Response and recovery of a pristine groundwater ecosystem impacted by toluene contamination - A meso-scale indoor aquifer experiment.

Microbial communities are the driving force behind the degradation of contaminants like aromatic hydrocarbons in groundwater ecosystems. However, little is known about the response of native microbial communities to contamination in pristine environments as well as their potential to recover from a contamination event. Here, we used an indoor aquifer mesocosm filled with sandy quaternary calciferous sediment that was continuously fed with pristine groundwater to study the response, resistance and resilience of microbial communities to toluene contamination over a period of almost two years, comprising 132days of toluene exposure followed by nearly 600days of recovery. We observed an unexpectedly high intrinsic potential for toluene degradation, starting within the first two weeks after the first exposure. The contamination led to a shift from oxic to anoxic, primarily nitrate-reducing conditions as well as marked cell growth inside the contaminant plume. Depth-resolved community fingerprinting revealed a low resistance of the native microbial community to the perturbation induced by the exposure to toluene. Distinct populations that were dominated by a small number of operational taxonomic units (OTUs) rapidly emerged inside the plume and at the plume fringes, partially replacing the original community. During the recovery period physico-chemical conditions were restored to the pristine state within about 35days, whereas the recovery of the biological parameters was much slower and the community composition inside the former plume area had not recovered to the original state by the end of the experiment. These results demonstrate the low resilience of sediment-associated groundwater microbial communities to organic pollution and underline that recovery of groundwater ecosystems cannot be assessed solely by physico-chemical parameters.

Prokaryotic community structure in deep bedrock aquifers of the Austrian Central Alps.

The bacterial and archaeal diversity of deep groundwater systems was investigated based on 16S rRNA-SSCP (single strand conformation polymorphism) fingerprints. The study site included five boreholes along the projected Brenner Base Tunnel in the central Alps of Tyrol, Austria. To obtain representative samples, packer-sealed fractures were sampled at specific depths between 105 and 780 m below surface. Sequence analysis of SSCP bands obtained from 13 samples showed that between 29 and 62 % of the phylotypes belonged to a variety of Proteobacteria including representatives of typical freshwater bacteria of the genera Acidovorax, Aquabacterium, and Sphingomonas. Bacteroidetes (especially Flavobacterium), Firmicutes (Acetobacterium), and candidate division OP3-related sequences were observed in the majority of the analysed groundwaters. On average, 14 % of the detected prokaryotic phylotypes were affiliated with Archaea, comprising the phyla Euryarchaeota, Crenarchaeota and Thaumarchaeota. Most of the archaeal sequences showed low similarities to known cultivated species, with exception of two sequences having 98 % similarity to Methanosaeta sp. A considerable number of thaumarchaeal sequences belonged to two groups related to Nitrososphaera and Nitrosopumilus phylotypes. An environmental clustering of the groundwater samples, based on the bacterial and archaeal phylogeny, revealed a clear distribution pattern of the samples (sites and depths) reflecting the hydrochemical characteristics and underlying geologies.

Fine-scale degrader community profiling over an aerobic/anaerobic redox gradient in a toluene-contaminated aquifer.

Hydrocarbon contaminants in groundwater can be degraded by microbes under different redox settings, forming hot spots of degradation especially at the fringes of contaminant plumes. At a tar-oil-contaminated aquifer in Germany, it was previously shown that the distribution of anaerobic toluene degraders as traced via catabolic and ribosomal marker genes is highly correlated to zones of increased anaerobic degradation at the lower fringe of the plume. Here, we trace the respective distribution of aerobic toluene degraders over a fine-scale depth transect of sediments taken at the upper fringe of the plume and below, based on the analysis of 16S rRNA genes as well as catabolic markers in intervals of 3-10 cm. Well-defined small-scale distribution maxima of typical aerobic degrader lineages within the Pseudomonadaceae, Comamonadaceae and Burkholderiaceae are revealed over the redox gradient. An unexpected maximal abundance of 9.2 × 106 toluene monooxygenase (tmoA) genes per g of sediment was detected in the strongly reduced plume core, and gene counts did not increase towards the more oxidized upper plume fringe. This may point towards unusual ecological controls of these yet unidentified aerobic degraders, and indicates that competitive niche partitioning between aerobic and anaerobic hydrocarbon degraders in the field is not yet fully understood. These findings demonstrate the potential of catabolic marker gene assays in elaborating the ecology of contaminant plumes, which is a prerequisite for developing integrated monitoring strategies for natural attenuation.

Comparative evaluation of prokaryotic 16S rDNA clone libraries and SSCP in groundwater samples.

A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries.