Date of Award

Winter 2003

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Louis S Tisa

Abstract

A culture-independent molecular phylogenetic approach was implemented to investigate spatial and temporal variations in microbial diversity and abundance in a chlorinated solvent-contaminated bedrock aquifer. Core and groundwater samples were collected from several boreholes and included the following samples: (1) groundwater that was associated with the core, (2) open fractures and (3) partially mineralized sealed fractures (PMSF) exposed by striking the core with a surface-sterilized geology hammer. DNA representing the attached and unattached microbial communities was extracted from these samples, and the 16S rRNA gene was amplified with primers specific for Bacteria, Archaea, sulfate reducers, Geobacteraceae family, Dehalorespirers (Dehalococcoides sp. and Desulfuromonas sp.), and anaerobic methane oxidizers. Molecular fingerprints of the samples were generated by the use of denaturing gradient gel electrophoresis (DGGE), and were analyzed to detect spatial and seasonal changes. DGGE fingerprints revealed significant compositional differences between the attached and the unattached microbial communities. Cluster analysis suggested that the key factor responsible for changes detected in the groundwater was time, not depth. PMSF fingerprint analysis revealed depth-dependent clustering trend. DGGE profiles for Desulfuromonas sp. revealed an unexpected level of diversity. Sequence analysis of about 400 DGGE bands indicated the predominance of a beta-Proteobacteria-like sequence in the groundwater. gamma-, alpha- Proteobacteria, Cyanobacteria-, Cytophaga/Flavobacterium/Bacterooides-, and Gram-positives including Actinobacteria-and Dehalococcoides-like sequences dominated the attached populations. A novel sulfate-reducing community was found in the groundwater, while hydrogen-utilizing archaea with similarity up to 94.6% to Methanospirillum hungatei dominated the PMSF samples. Most of the groundwater archaeal sequences were affiliated with the thermophilic Crenarchaeota and Methanosarcinales. The relative abundance of several microbial groups, which was determined by the use of nested Real Time-PCR (nRT-PCR), did not show any significant changes in the groundwater in relationship to depth or time. However, the relative abundance of Bacteria, Archaea, Geobacteraceae, and sulfate reducers was higher for the attached microbial communities compared to the unattached communities in at least one of the wells studied (BBC5). Molecular evidence also suggests the existence of anaerobic methane oxidation at this site and that hydrogen was acting as an interspecies electron shuttle.

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