Brian Kakuk peering over the entrance to Magical Blue Hole. Photo by Tamara Thomsen.

Caves provide untapped opportunities for scientific research, yielding remarkable biodiversity and evolutionary novelties.  Recently, caves have become of special interest to microbiologists in understanding the complexity of uncharted subterranean ecosystems.  Investigations from Guerrero Negro Cave in Baja California and the Frasassi Caves in Italy have led to unprecedented microbial discoveries constituting several new candidate phyla.  Results from their findings reiterate the diversity and complexity of the subterranean environment.

	Divers Brett Gonzalez and Gregg Stanton descend into Magical Blue Hole.  Photo by Tamara Thomsen.

Inland (anchialine) blue holes are of  particular interest since they constitute extreme lightless, food-poor and low dissolved oxygen environments.  Blue holes are water-filled sinkholes commonly found in the Bahamas Archipelago,    occurring both on land and submerged on the seabed. These depressions are formed by limestone dissolution during glacial periods of lowered sea level.  Today, these systems contain tidally influenced layers of fresh and marine waters which are separated by a halocline.  Occasionally hydrogen sulfide (H₂S) may form between the two water masses when outside input of organics is high and naturally occurring bacteria deplete the already low levels of dissolved oxygen.  The high degree of vertical stratification within these caves gives rise to numerous microhabitats, each with their own potentially unique microbiology. 

Rebreather diver Gregg Stanton towing plankton net alongside orange bacterial mats on cave wall.  Photo by Tamara Thomsen.

Indirect evidence of microbial activity in anchialine caves such as the presence of sulfide and nitrifying bacteria has been indicated by water chemistry and stable isotope data from Yucatán caves in southern Mexico. It has been suggested that chemoautotrophic bacteria, able to independently produce organic matter, constitute the base of the anchialine cave food chain. Thus, anchialine caves, like deep sea hydrothermal vents, may be one of a few ecosystems on Earth capable of existing in the absence of solar energy and photosynthesis.

	Rebreather diver Tom Iliffe passing through dense cloud of hydrogen sulfide in Magical Blue Hole.  Photo by Tamara Thomsen.

To date, relatively few investigations has been conducted to identify the bacterial biodiversity in caves, and even less is known concerning underwater caves.  This investigation will use culture-independent molecular means to characterize the microbial communities that make up the dense gelatinous biofilms occurring on cave walls below the halocline in Magical Blue Hole on Abaco Island, Bahamas.  Bacteria specific primers will be used in polymer chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to quantify bacterial diversity.  Clone libraries will be constructed with the resulting DGGE data.  Abundance and distribution of bacteria will be revealed with fluorescence in situ hybridization (FISH) using existing DNA probes with the potential to create new probes for dominant and or new and interesting bacteria.

ARDRA gel showing diversity of cloned DNA.

The goal of this research is to further enhance our understanding of biological processes within the subterranean aquatic ecosystems.  The ability to identify and characterize the diversity of bacterial assemblages inhabiting underwater caves will finally permit “bottom-up” designs of the cave food web.

	Unknown crystals embedded in the cellular matrix of the bacterial biofilm.

Predator-prey interactions between higher cave invertebrates are better understood than events and organisms occurring at the base of the food chain. Anchialine caves contain an unprecedented array of invertebrate diversity suggesting the potential for discoveries in the microbial realm will be equal, if not greater.   Further studies of the biofilms in Magical Blue Hole aim to identify the crystal structure, mineral composition, and origin of pyramidal inclusions within the cellular matrix.

Continued research in the microbiology of anchialine caves promises to determine if bacterial assemblages are microhabitat, cave, island, or even region specific.  Additional sampling sites include caves on other Bahamian islands and in the Yucatán Peninsula.