Molecular Surveys of Microbial Communities in Mammoth Cave National Park

Rick Fowler
Applied Research and Technology Program
Ogden College of Science and Engineering
Western Kentucky University, 1906 College Heights Blvd., Bowling Green, KY 42101 USA

 

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Amos Hawkins Formation on the route to Hawkins-Logsdon Confluence to retreive artificial substrates in 2005. Left to right: Norm Waddell, Rick Fowler, and Kathy Leech (not visible, Rick Olson). Photo by Gary Berdeaux.

 

These pages summarize data collected between 2000 and 2006 during the first systematic attempt to monitor microbial communities in Mammoth Cave National Park (MACA) using modern methods of DNA analysis. Sediment samples were collected in October, 2000; June, 2003; and January, 2005. Artificial substrates were in place from March 2004 until September 2005. The study employed a combination of Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiles, quantitative Real-Time PCR (qRT-PCR), clone libraries of eubacterial 16S SSU-DNA sequences, phylogenetic analysis, and Phospholipid Fatty Acid analysis (PLFA) of membrane components. These techniques were used to estimate the microbial biomass in selected aquifers at base level of the Flint-Mammoth Cave system, and to gather information about the bacteria and other microorganisms that inhabit interstitial fluids and biofilms in karst aquifers.

Bacterial biofilms support the growth of higher organsims such as these cave-adapted invertebrates including flatworms, copepods and isopods. Photo by Rick Fowler.

 

Primary funding for the project was provided by the NPS Long-Term Ecological Monitoring Program (LTEM) through cooperative agreements established by WKU with MACA and the National Cave and Karst Research Institute (NCKRI). Rick Fowler was principal investigator of this study at Western Kentucky University (WKU) and Rick Olson, Chief Ecologist at MACA, acted as the principal scientific advisor and expedition coordinator for the National Park Service (NPS). Permits MACA-2001-SCI-001 and MACA-2003-SCI-0321 were issued by the MACA Division of Science and Resources Management (MACA-SRM) for access to remote study sites in the cave, to remove geological and biological specimens, and to deploy experimental artificial substrates for future retrieval. Annual reports for these permits were filed in the NPS system. Laboratory operations were performed at WATERS Laboratory and the WKU Biotechnology Center approximately 40 km from MACA on the main campus of WKU.

Shivendra Sahi, Assistant Director of the Applied Research and Technology Program (ARTP) and former Director of the WKU Biotechnology Center; Chris Groves, Director of the Hoffman Environmental Research Institute (HERI); with Andrew Ernest, Jana Fattic, and Marsha Wallace of the Center for Water Resource Studies (CWRS) all served as adminstrators for the project through various capacities at WKU.

Rick Fowler dropping in through the Doyle Valley entrance . Photo by Norm Waddell.

Other significant personnel included Mark DePoy, Kurt Helf, Joe Meiman, Steve Thomas, and Bob Ward at MACA-SRM. Support from NCKRI was granted by Zelda Chapman-Bailey and Ron Kerbo in 2001 to fund a cooperative agreement between WKU and NCKRI. Lipid analyses were carried out at the Center for Biomarker Analysis of the University of Tennessee by David C. White, Aaron Peacock, and David Hedrick. John Andersland of the WKU Biology Department provided electron microscopy services. Additional consultants and advisors were Art Palmer, Peg Palmer, Roger Brucker, Will White, Nick Crawford, and Pat Kambesis.

Photographs of the Amos Hawkins Formation and the panorama of the Hawkins-Logsdon Confluence were taken by Gary Berdeaux in 2005. Cavers of the NSS and CRF contributed their skills during the project on numerous sampling expeditions.

Hazel Barton of and Brandon Iker of Northern Kentucky Univeristy (NKU) performed phylogenetic analyses for Charon's Cascade sediments collected in 2005 and for all bead biofilm eubacterial sequences. WKU students who made significant contributions in the laboratory include Wes Braden, Luann Breeding, Jamie Burch, Ben Fowler, Roger Greenwell, Matt Honaker, Afnan Kahn, Jerry Ovesen, Paulette Reneau, Eli Roberson, Jyoti Sahi, John Schrimsher, and Christal Wade.