Molecular analysis of microbial community structures in pristine and contaminated aquifers: field and laboratory microcosm experiments

Abstract

This study used phylogenetic probes in hybridization analysis to (i) determine in situ microbial community structures in regions of a shallow sand aquifer that were oxygen depleted and fuel contaminated (FC) or aerobic and noncontaminated (NC) and (ii) examine alterations in microbial community structures resulting from exposure to toluene and/or electron acceptor supplementation (nitrate). The latter objective was addressed by using the NC and FC aquifer materials for anaerobic microcosm studies in which phylogenetic probe analysis was complemented by microbial activity assays. Domain probe analysis of the aquifer samples showed that the communities were predominantly Bacteria; Eucarya and Archaea were not detectable. At the phylum and subclass levels, the FC and NC aquifer material had similar relative abundance distributions of 43 to 65% beta- and gamma-Proteobacteria (B+G), 31 to 35% alpha-Proteobacteria (ALF), 15 to 18% sulfate-reducing bacteria, and 5 to 10% high G+C gram positive bacteria. Compared to that of the NC region, the community structure of the FC material differed mainly in an increased abundance of B+G relative to that of ALF. The microcosm communities were like those of the field samples in that they were predominantly Bacteria (83 to 101%) and lacked detectable Archaea but differed in that a small fraction (2 to 8%) of Eucarya was detected regardless of the treatment applied. The latter result was hypothesized to reflect enrichment of anaerobic protozoa. Addition of nitrate and/or toluene stimulated microbial activity in the microcosms, but only supplementation of toluene alone significantly altered community structures. For the NC material, the dominant subclass shifted from B+G to ALF, while in the FC microcosms 55 to 65% of the Bacteria community was no longer identifiable by the phylum or subclass probes used. The latter result suggested that toluene exposure fostered the proliferation of phylotype(s) that were otherwise minor constituents of the FC aquifer community. These studies demonstrated that alterations in aquifer microbial communities resulting from specific anthropogenic perturbances can be inferred from microcosm studies integrating chemical and phylogenetic probe analysis and in the case of hydrocarbon contamination may facilitate the identification of organisms important for in situ biodegradation processes. Further work integrating and coordinating microcosm and field experiments is needed to explore how differences in scale, substrate complexity, and other hydrogeological conditions may affect patterns observed in these systems.

FIG. 1

(A) Agarose gel analysis of DNA extracted from aquifer field samples and standards. Lanes 1 to 3, Pseudomonas genomic DNA loaded at 0.59, 1.18, and 2.47 μg; lanes 4 and 5, aliquots (10 μl) of purified DNAs extracted from the NC or FC sediment (total volumes of the DNA extracts were 100 and 600 μl for the NC and FC extracts, respectively). (B) Relative abundance (universal probe-normalized signals) of domains and subgroups (phylum and subclass levels) in the NC and FC aquifer samples. The sums of domain and subgroup hybridization signals (± standard deviations) are given in the box above the graph. Legend for probe target groups: ■, universal; □, Bacteria; , Eucarya; , ALF; , B+G; , SRB; , HGC. Data are means of duplicate measurements (± standard deviations). An asterisk indicates that the hybridization signal from a given probe differed significantly (P ≤ 0.05) from that for the nonamended control.

FIG. 1

(A) Agarose gel analysis of DNA extracted from aquifer field samples and standards. Lanes 1 to 3, Pseudomonas genomic DNA loaded at 0.59, 1.18, and 2.47 μg; lanes 4 and 5, aliquots (10 μl) of purified DNAs extracted from the NC or FC sediment (total volumes of the DNA extracts were 100 and 600 μl for the NC and FC extracts, respectively). (B) Relative abundance (universal probe-normalized signals) of domains and subgroups (phylum and subclass levels) in the NC and FC aquifer samples. The sums of domain and subgroup hybridization signals (± standard deviations) are given in the box above the graph. Legend for probe target groups: ■, universal; □, Bacteria; , Eucarya; , ALF; , B+G; , SRB; , HGC. Data are means of duplicate measurements (± standard deviations). An asterisk indicates that the hybridization signal from a given probe differed significantly (P ≤ 0.05) from that for the nonamended control.

FIG. 2

Molecular analysis of DNAs extracted from microcosms established with the NC aquifer material. Relative abundance of the domain or subgroups was determined by hybridization to the indicated probe (see the legend to Fig. 1 for the key to probe abbreviations). Bars marked with an asterisk were significantly different (P ≤ 0.05) from those for the nonamended microcosms. Probes for which data are not plotted gave hybridization signals that were below background.

FIG. 3

Molecular analysis of DNA extracted from microcosms established with the FC aquifer material. Relative abundance of the domain or subgroups was determined by hybridization to the indicated probe (see the legend to Fig. 1 for the key to probe abbreviations). Bars marked with an asterisk were significantly different (P ≤ 0.05) from those for the nonamended microcosms. Probes for which data are not plotted gave hybridization signals that were below background.