Bacterial and Archaeal Phylogenetic Diversity of a Cold Sulfur-Rich Spring on the Shoreline of Lake Erie,Michigan

Studies of sulfidic springs have provided new insights into microbial metabolism, groundwater biogeochemistry, and geologic processes. We investigated Great Sulphur Spring on the western shore of Lake Erie and evaluated the phylogenetic affiliations of 189 bacterial and 77 archaeal 16S rRNA gene sequences from three habitats: the spring origin (11-m depth), bacterial-algal mats on the spring pond surface, and whitish filamentous materials from the spring drain. Water from the spring origin water was cold, pH 6.3, and anoxic (H₂, 5.4 nM; CH₄, 2.70 μM) with concentrations of S²⁻ (0.03 mM), SO₄²⁻ (14.8 mM), Ca²⁺ (15.7 mM), and HCO₃⁻ (4.1 mM) similar to those in groundwater from the local aquifer. No archaeal and few bacterial sequences were >95% similar to sequences of cultivated organisms. Bacterial sequences were largely affiliated with sulfur-metabolizing or chemolithotrophic taxa in Beta-, Gamma-, Delta-, and Epsilonproteobacteria. Epsilonproteobacteria sequences similar to those obtained from other sulfidic environments and a new clade of Cyanobacteria sequences were particularly abundant (16% and 40%, respectively) in the spring origin clone library. Crenarchaeota sequences associated with archaeal-bacterial consortia in whitish filaments at a German sulfidic spring were detected only in a similar habitat at Great Sulphur Spring. This study expands the geographic distribution of many uncultured Archaea and Bacteria sequences to the Laurentian Great Lakes, indicates possible roles for epsilonproteobacteria in local aquifer chemistry and karst formation, documents new oscillatorioid Cyanobacteria lineages, and shows that uncultured, cold-adapted Crenarchaeota sequences may comprise a significant part of the microbial community of some sulfidic environments.Cold, sulfidic springs upwelling into caves (1, 16-19) or exposed at the land surface (14, 15, 31, 39, 47, 50, 51) have recently been shown to harbor unique microbial communities, reflective of the aqueous sulfur chemistry of the upwelling groundwater or of unique cave conditions. Within these spring and cave ecosystems, new and unique Epsilonproteobacteria 16S rRNA gene sequences associated with a limited number of cultured isolates that carry out oxidation of sulfur compounds have been discovered (7). The abundance of Epsilonproteobacteria sequences in these settings and associated biogeochemical research have led to new interest in the role of microbially mediated sulfuric acid speleogenesis as an important limestone dissolution process that may contribute to the development of karst features in limestone bedrock (19). Additionally, in streamlets from sulfidic springs, unique symbioses between uncultured Euryarchaeota, Crenarchaeota, and Epsilonproteobacteria spp. that grow in whitish, macroscopically visible filaments have been described (31, 51). Sulfur cycling was identified as a major means of energy production and maintenance of microbial communities in cold, saline, perennial springs emanating from permafrost in the Arctic (47). Studies of cold, sulfidic springs have therefore provided new insights into microbial metabolism, ecology, and evolution as well as groundwater biogeochemistry and geologic processes.All studies of sulfidic springs to date have focused on terrestrial landscapes typically associated with limestone (CaCO₃) bedrock. Limestone is one of several carbonate sedimentary rocks deposited by ancient seas, which may contain significant amounts of gypsum (CaSO₄·2H₂O) as well as pockets of hydrocarbon deposits, both a source of sulfur. Water that moves for long distances through such rocks evolves through sequential dissolution and precipitation reactions to a geochemistry that bears little resemblance to freshwater. SO₄²⁻ becomes available for microbial reduction to sulfide in aquifer zones where conditions are appropriate. Where spring waters rich in CaCO₃, CO₂, and sulfide emerge at the surface, carbonate deposition and microbially mediated sulfide oxidation occur. These processes result in tufa deposits and the whitish crusts often noted in sulfidic spring outflows (12, 13). Carbonate bedrock underlies large portions of the lower Laurentian Great Lakes. Caves in contact with lake water occur on islands in Lake Erie and along the Bruce Peninsula in Ontario, Canada. A cold, sulfidic spring is located in Ancaster, Ontario, about 5 km from the Lake Ontario shoreline (13). Recently, plumes of high-conductivity sulfidic groundwater, surrounded by whitish filamentous materials and variously colored microbial mats, were reported to occur at a 93-m depth in Lake Huron (2, 49). However, there have been few molecular surveys of Bacteria or Archaea in any Great Lakes environment, and no reports focusing on the molecular phylogenetic diversity of microorganisms associated with these Great Lakes sulfidic environments.Along the western shoreline of Lake Erie and within Monroe County, MI, sinkholes and springs are abundant in the Silurian-Devonian carbonate bedrock, and Ca²⁺ and Mg²⁺ with SO₄²⁻ or HCO₃⁻ dominate groundwater composition (43). In some areas of Monroe County, sulfide in groundwater prohibits its use as a drinking water source. Great Sulphur Spring (GSS) was first described by Sherzer in 1900 (53) and was named for its sulfide-rich water. The spring arises from Silurian-Devonian carbonate bedrock within 0.5 km of the Lake Erie shoreline and is a convenient location for accessing sulfide-rich groundwater and for exploring potential interactions between groundwater and lake water. As part of a larger study of nearshore groundwater interactions with Lake Erie (27) and to better understand the potential role of microorganisms in sulfur chemistry of nearshore groundwater, we evaluated the chemistry and bacterial and archaeal 16S rRNA gene diversity of GSS. Our study documents a unique microbial community for the Laurentian Great Lakes, comprised in large part of new lineages and uncultivated members of the Archaea, Deltaproteobacteria, Epsilonproteobacteria, and Cyanobacteria. These sequences suggest a microbial community structure driven by (possibly H₂S-based) carbon fixation and chemolithotrophy of reduced compounds such as H₂, H₂S, or reduced nitrogen compounds, all consistent with spring geochemistry.