Deep subsurface mine stalactites trap endemic fissure fluid Archaea, Bacteria, and Nematoda possibly originating from ancient seas

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.

Keywords: Monhystrella parvella; diversity; stalactites; subsurface sea.

Figure 1

Phylogenetic tree of the Archaea extracted from the salt stalactites.

Figure 2

Phylogenetic tree of the Bacteria extracted from the salt stalactites.

Figure 3

Micro CT scan of CaCO₃ stalactite. (A) General habitus of CT scan of CaCO₃ stalactite that contained nematodes. The area shown is near the tip of the soda straw at the bottom of the stalactite away from the ceiling. The bent laminae are clearly visible as is the knob like outgrowths on them. Cotton (C) was used to stabilize the stalactite for scanning. (B) Longitudinal section inside the lower part of image (A). The lamellar built of the stalactite and the outgrowths are plainly visible. (C,D) Cross sections of the stalactite in an area close to the ceiling (C) and near the end (D). Outgrowths can become very dominant and the laminae can form complete or partially enclosed spaces (^*) within the inner stalactite. Scale bars: (A) 4 cm, (B): 0.1 cm, (C): 7cm, (D): 5 cm.

Figure 4

SEM of the CaCO₃ stalactite. (A) General built. Image identical to Figure 3A but non-linearly compacted as it only serves to highlight the position of image (B). (B) Detail of the outer, smooth surface of the stalactite showing the much coarser built of the more interior layers inside. Low resolution of an area covered in the shrub called “pine forest.” (C–F) The different types of shrubs identified in the CaCO₃ stalactite. (C) The “cauliflower” the most dominantly present. (D) The “Chinese fan” (dark blue) (E) the “pine forest” (light blue). (F) The “amphora” (blue). No logical pattern of presence could be deduced for any of these shrubs. Scale bars (B) and (F): 100 μm, (C–E): 10 μm.

Figure 5

SEM of Monhystrella parvella inside a CaCO₃ stalactite after longitudinal sections with a scalpel. (A) Composite image made of four images taken at different focal points and assembled using Adobe Photoshop. Nematodes (blue) reside in the amorph mass within the confines of the CaCO₃ outer stalactite wall (pink). (B) Detail of M. parvella, arrow highlights the fovea. Scale bars: (A,B): 200 μm.