated aquifer. However, this study demonstrates that a combination of several methods is needed for an adequate characterization of methanogenesis and involved microorganisms.