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Planktonic microbes in anaerobic fracture water and biofilm microbial communities on aerobic rock surfaces were compared from the deep subsurface of the Witwatersrand Basin, South Africa. A deep-branching clade of nearly identical Firmicutes 16S rDNA sequences (>99% homology) has been identified as the dominant microorganism in fracture water from multiple gold mines of the Witwatersrand Basin, South Africa. This Firmicutes bacterium is only the dominant form in the planktonic phase of the deepest (2 — 3 km depth), most saline fracture water.

DNA from fissure water and rock biofilm samples collected from 3m, 6m and 9m into a vertical borehole with fracture water was amplified, fragmented and hybridized to our high density 16S microarrays containing 300,000 probes capable of detecting 8,735 prokaryotic operational taxonomic units (OTUs). Clone libraries of 16S rDNA were also generated in order to validate array results. 16S rRNA microarray analysis indicated that the fracture water where the Firmicutes type bacterium dominated the clone library was more complex than clone libraries had indicated (19 OTUs). No archaea were detected in the fracture water, however, which is consistent with the high concentrations of abiotic methane in fissure water. Biofilm samples showed far greater diversity with 195, 224 and 97 OTU’s detected, respectively. Significantly, the Firmicutes type bacterium was present, but the relative proportion of this bacterium was substantially lower in all biofilm samples. However, both Crenarchaeota and methanogenic Euryarchaeota were detected in biofilms, consistent with the methanogenic isotopic signature of the methane. Other functional groups of significance to this sulfate, hydrogen, methane dominated system detected in biofilms but not in the planktonic phase, included, methanotrophs, sulfur oxidizers, and syntrophs. This is also consistent with the trace levels of oxygen in the open borehole.

Biofilm microbial communities in open boreholes are considerably more diverse than planktonic communities present in fracture water and appear to contain many of the functional groups expected given the geochemistry of an ecosystem where highly reduced, metal, sulfide, H2 and CH4 rich water encounter oxygenated mine air. Although clone library analyses indicated that this biome was dominated by a single bacterial species forming a deep-branching clade within the Firmicutes, high density array analyses also demonstrated the presence of other relatively abundant Firmicutes (Bacillus) and less abundant Alpha-, Beta-, Gamma and Delta-proteobacteria, Cyanobacteria, Chloroflexi, Acidobacteria, Bacteroidetes, Actinobacteria, Spirochaetes, Verrucomicrobia and Planctomycetes.

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