Type of Document	Thesis
Author	North, Nadia
URN	etd-09182003-152635
Title	Molecular Characterization Of A Microbial Community From Uranium-Contaminated Acidic Sediment
Degree	Master of Science
Department	Oceanography, Department of
Advisory Committee	Advisor Name Title David Balkwill Committee Chair Joel Kostka Committee Member Nancy Marcus Committee Member Sherry Dollhopf Committee Member
Keywords	Strategies For Bioremediation Contaminant Metals A
Date of Defense	2003-08-02
Availability	unrestricted
Abstract The Field Research Center (FRC) in Oak Ridge, TN, was established by the U.S. Department of Energy’s Natural and Accelerated Bioremediation Research (NABIR) Program to develop strategies for bioremediation of contaminant metals and radionuclides. The FRC is centered on groundwater plumes that originate from former S-3 Waste Disposal Ponds located at the Y-12 Plant where acidic nitrate- and uranium-contaminated waste was disposed. The groundwater and sediment surrounding this area are in need of remediation. To test the bioremediation potential of microorganisms in the contaminated FRC sediment, Fe(III)-reducing bacteria (FeRB) were enriched for, as they represent indigenous microorganisms capable of immobilizing uranium in situ. DNA was extracted from more than 20 Fe(III)-reducing enrichment cultures at both neutral and low pH. Through cloning and sequencing of 16S rRNA genes, considerable differences in phylotypes found in Fe(III)-reducing enrichments from background and contaminated FRC sediment were discovered. In the background Fe(III)- reducing enrichments (pH 7), over two-thirds of the 16S rRNA gene sequences obtained were closely related to members of the Geobacteraceae family. The cultivated isolates from this family are capable of Fe(III)-reduction, and are commonly found in sedimentary environments. However, sediment from the contaminated neutrophilic Fe(III)-reducing enrichments revealed that almost half of the 16S rRNA gene sequences were 96% similar to the newly described Fe(III)-reducing species Anaeromyxobacter dehalogenans, but no Geobacteraceae sequences were detected. In Fe(III)-reducing enrichments cultured at low pH (4 to 5), only 16S rRNA gene sequences closely related to Gram positive organisms were detected. From the contaminated sediment enrichment cultures at low pH, the most predominant 16S rRNA gene sequences retrieved were closely related to Gram positive organisms Brevibacillus and Paenibacillus. T-RFLP analysis of enrichment cultures strongly supported the sequencing results. Following the experiments with Fe(III)-reducing enrichment cultures, cultivation-independent studies were conducted using a quantitative method. Quantitative molecular techniques provided a direct determination of the abundance of selected groups of FeRB before and after sediment biostimulation. Using DNA extracted directly from the sediment, MPN-PCR was conducted to quantify the differences in abundance of Geobacter-, Anaeromyxobacter-, Paenibacillus-, and Brevibacillus- type sequences using primer sets specifically designed for these groups. The only sequences that increased in abundance after biostimulation were Geobacter-type 16S rRNA gene sequences. To account for other groups of stimulated organisms, cloning and sequencing was conducted in parallel with the quantitative PCR experiments. A large diversity of microorganisms from FRC sediment were revealed, including species from alpha, beta, delta, and gamma subdivisions of the Proteobacteria, as well as low and high G+C Gram positive species. Phylogenies suggesting certain physiologies, such as nitrate reduction, metal reduction, dechlorination, and degradation of metal-chelator complexes and fuel hydrocarbons were identified. Obvious trends in 16S rRNA gene sequences following the biostimulation of FRC sediments included an increase in sequences within the delta Proteobacteria, as well as the maintenance of a large abundance of sequences within the genus Methylobacterium, in the alpha Proteobacteria. Due to the diversity of organisms detected after biostimulation of contaminated sediments, we suggest that new model organisms should be pursued to aid in the ongoing development of bioremediation strategies for uranium contamination.
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