This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.BackgroundSoil and groundwater systems contaminated with toxic heavy metals and radionuclides remain a legacy of Cold War nuclear weapons development. As a result, the United States Department of Energy (DOE) is charged with the remediation and long-term stewardship of such contaminated sites at many U.S. national laboratories. The focus of DOE funded bioremediation research aims at utilizing naturally occurring bacterial strains, obtained from contaminated soils, for the in situ immobilization of soluble uranium. Two distinct strategies for the removal of radionuclides such as uranium from contaminated soils and groundwater are known to be directly mediated by bacteria: 1. Bio-reduction: in the absence of oxygen, uranium is utilized as a terminal electron acceptor for respiration. Soluble U(VI) is reduced to the insoluble U(IV) oxidation state. 2. Bio-mineralization: in the presence of oxygen, uranium precipitation occurs when bacterially liberated phosphate yields an insoluble mineral phosphate (Fig.1). Fig.1. Three potential cell associated locations (i.e. cell surface, cytoplasm and periplasm) of bacterial phosphatase activity hypothesized to occur in heavy-metal and radionuclide contaminated soils and groundwater (A). The hypothesized mechanism of microbial phosphatase activity which yields a uranium phosphate precipitate (B).Current ResearchOur research focuses on the aerobic bio-mineralization strategy of uranium via phosphate-liberating bacterial strains that we have isolated from heavy metal and radionuclide contaminated soils from the Oak Ridge National Laboratory (ORNL) in Oak Ridge, Tennessee. We hypothesize that such phosphate liberation into the surrounding environment is a result of either: 1) Bacteria that constitutively express a sub-class of phosphatase enzymes known as non-specific acid phosphatases (NSAP) or 2) Bacterial strains that are able to secrete de-polymerized cytoplasmic polyphosphate granules. Our work has identified two ORNL bacterial strains belonging to the genera, Rahnella and Bacillus that we have used as model bio-mineralizing strains. Both strains exhibit a phosphate-liberating phenotype when grown on both solid and liquid media. The Rahnella and Bacillus strains demonstrate great promise as they represent naturally occurring bacteria that have been shown to liberate sufficient phosphate from organophosphate substrates such as glycerol-3-phosphate and subsequently precipitate uranium in the form of calcium autinite, Ca(UO2)2(PO4)2 (Martinez et al.; Beazley et al., in preparation). Our ORNL Rahnella and Bacillus strains are representatives of the two fundamental classifications of bacterial cell envelopes (i.e., gram-negative and gram-positive cell envelopes) (Fig 2). The identification of these cells was done through DNA sequencing rather than the traditional method of staining with the use basic dyes. The distinctions between these two cell envelopes are based upon the presence of a single peptidoglycan layer located between two concentric lipid membranes (i.e., periplasmic domain) for gram-negative bacteria. Gram-positive bacteria are characterized as having a single cytoplasmic lipid membrane and a large peptidoglycan layer. Until the recent application of energy filtering electron microscopy on frozen hydrated sections, it was believed that a periplasmic domain was absent in gram-positive bacteria. Recently, the Beveridge laboratory has demonstrated through the use of this technique as well as freeze substituted fixation that gram-positive bacteria do indeed have a periplasmic space similar to gram-negative bacteria (Matias et al. 2006). This finding suggests that, like gram-negative bacteria, gram-positive bacteria may have localized chemistry in this previously unseen periplasmic domain. The insight gained through the visualization of this new structure can further the current understanding of gram-positive physiology. Specifically, phosphate accumulation via the activity of NSAPs, which have been previously shown to be localized in the periplasm of gram-negative bacteria, may be functioning in a comparable fashion in our ORNL gram-positive bacterial strain. A second mechanism by which both gram-negative and gram-positive bacteria liberate phosphate and sequester heavy metals is through the depolymerization of cytoplasmic polyphosphate granules (Fig. 2A, 2B). The storage of polymerized phosphate acts as both a reservoir for growth under phosphate-limiting conditions and as a metal detoxification system. The depolymerization of polyphosphate has been shown to be involved in the extracellular precipitation of heavy metals (Boswell et al., 2001). Additionally, polyphosphate granules have also been shown to sequester radionuclides that have been transported into the cytoplasm of gram-positive bacteria (Suzuki and Banfield, 2004). Fig.2. Generalized composition of the two fundamental bacterial cell envelops (gram-negative and gram-positive) both containing polyphosphate storage granules (A). The three hypothesized uranium phosphate localization sites (i.e., outer membrane, periplasmic domain and polyphosphate granule sequestration) predicted in our model ORNL bacterial strains (B). Presently, our findings have demonstrated the capability of bacteria extant within the ORNL soils to bio-precipitate soluble uranium under similar in situ growth conditions. To understand the effect uranium phosphate precipitation has on ORNL Rahnella and Bacillus strains that have never been visualized on the nanometer scale, we would like to address the following questions:1. Do the cell envelopes of ORNL Rahnella and Bacillus strains exposed to uranium differ from those unexposed to uranium? 2. For ORNL Rahnella and Bacillus strains exposed to uranium, is phosphorus and uranium localized on the outer membrane, periplasmic space or associated with polyphosphate granules within the cytoplasm? The visualization of the cell envelopes as well as the localization of both phosphorus and uranium in these strains will allow us to definitively determine the source of accumulating phosphate (i.e., NSAP activity within the periplasm or cytoplasmic depolymerization of polyphosphate granules). As bacterial viability is crucial for optimal bio-precipitation, the localization pattern of phosphorus and uranium is important. Cytoplasmic polyphosphate sequestration of uranium would be less detrimental to cell viability than a periplasmic accumulation. Due to the inhibition of electron and nutrient transport, periplasmic mineralization of uranium would contribute to a loss in cell viability.Goals at the RVBCThe microanalysis and ultra-structure imaging will be critical for our understanding of heavy-metal and radionuclide toxicity. Knowledge gained by such analysis will allow optimal utilization of bacterial strains for future bioremediation experiments. Therefore, we feel the imaging and spectroscopic capabilities of the Resource for the Visualization of Biological Complexity (RVBC) will further our understanding of microbes capable of metal and radionuclide precipitation. The proven expertise of the RVBC staff will allow us to conduct imaging analysis of dehydrated cells, freeze substitution fixed cells and frozen hydrated cells. Additionally, the energy filtering capabilities of the RVBC allow for EELS elemental microanalysis as well as improved resolution at the nanometer scale. ConclusionThe microanalysis and ultra-structure imaging of the ORNL Rahnella and Bacillus strains has never been conducted. The knowledge gained from the microanalysis and ultra-structure imaging will yield new insights applicable to future in situ bioremediation strategies. Such strategies will aim to stimulate soil microbes capable of bio-precipitating metals found in contaminated soils and groundwater systems. This research is conducted at the Georgia Institute of Technology in the laboratory of Dr. Patricia A. Sobecky and supported by the Office of Science (BER), U.S. Department of Energy Grant No. DE-FG02-04ER63906.ReferencesBeazley, M., Martinez, R.J., Webb, S.M., Sobecky, P.A., Taillefert, M. Environmental Science and Technology (in preparation). Boswell, C. D., Dick, R. E.,Eccles, H. 2001. Journal of Industrial Microbiology & Biotechnology. 26, 333-340.Martinez, R.J., Beazley, M., Taillefert, M., Arakaki, A.K., Skolnick, J. and Sobecky, P.A. Environmental Microbiology (in preparation).Matias, V. R. F., Beveridge, T. J. 2006. Journal of Bacteriology. 3, 1011-1021.Suzuki, Y., Banfield, J. F. 2004. Geomicrobiology Journal. 21, 113-121.
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