Abstract
Stalactites (CaCO3 and salt) from water seeps are frequently encountered in ceilings of mine tunnels whenever they intersect water-bearing faults or fractures. To determine whether stalactites could be mineralized traps for indigenous fracture water microorganisms, we analyzed stalactites collected from three different mines ranging in depth from 1.3 to 3.1 km. During sampling in Beatrix gold mine (1.4 km beneath the surface), central South Africa, CaCO3 stalactites growing on the mine tunnel ceiling were collected and observed, in two cases, to contain a living obligate brackish water/marine nematode species, Monhystrella parvella. After sterilization of the outer surface, mineral layers were physically removed from the outside to the interior, and DNA extracted. Based upon 16S and 18S rRNA gene sequencing, Archaea, Bacteria, and Eukarya in different combinations were detected for each layer. Using CT scan and electron microscopy the inner structure of CaCO3 and salt stalactites were analyzed. CaCO3 stalactites show a complex pattern of lamellae carrying bacterially precipitated mineral structures. Nematoda were clearly identified between these layers confirming that bacteria and nematodes live inside the stalactites and not only in the central straw. Salt stalactites exhibit a more uniform internal structure. Surprisingly, several Bacteria showing highest sequence identities to marine species were identified. This, together with the observation that the nematode M. parvella recovered from Beatrix gold mine stalactite can only survive in a salty environment makes the origin of the deep subsurface colonization enigmatic. The possibility of a Permian origin of fracture fluids is discussed. Our results indicate stalactites are suitable for biodiversity recovery and act as natural traps for microorganisms in the fissure water long after the water that formed the stalactite stopped flowing.
Highlights
Studying deep subsurface terrestrial biodiversity can only be achieved through deep drilling and mining or exploration of caverns
The X-Ray Diffraction (XRD) analysis of the Moab Khotsong salt stalactite determined the composition as ∼98% halite + ∼1% sylvite + ∼1% quartz
After sterilization none of the outer layers that were tested on Potato dextrose Agar (PDA), Luria Bertani (LB) or nutrient agar (NA) agar showed any growth indicating that sterilization worked satisfactorily
Summary
Studying deep subsurface terrestrial biodiversity can only be achieved through deep drilling and mining or exploration of caverns. Access to fracture water is unpredictable, irregular and the volume available for collection may be too limited The latter is important as cell counts of bacteria in deep subsurface fracture water is typically low, requiring large volumes for deriving sufficient DNA for characterization of the taxonomic diversity. In many caverns a large variety of communities comprised of Algae, Bacteria, and Fungi are found embedded in stalactites and in some cases, these microorganisms have been implicated in the precipitation of carbonate soda-straw (“straws”) stalactites in caves. Underground mine stalactites raised the possibility that they may act as natural traps for microorganisms in the fissure water More importantly they could serve as enrichment sites allowing sampling drip waters that defy more traditional sampling methods due to the low water volume. The small size of the stalactites and the enrichment potential would only allow studying partial biological/chemical content
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