Date on Master's Thesis/Doctoral Dissertation

5-2005

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Committee Chair

Atlas, Ronald M., 1946-

Author's Keywords

Microbial communities; Karstic; Sediments; Cave bacteria; Kentucky

Subject

Cave ecology--Kentucky; Bacteria--Ecology

Abstract

Few previous studies have compared microbial communities in subterranean and surface environments. Chemical analyses used to characterize the surface and cave microbial environments indicated limited exchange between surface and subsurface waters. Here, bacterial communities from four distinct cave sediments collected in central Kentucky and adjacent surface streams were compared using a combination of culture and molecular methods. Predictions were that: 1) Cave communities will have unique community structures when compared to those of nearby surface sediments, and 2) Genetic diversity of bacterial communities in cave sediments will be higher than those in nearby surface sediments. Since diversity and community structure influence the ability of biological communities to respond to environmental disturbance, it is important to understand the ecological role of bacterial communities in cave sediments. A combination of plate counts and direct counts by epifluorescent staining were used to enumerate bacteria. When cell numbers were compared to sediments, average particle size was positively correlated with plate counts but had a higher indirect relationship to direct cell counts using DAPI stain and epifluorescent microscopy. Cell counts varied between sites. Visual characterization of the cultured bacteria revealed observed higher proportions of pigmented bacteria in surface sediments. This is likely light influenced as cave sediment bacteria were mostly colorless. Levels of the pigments chlorophyll a and pheophytin were also negligible in the cave waters sampled. Amplified ribosomal DNA restriction analysis (ARDRA) and reassociation kinetics were used for molecular analyses of DNA extracted from the surface and cave communities. High calcium concentrations in the karstic sediments sampled made the molecular analyses by these methods complicated. It is likely that calcium and other compounds interfered with sphectrophotometric methods as well. In addition to purification to reduce calcium and other inhibitors, dilution of extracts to 1/100 or 1/1000 was necessary for amplification of in-situ extracted DNA. However, DNA extracted from cultured communities was easily amplifiable. Conclusions from the community structure analyses are that the cave microbial communities sampled contained different members than the adjacent surface sediments as detected by digestion of 16S rDNA from the cultured and in-situ extracted bacterial communities. In addition, cave sites sampled had unique community structures when compared to each other. Cultured community signatures were less complex when compared to the total community 16S amplicons by ARDRA. This method is, and will continue to be, useful for comparing bacterial community structure in different habitats, over time, or in response to environmental changes. Comparison of diversity by reassociation indicated that cave microbial communities contained similar genetic complexity as those in adjacent surface sediments. Rates of reassociation were different for 16S amplicons from cultured and in-situ extracts. With careful selection of methods for extraction and purification, and proper interpretation of results for reassociation rates of 16S rDNA amplicons, this method can provide useful genetic and taxonomic information about bacterial communities.

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