High Microbial Diversity Despite Extremely Low Biomass in a Deep Karst Aquifer

Abstract

Despite the importance of karst aquifers as a source of drinking water, little is known about the role of microorganisms in maintaining the quality of this water. One of the limitations in exploring the microbiology of these environments is access, which is usually limited to wells and surface springs. In this study, we compared the microbiology of the Madison karst aquifer sampled via the potentiometric lakes of Wind Cave with surface sampling wells and a spring. Our data indicated that only the Streeter Well (STR), which is drilled into the same hydrogeologic domain as the Wind Cave Lakes (WCL), allowed access to water with the same low biomass (1.56-9.25 × 10³ cells mL^-1). Filtration of ∼300 L of water from both of these sites through a 0.2 μm filter allowed the collection of sufficient cells for DNA extraction, PCR amplification of 16S rRNA gene sequences, and identification through pyrosequencing. The results indicated that bacteria (with limited archaea and no detectable eukaryotic organisms) dominated both water samples; however, there were significant taxonomic differences in the bacterial populations of the samples. The STR sample was dominated by a single phylotype within the Gammaproteobacteria (Order Acidithiobacillales), which dramatically reduced the overall diversity and species richness of the population. In WCL, despite less organic carbon, the bacterial population was significantly more diverse, including significant contributions from the Gammaproteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Planctomycetes, Fusobacter, and Omnitrophica phyla. Comparisons with similar oligotrophic environments suggest that karst aquifers have a greater species richness than comparable surface environs. These data also demonstrate that Wind Cave provides a unique opportunity to sample a deep, subterranean aquifer directly, and that the microbiology of such aquifers may be more complex than previously anticipated.

Keywords: aquifer; bacteria; cave; deep; freshwater; karst; ultra-oligotrophic.

FIGURE 1

(A) Image illustrating some of the passages that must be traversed en route to the Wind Cave Lakes (it should be noted that this is not the smallest passage that researchers must navigate with equipment). (B) Location map of South Dakota, the Black Hills, and Wind Cave. The exposed Madison limestone, where some of the Madison aquifer water recharge occurs is indicated in blue, the location of Wind Cave National Park (red), Wind Cave (black star), and Streeter well (black triangle). (C) The survey line plot of the passages within Wind Cave, demonstrating the location of the lakes in relation to the natural entrance to the cave. (D) Location of the sample site within the lakes area. The lakes are indicated (in blue) along with dry cave passages (brown). The named areas of the cave are indicated. All arrows indicate true north. Cave data compiled by, and with permission of, Wind Cave National Park.

FIGURE 2

Chao 1 estimate of community species richness of WCL samples (red), STR samples (blue), and PCTL (green). Chao 1 points were generated using median abundance values of sample replicates, randomly subsampled (with replacement) at even sampling depths over 10 iterations. A best fit curve was generated using a robust linear model.

FIGURE 3

Phylum level 454-pyrosequencing data from the Wind Cave Lakes (WCL), Streeter Well (STR), and the processing control (PCTL). Three PCR replicates were performed for each sample. The relative contribution of each phyla is shown; phyla composing less than 1% of the microbial community were not included for clarity (a more detailed breakdown of all represented clades at the Order level is shown in Supplementary Table S1).

FIGURE 4

(A) Reciprocal Simpson rarefaction plot of WCL, STR, PCTL, and demonstrating community evenness. Data were randomly subsampled (with replacement) at even intervals to generate reciprocal Simpson curve (the 95% confidence intervals are indicated by gray shading). (B) Principle Coordinate Analysis (PCoA) of weighted Unifrac distances between WCL (red), STR (blue), and PCTL (green). Ellipses depict the 95% confidence interval of the sample cluster, with centroids determined by source mean values.

FIGURE 5

(A) Principle Coordinate Analysis plot of unweighted Unifrac distances between WCL, STR, PCTL, and similarly oligotrophic environments. Comparative data include the Edwards aquifer (SRP010407), pelagic microbial populations from oligotrophic Lake Brienz, Switzerland (SRP021556), a limestone aquifer (ERP020663), and stream water in a Kentucky cave (upstream and downstream; SRP058014). Ellipses depict the 95% confidence interval. (B) Comparative reciprocal Simpson rarefaction plot of WCL and STR, with the Edwards aquifer, Lake Brienz, a limestone aquifer, and the Kentucky cave stream, demonstrating community evenness.