Assessment of microbial in situ activity in contaminated aquifers

Microbial ecologists and environmental engineers share the interest in identifying the key microorganisms responsible for compound turnover in the environment and in estimating the respective transformation rates. For the successful application of Natural Attenuation processes, a reliable assessment of the in situ turnover of a contaminant in an aquifer is essential. Here, we review and present new details of two recently developed approaches concerning the assessment of in situ biodegradation: (i) determination of biodegradation caused by microbial metabolism in a contamination plume by stable isotope fractionation analysis (SIFA) and (ii) determination of the actual degradation under the respective environmental conditions in the aquifer by using in situ microcosms (BACTRAPS (R)) amended with C-13-labeled substrates as tracer compounds. Based on stable isotope fractionation analysis, the degradation occurring under anoxic biogeochemical conditions at a respective site can be calculated for the entire plume. This has been shown for benzene and toluene at the Zeitz site and partly for chlorobenzene at the Bitterfeld site. By use of the in situ microcosm approach with C-13-labeled compounds, the microbial in situ degradation under strictly anaerobic conditions could be proven for benzene and toluene in Zeitz and for chlorobenzene in Bitterfeld. The transformation of C-13-carbon of the labeled substrate into microbial fatty acids confirmed the assimilation of the pollutant resulting in the formation of biomass. In addition, metabolites such as benzylsuccinic acid were found in the toluene-amended microcosms indicating anaerobic degradation of toluene. This result corresponds to the geochemical conditions found at the field site and therefore, the microcosm approach with C-13-labeled compounds can be used to assign the predominant in situ degradation pathways in a contaminated aquifer. Since fatty acids profiles alone are often too unspecific for a community analysis at species level, the composition of the microbial communities was analyzed by genetic profiling and sequencing of partial 16S rRNA genes PCR-amplified from total DNA extracted directly from the microcosms. Sequences retrieved from the microcosms indicated a dominance of not yet cultivated bacteria. Several sequences were phylogenetically closely related to sequences of bacteria known to be iron and sulfate reducers, typically found at sites polluted with BTEX and/or mineral oil. The results show that the current methods for monitoring microbial in situ activity at present stage are valuable tools for improving environmental control of compound turnover and will speed up engineering approaches.