Type of Document	Dissertation
Author	Edwards, Ellen McLain
Author's Email Address	lpetrie@ocean.fsu.edu
URN	etd-12222004-153409
Title	Characterization of Metal-Reducing Microbial Communities from Acidic Subsurface Sediments Contaminated with Uranium(VI)
Degree	Doctor of Philosophy
Department	Oceanography, Department of
Advisory Committee	Advisor Name Title Joel Kostka Committee Chair Bill Burnett Committee Member David Balkwill Committee Member Jeff Chanton Committee Member Lee Krumholz Committee Member
Keywords	Iron-reducing Microorganisms Biostimulation Bioremediation
Date of Defense	2004-12-10
Availability	unrestricted
Abstract Extraction and processing of uranium ore during the Cold-War era have left many sites around the world contaminated with uranium. Leaching of uranium into the groundwater is of major concern because oxidized uranium, U(VI), is toxic, soluble, and therefore mobile in subsurface environments where the majority of contamination resides. Uranium [U(VI)] can be immobilized from water by its reduction from UO22+ to insoluble U(IV) oxide, and biostimulation by the addition of carbon substrates has been shown to stimulate the microbial reduction of U(VI) in contaminated subsurface environments. However, in order to develop effective bioremediation strategies for contaminant metals, the microbial communities and mechanisms controlling metal reduction need to be better understood, especially in acidic subsurface environments. This dissertation research used an array of microbiological and geochemical techniques to examine metal reduction in materials from the U.S. Department of Energy (DOE) Natural and Accelerated Bioremediation Research (NABIR)'s Field Research Center (FRC), in Oak Ridge, Tennessee, where subsurface sediments are cocontaminated with high levels of U(VI) and nitrate. The combination of low pH and high concentrations of nitrate and radionuclides in an aerobic subsurface environment is representative of many sites within the U.S. nuclear weapons complex managed by the DOE. Thus, results are not only important for bioremediation research at the FRC but can also be applied to other sites. Iron(III)-reducing bacteria (FeRB) have been demonstrated to rapidly catalyze U(VI) reduction and Fe(III) is an abundant electron acceptor in uranium-contaminated subsurface sediments. Thus, FeRB communities were the focus of this dissertation. The abundance, diversity, and activity of indigenous metal-reducing microbial communities likely to contribute to uranium reduction was examined in the field and under more controlled conditions in the laboratory. In chapter one, a combination of cultivation-dependent and cultivation-independent microbiological techniques were utilized to characterize metal-reducing bacteria in FRC subsurface sediments. Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH (4-5). In selected enrichments, nitrate contamination was removed from the sediment by washing. Using a most probable number (MPN) approach and a range of different carbon sources (glycerol, acetate, lactate, or glucose), sediments of lower pH typically yielded lower counts of FeRB except when glucose was utilized as an electron donor in acidic enrichments. Phylogenetic analysis of the 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia cultured from pristine background sediments were closely related to the family Geobacteraceae, whereas a recently characterized Fe(III)-reducer (Anaeromyxobacter) and organisms not previously shown to reduce Fe(III) (Paenibacillus, Brevibacillus) predominated the Fe(III)-reducing consortia of contaminated sediments. Analysis of enrichment cultures using terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results. Enrichment cultures of Fe(III)-reducers from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls. Using DNA extracted directly from the subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated that Geobacteraceae sequences were one to two orders of magnitude less abundant in contaminated as compared to pristine environments. In contrast, Anaeromyxobacter sequences were more abundant in contaminated sediments. Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/ community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and microorganisms capable of producing spores (gram positives) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments. In chapter two, microbial communities were studied in sediment microcosms under near in situ conditions in order to establish rates of respiration and to assess which environmental parameters might be governing activity. Rates of nitrate reduction, metal reduction, and electron donor utilization were measured in acidic subsurface sediments across a range of environmental variables (pH, nitrate) relevant to bioremediation. Microbial activity was minimal at pH 5 or below and in the absence of added electron donor, indicating that acidity is a master variable controlling microbial metabolism in FRC sediments, while high nitrate concentrations were not found to be toxic to microorganisms. In microcosms of neutral pH sediment and neutralized acidic sediment, similar, rapid rates of terminal-electron-accepting pathways were observed. The pathways of nitrate reduction were dictated by sediment pH, as denitrification predominated in glucose-amended sediments originating from neutral pH zones, whereas in neutralized acidic microcosms, metabolism shifted to dissimilatory nitrate reduction (to ammonium). Electron donors were determined to stimulate microbial metabolism leading to metal reduction in the following order: glucose > ethanol > lactate > hydrogen. A mass balance of carbon equivalents was obtained in glucose- and ethanol-amended microcosms. In neutralized acidic sediments amended with glucose, 50 to 60 % of carbon equivalents were recovered as fermentation products (mainly as acetate) and glucose-amended microcosms showed the highest iron reduction activity, while the extended presence of ethanol seemed to hinder iron reduction. The presence of bicarbonate greatly increased both nitrate and iron reduction activity in glucose-amended microcosms, more so than raising the pH by washing. Washing did increase iron reduction in glucose-amended microcosms as compared to neutralized acidic sediments, indicating that soluble toxins may somehow decrease iron reduction potential. For the first time, rates of metal reduction and electron donor utilization were measured in acidic subsurface sediments across a range of environmental variables (pH, nitrate) relevant to bioremediation. In concurrence with previous studies of neutrophilic uranium-contaminated subsurface environments, metal reduction in the acidic subsurface did not occur until after nitrate was depleted to low levels in response to pH neutralization and carbon substrate addition. Through quantification of the rates and pathways of terminal-electron-accepting pathways in acidic subsurface sediments, we provide important inputs for reaction-based biogeochemical models that will greatly aid in the development of in situ radionuclide remediation strategies. In chapter 3, a pure culture of Fe(III)-reducing bacteria isolated from the FRC subsurface was further examined for its ability to reduce U(VI). Uranium measurements were conducted using a Kinetic Phosphorescence Analyzer, which was cross-calibrated using alpha spectrometry. The uranium reduction ability of isolate FRC32, was compared to a known uranium-reducing organism, Geobacter metallireducens. FRC32 was tested under various cultivation conditions, including a range of uranium and cell concentrations and up to 90% of 0.1-5 mM uranium was reduced. However, reduction in killed-control cultures suggests either a strong potential for abiotic reduction or the ability to form spores. Thus, the potential for uranium reduction was observed, but further research is necessary to determine which environmental parameters are controlling uranium transformation by this organism.
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