Type of Document Dissertation Author Akob, Denise Marie URN etd-08242008-163320 Title Structure and Function of Microbial Communities Controlling the Fate and Transformation of U(VI) in Radionuclide Contaminated Subsurface Sediments Degree Doctor of Philosophy Department Oceanography, Department of Advisory Committee
Advisor Name Title Joel E. Kostka Committee Chair Kirsten Kuesel Committee Member Markus Huettel Committee Member Thomas DiChristina Committee Member Vincent Salters Outside Committee Member Keywords
- SSU rRNA
Date of Defense 2008-07-22 Availability unrestricted AbstractUranium contamination is widespread in subsurface sediments at mining and milling sites across North America, South America, and Eastern Europe. In the U.S. alone, the Department of Energy (DOE) is responsible for the remediation of 7,280 km2 of soils and groundwater contaminated due to processes associated with uranium extraction for nuclear weapons production. As a result of waste disposal practices, subsurface sediments at these sites are often co-contaminated with nitric acid, sulfuric acid, and toxic metals. Oxidized uranium, U(VI), is highly soluble and toxic, and thus is a potential contaminant to local drinking water reservoirs. The most promising strategy for in situ uranium bioremediation is immobilization through the biological reduction of U(VI) to insoluble U(IV) by indigenous microbial communities. However, the development of effective U(VI) bioremediation strategies is limited by our current understanding of the composition, metabolic potential and physiological requirements of in situ microbial communities.
A polyphasic approach employing microbiological and geochemical techniques was used in this dissertation to link the structure and function of microbial communities in subsurface sediments of the U.S. Department of Energy’s Oak Ridge Field Research Center (ORFRC), in Oak Ridge, Tennessee. Subsurface sediments at the ORFRC site are cocontaminated with high levels of U(VI) and nitrate and microbial activity is limited by carbon availability and variable pH. The conditions at the ORFRC site are representative of many radionuclide-contaminated sites; therefore, results from this dissertation will have broader significance for development of bioremediation strategies that can be employed worldwide.
To develop effective bioremediation strategies for radionuclide contaminants, the composition and metabolic potential of microbial communities need to be further understood, especially in highly contaminated subsurface sediments for which little cultivation-independent information is available. Thus, the metabolically active and total microbial communities associated with uranium contaminated subsurface sediments were characterized along geochemical gradients (Chapter 1). DNA and RNA were extracted and amplified from four sediment depth intervals representing moderately acidic (pH 3.7) to near neutral (pH 6.7) conditions. Phylotypes related to the Proteobacteria (α−, β−, δ−, and γ−Proteobacteria), Bacteroidetes, Actinobacteria, Firmicutes and Planctomycetes were detected in DNA- and RNA-derived clone libraries. Diversity and numerical dominance of phylotypes were observed to correspond with changes in sediment geochemistry and rates of microbial activity, suggesting geochemical conditions have selected for well-adapted taxa. Sequences closely related to known nitrate-reducing bacteria, comprised 28 and 43% of clones from the total and metabolically active fractions of the microbial community, respectively. Chapter 1 provides the first detailed analysis of total and metabolically active microbial communities in radionuclide contaminated subsurface sediments. The microbial community analysis, in conjunction with rates of microbial activity, points to several groups of nitrate-reducers that appear to be well adapted to environmental conditions common to radionuclide-contaminated sites.
To elucidate the potential mechanisms of U(VI) reduction for optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate (Chapter 2). A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in-situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped with time in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (NRB and FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended as compared to glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon amended microcosms with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. The results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.
Metabolic activity and phylogenetic structure of microbial communities mediating U(VI) bioimmobilization were directly linked in microcosms constructed with contaminated subsurface sediments using a stable isotope probing (SIP) approach (Chapter 3). Ethanol and acetate are the electron donors that have been shown in previous bioremediation studies to promote microbially mediated U(VI) reduction. Therefore, microcosms were amended with 13C-labeled ethanol or acetate, as supplemental electron donors, and molybdate was added to select treatments as an inhibitor of sulfate reduction. Activity was assessed by monitoring terminal electron accepting processes (TEAPs) (e.g., nitrate-, sulfate-, Fe(II) and U(VI) concentrations) and electron donor utilization. 13C incorporation into community DNA was examined by density gradient centrifugation along with PCR amplification and terminal restriction fragment length polymorphism (TRFLP) analysis. Incorporation of 13C into microbial DNA was detected by day 3, corresponding with the onset of TEAPs and carbon utilization. Metal reduction commenced only with removal of nitrate and U(VI)-reduction preceded Fe(III)-reduction. Fe(III)-reduction occurred in all treatments, regardless of electron donor or presence of molybdate. Sulfate reduction rates were most rapid in the ethanol-amended treatments, whereas little to no sulfate reduction occurred in the acetate- or molybdate- amended treatments. Microbial community composition was affected by treatment and changed with shifts in TEAPs. The metabolically active denitrifying microorganisms were identified as members of the Betaproteobacteria, whereas, members of the Deltaproteobacteria, Actinobacteria, Firmicutes and Bacteroidetes were active during sulfate- and/or metal-reduction. Our data showed that carbon from supplemental ethanol is coupled to microbial growth either by direct utilization or secondary carbon flow from oxidation end products or dead 13C-biomass. Application of the SIP technique allowed for the definitive identification of metabolically active microbial populations and identification of their role in the separate TEAPs occurring in uranium-contaminated subsurface sediments.
A combination of biogeochemical rate measurements and robust microbial community characterization was used to determine the metabolic potential of microbial groups affecting U(VI) reduction in the subsurface. The composition, distribution, and metabolic potential of in situ microbial communities at the ORFRC site varies with subsurface geochemistry and will impact the success of U(VI) bioremediation. Ethanol is an effective electron donor for biostimulation of indigenous microbial communities at the ORFRC site and promotes complete nitrate-reduction and U(VI) removal. U(VI) reduction was observed to overlap with alternative TEAPs, e.g., sulfate- or Fe(III)-reduction and microbial populations involved in these TEAPs included members of the Firmicutes and Deltaproteobacteria. This research provides necessary data for the future optimization of in situ bioremediation practices in uranium-contaminated sediments at the ORFRC.
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