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Subsurface Science
The Molecular Environmental Science Group (MESG)

The MESG is part of the Biosciences Division at Argonne National Laboratory.  One of the main foci during the creation and growth of the MESG has been the development of an internationally recognized integrated multidisciplinary scientific team focused on the investigation of fundamental biogeochemical questions.  Presently, expertise that is represented by members of the MES Group includes x-ray Physics, Environmental Chemistry, Environmental Microbiology, (Bio)geochemistry, and radiolimnology. Additional expertise in electron microscopy, x-ray microscopy, Microbial Ecology, and Bioinformatics often is provided by collaborations with scientists outside of our group.

Partial support of the Molecular Environmental Science Group is provided by the Argonne Subsurface Science Scientific Focus Area (SFA) which is supported by the US DOE Subsurface Biogeochemistry Research Program. In addition to investigating transformations resulting from biological, physical, and chemical processes in the subsurface that affect the mobility of contaminants, carbon/nutrient forms, and the geochemical character of groundwater, members of the Argonne National Laboratory MES Group facilitate the use of the Advanced Photon Source and other synchrotrons by scientists funded to do work closely related to research within the MES Group. Information about how to apply for General User Beam time at the Advanced Photon Source can be found at the Advanced Photon Source web site or by contacting Ken Kemner.

It is currently difficult to predict the biogeochemical cycling of elements in the subsurface. Understanding the coupled biological, chemical, and physical processes controlling elemental cycling in the environment is of fundamental importance to advance a robust predictive understanding of Earth’s climate and environmental systems and to inform the development of sustainable solutions to the Nation’s energy and environmental challenges. Bacteria and the extracellular material associated with them are thought to play key roles in determining an element’s chemical speciation and its mobility in the environment. Additionally, the microenvironment at and adjacent to actively metabolizing cells can be significantly different from the bulk environment. Our group uses a number of analytical techniques (i.e. ICP-AES, HPLC, kinetic phosphorescence analysis, x-ray diffraction, electron microscopy, etc.) and “now” generation high-throughput sequencing and bioinformatics approaches to better understand the role of minerals, microbes, and microbial communities in determining elemental cycling in the environment. Additionally, we make use of a number of synchrotron-based x-ray techniques to further our understanding of the processes occurring at physical, geological, chemical, and biological interfaces that affect these transformations. Hard x-ray absorption spectroscopy techniques such as extended x-ray absorption fine structure (EXAFS) spectroscopy, x-ray absorption near edge spectroscopy (XANES), and nonresonant inelastic x-ray scattering (NIXS) can provide information on the local chemical environment, coordination, and valence of individual elements in soils and sediments. Additionally, hard x-ray micro-imaging techniques (i.e. x-ray fluorescence microscopy and x-ray microtomography) enable investigation of complex environmental samples at the needed micron and submicron length scales. An important advantage of these techniques results from their utility in investigating environmental materials in their natural, and often hydrated, state.

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> Research Highlights

Effects of bound phosphate on the bioreduction of lepidocrocite (γ-FeOOH) and maghemite (γ-Fe2O3) and formation of secondary minerals

Figure for Effects of bound phosphate on the bioreduction of lepidocrocite (γ-FeOOH) and maghemite (γ-Fe<sub>2</sub>O<sub>3</sub>) and formation of secondary minerals
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The US Department of Energy has been investigating ways to use bacteria that are naturally present in soils and aquifers to help control the movement of pollution in contaminated groundwater and to better understand the role bacteria play in affecting the biogeochemical cycling of C and, hence, global climate change. The activity of these bacteria can create products that change the chemical form of pollutants such that they are less toxic or less mobile. One example of this is the use of iron-“breathing” bacteria (known as iron-reducing bacteria (IRB)) to transform iron oxides (i.e., “rust”) commonly found in soils and sediments to reactive iron minerals; these reactive iron minerals can be effective in treating contaminants such as chlorinated solvents, heavy metals, and radionuclides and play an important role in the biogeochemical cycling of C. However, many aspects of how IRB make specific reactive iron minerals are not well understood. Researchers in the Biosciences Division at Argonne National Laboratory in collaboration with scientists at the University of Iowa, The Pennsylvania State University, and Hamilton College, identified the importance of phosphorous (an element commonly found in association with iron oxides) as a factor in controlling which reactive iron minerals are made by IRB. The researchers examined the effect of phosphate bound to iron oxides on reactive iron mineral formation by the IRB Shewanella putrefaciens CN32. They found that in the absence of phosphate, CN32 transformed some types of iron oxides to a mineral called magnetite. However, when phosphorous is present in some of these iron oxides, CN32 produced a reactive iron mineral called “green rust”. The mineral green rust is an effective treatment for many common pollutants and has been shown by Argonne scientists to be much more reactive than the mineral magnetite. These results provide key information for understanding how IRB make reactive iron minerals that may lead to improvements in the effectiveness of the use of IRB for treatment of pollution in below ground environments such as contaminated aquifers.

O'Loughlin, E. J.; Boyanov, M. I.; Flynn, T. M.; Gorski, C.; Hofmann, S. M.; McCormick, M. L.; Scherer, M. M.; Kemner, K. M. Effects of bound phosphate on the bioreduction of lepidocrocite (γ-FeOOH) and maghemite (γ-Fe2O3) and formation of secondary minerals. Environ. Sci. Technol. 2013, DOI 10.1021/es400627j.

Deep Underground, a Potential Sink for Methane

Diagram showing a cross section of a hypothetical aquifer
Figure 1. Diagram showing a cross section of a hypothetical aquifer. Methane gas is produced by methanogenic archaea upgradient of where sulfate infiltrates the aquifer from the bedrock. The mixture of methane-rich and sulfate-rich groundwater allows the microbially-mediated anaerobic oxidation of methane to occur, transforming the volatile methane gas into bicarbonate. Click image to view larger version.

The Mahomet Aquifer in central Illinois contains nearly four trillion gallons of groundwater, more than enough to provide clean drinking water for the nearly 800,000 people who live there. Yet the Mahomet is not merely a resource to be exploited by thirsty Illinois residents, it is also home to a diverse ecosystem of microorganisms. A recent study by a team of researchers from the University of Illinois at Urbana-Champaign, the United States Environmental Protection Agency, and Argonne National Laboratory suggests that besides maintaining the high quality of the drinking water, the bacteria and archaea that dwell in the Mahomet Aquifer may play an important role in preventing the greenhouse gas methane from reaching our atmosphere.

Methane is a much more potent greenhouse gas than carbon dioxide, and its release into the atmosphere has the potential to accelerate global climate change. Much of the methane that is released into Earth’s atmosphere is produced by microscopic organisms known as methanogenic archaea. While these microbes are perhaps better known for the methane they produce inside (and its subsequent emission from) mammalian guts, they are also found in sedimentary environments across the globe, including the Mahomet Aquifer. Groundwater in some areas of the Mahomet is so charged with methane, in fact, that when pumped to the surface it effervesces bubbles of the gas that can be ignited.

What Flynn et al. found, however, was that the Mahomet is home not only to methane-producing archaea but also to closely-related cousins who consume, rather than produce, methane. What is unique about these methane consumers (often referred to as methane oxidizers) is that they do so anaerobically, meaning in the absence of oxygen. While these organisms are often detected in marine sediments where methane seeps from cracks in the ocean floor, they are only rarely observed in terrestrial aquifers. Flynn et al. hypothesize that these anaerobic methane oxidizers, as they are called, may represent a critical piece of the microbial ecosystem in aquifers that provide a sink to prevent microbially-generated methane from reaching the atmosphere (Figure 1).

Microorganisms capable of anaerobically oxidizing methane may also play an important role in mitigating the release of methane leaked from wells used to mine natural gas from hydraulically-fractured shales via the process known as “hydrofracking.” The activity of anaerobic methane oxidizers can short-circuit the release of methane into the atmosphere by transforming methane into bicarbonate, an ion which is much less volatile. Flynn et al. found that the abundance of these organisms in the Mahomet was tied to the chemistry of groundwater there, in particular the concentration of sulfate. This insight may help us better understand their distribution in other aquifers and, perhaps, how best to stimulate their activity to clean up after leaky natural gas wells.

“Functional microbial diversity explains groundwater chemistry in a pristine aquifer,” T. M. Flynn, R. A. Sanford, H. Ryu, C. M. Bethke, A. D. Levine, N. J. Ashbolt, J. W. Santo Domingo, BMC Microbiology 2013, 13:146 doi:10.1186/1471-2180-13-146

Bioreduction of solid uranyl phosphate occurs through U(VI) dissolution and results in non-uraninite U(IV) products

Figure for Bioreduction of solid uranyl phosphate occurs through U(VI) dissolution and results in non-uraninite U(IV) products
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The mobility of uranium in subsurface environments is controlled by interrelated adsorption, reduction/oxidation, and precipitation reactions. Reductive bioremediation approaches, whereby aqueous UVI is reduced to solid-phase UIV and thus removed from groundwater, have been widely investigated. An alternative control on U in groundwater is the presence of either native or amended phosphate, which can cause the precipitation of UVI-phosphate minerals. UVI-phosphate precipitation may occur before or during reductive processes, altering the source form of UVI for reduction and potentially affecting its reduceability and stability. In this study, researchers from Argonne National Laboratory, in collaboration with scientists from the University of Notre Dame, have used the Advanced Photon Source at Argonne National Laboratory to demonstrate that UVI-phosphates can be bioreduced by three bacteria relevant to field remediation efforts at Department of Energy sites. Unlike bioreduction of dissolved UVI, reduction of UVI-phosphate occurred at a much slower rate. Using a combination of approaches the researchers were able to show that the bacteria did not reduce UVI in the solid mineral; instead, bacteria reduced the small amount of UVI that was released by dissolution from the mineral. In this dynamic dissolution-reduction-reprecipitation process, the bacteria produced an unexpected form of UIV—while bioreduction of aqueous UVI commonly produces the lowest solubility mineral UO2, reduction of UVI-phosphate resulted in U being present as isolated, complexed UIV atoms in the solids. These findings enhance our understanding of U fate in contaminated environments by contributing to our ability to predict U migration using transport models and to prevent ecological disasters by designing effective remediation strategies.

“Bioreduction of hydrogen uranyl phosphate: mechanisms and U(IV) products” by Xue Rui, Man Jae Kwon, Edward J. O’Loughlin, Sarrah Dunham-Cheatham, Jeremy B. Fein, Bruce Bunker, Kenneth M. Kemner, and Maxim I. Boyanov; Environ. Sci. Technol. 2013, DOI: 10.1021/es305258p

Influence of Al-substitution and anion sorption on Fe electron transfer and atom exchange in goethite

Figure for Influence of Al-substitution and anion sorption on Fe electron transfer and atom exchange in goethite
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Iron is a common element in the subsurface, and its several chemical forms play a key role in the fate and transport of environmental contaminants. As iron cycles between its Fe(III) and Fe(II) oxidation states (commonly known as ferric and ferrous iron) it interacts with contaminants, often determining whether they are mobile or immobile in the subsurface. New research has provided a better understanding of how Fe(II) in solution interacts with the Fe(III)-containing mineral goethite under natural environmental conditions. Researchers at the University of Iowa and Argonne National Laboratory have shown that electron transfer between the two forms of iron occurs even when common species such as phosphate and carbonate ions and the humic acids representative of the natural organic matter found in the subsurface coat the mineral surface. Similarly, electron transfer was observed when aluminum substituted for Fe(III) in goethite. Substitution of even a moderate amount of aluminum for Fe(III) in goethite, however, reduced the atom mixing between aqueous Fe(II) and goethite driven by the electron transfer process. This observation is significant since the electron transfer and atom mixing process has a major influence on incorporation of contaminants into minerals such as goethite and their subsequent immobilization. The new findings will help explain the immobilization (and potential for remobilization) of the contaminants and will be helpful in the development of models of contaminant transport at DOE cleanup sites.

Drew E. Latta, Jonathan E. Bachman, and Michelle M. Scherer, “Fe electron transfer and atom exchange in goethite: Influence of Al-substitution and anion sorption” (2012) Environmental Science & Technology, 46, 10614–10623. DOI: 10.1021/es302094a

Effects of dissimilatory sulfate reduction on FeIII (hydr)oxide reduction and microbial community development

The observed reduction of iron was driven primarily by chemical reduction of iron(III) oxides to iron(II) sulfide by sulfide produced by sulfate-reducing bacteria oxidizing propionate produced by lactate-fermenting bacteria; direct microbial reduction of iron(III) oxide played only a minor role.
The observed reduction of iron was driven primarily (bold arrows indicate dominant pathways) by chemical reduction of iron(III) oxides to iron(II) sulfide by sulfide produced by sulfate-reducing bacteria oxidizing propionate produced by lactate-fermenting bacteria; direct microbial reduction of iron(III) oxide played only a minor role. Click image to view larger version.

Aquatic and terrestrial environments are dynamic systems where coupled microbiological, geochemical, and hydrological processes define the complex interactions that drive the biogeochemical cycling of the major and minor elements. For example, microbial iron and sulfate reduction profoundly affect the biogeochemical cycling of carbon, iron, and sulfur in natural systems; however, the dynamics of microbial iron and sulfate reduction in the presence of both iron(III) oxides (i.e., “rust”) and sulfate (forms of iron and sulfur commonly found in nature) are not well-understood in systems with mixed microbial populations. Researchers in the Biosciences Division and the Institute for Genomics and Systems Biology at Argonne National Laboratory used an integrated approach combining laboratory-scale model experimental systems with synchrotron-based X-ray spectroscopic techniques and high-throughput DNA sequencing to determine the response of native microbial communities in subsurface sediment from the U.S. Department of Energy’s Integrated Field Research Challenge site in Rifle, CO to sulfate and specific iron(III) oxides when provided with carbon in the form of lactate, a product of microbial fermentation of biomass. They found that instead of promoting microbial iron and sulfate reduction directly, lactate was consumed by populations of lactate-fermenting bacteria and converted to acetate and propionate. Following the fermentation of lactate, sulfate-reducing bacteria coupled the oxidation of propionate to carbon dioxide with the reduction of sulfate to sulfide. The sulfide from microbial sulfate reduction then chemically reduced the iron(III) oxides to form iron(II) sulfide, as shown in Figure 1. 16S rRNA-based microbial community analysis revealed the development of distinct communities in the presence of specific iron(III) oxides. These results improve our understanding of the role of microbial sulfate reduction in coupling the biogeochemical cycles of carbon, iron, and sulfur while providing new insight into the effects of carbon utilization and iron(III) oxide mineralogy on microbial community development. More broadly, these results improve our understanding of complex environmental systems, which is critical for predicting the biogeochemical cycling of carbon, nutrients, heavy metals, radionuclides, and other contaminants; managing water quality; and understanding the interactions between Earth’s terrestrial and atmospheric components.

Kwon, M. J., M. I. Boyanov, D. A. Antonopoulos, J. M. Brulc, E. R. Johnston, K. Skinner, K. M. Kemner, and E. J. O’Loughlin. (Accepted). Effects of sulfate reduction on FeIII (hydr)oxide reduction and microbial community development. Geochimica et Cosmochimica Acta.

Impurities in Natural Minerals Can Affect Uranium Mobility

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Contamination of groundwater with uranium has resulted from its mining for use as an energy source, as well as from past enrichment and weapons production activities at sites managed by the U.S. Department of Energy. Understanding the impact of such contamination on water sources and developing appropriate remediation strategies is paramount in protecting public safety and in the continued use of uranium in a balanced energy production portfolio. Groundwater travels underground in the subsurface through a complex mixture of soils and sediments. A magnetic iron oxide mineral known as magnetite is commonly found within these sediments. Magnetite can significantly slow down uranium migration and act like a “rechargeable battery” for continued removal of uranium from groundwater by sequestering it as nanoparticles of uranium dioxide within underground sediments. Using the Advanced Photon Source at Argonne, IL, one of the brightest synchrotron x-ray sources in the world, new information was discovered on how uranium interacts with magnetite and behaves within the complex chemical environment of the subsurface. In a collaborative effort, researchers at Argonne National Laboratory and Pacific Northwest National Laboratory have found that titanium, a common impurity in these natural magnetic iron minerals, obstructs the formation of the uraninite nanoparticles which results in the formation of novel molecular-sized uranium-titanium structures. This previously unknown association of uranium with titanium affects the mobility of uranium within groundwater in the subsurface. Incorporation of this knowledge into ongoing modeling efforts will improve scientists' ability to predict future migration of contaminant plumes in the subsurface and help to provide the detailed information necessary for the long-term stewardship of U.S. Department of Energy legacy sites.

D.E. Latta, C.I. Pearce, K.M. Rosso, K.M. Kemner, M.I. Boyanov, “Reaction of UVI with Titanium-substituted Magnetite: Influence of Ti on UIV Speciation.” Environ. Sci. Technol., 2013, 47(9), 4121–4130. DOI: 10.1021/es303383n

Influence of Chloride and FeII Content on the Reduction of HgII by Magnetite

Figure for Influence of Chloride and FeII Content on the Reduction of HgII by Magnetite

Mercury (Hg) is a highly toxic element. In its elemental form (Hg0), it is volatile under ambient conditions and can travel long distances in the atmosphere and become oxidized to ionic Hg (HgII) via interactions in the ozone layer. Methylation of HgII by bacteria creates methylmercury, the most toxic form of Hg. Instead of methylation, HgII also can be reduced to Hg0 by 1) microbial respiration processes, 2) photoreduction, and 3) organic matter. Recently, reduction of HgII by ferrous Iron (FeII) bearing minerals (green rust, magnetite, and Fe sulfides) that are found in soils and sediments has gained significant attention. Microbial processes within soils often control the FeII content within these minerals, and the ability of these minerals to reduce HgII to Hg0 is thought to be dependent upon the FeII content within them. In this study, researchers from Argonne National Laboratory, in collaboration with scientists from the University of Iowa and Illinois Institute of Technology, have used the Advanced Photon Source at Argonne National Laboratory to demonstrate that the FeII content of magnetite influences the rate of HgII reduction and formation of reduced Hg products. Additionally, the research has shown that, in the presence of a ubiquitous inorganic ligand such as chloride (Cl-), reduction of HgII by magnetite with less FeII content results in the formation of a metastable HgI species (Hg2Cl2). Interestingly, reduction of HgII by magnetite with less FeII content resulted in only 60% Hg0 (and 40% HgI) even after 4 months of reaction. This study illustrates how the changes in magnetite FeII content, brought about by biogeochemical reactions, or the presence of environmental constituents such as Cl- can have significant implications for the global cycling of Hg, bioremediation approaches, and Hg toxicity.

T. S. Pasakarnis, M. I. Boyanov, K. M. Kemner, B. Mishra, E. J. O'Loughlin, G. Parkin, and M. M. Scherer, “Influence of Chloride and Fe(II) Content on the Reduction of Hg(II) by Magnetite,” Environ. Sci. Technol. 47 6987-6994, 2013. DOI: 10.1021/es304761u

How bacteria influence speciation of mercury in the environment

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Mercury is a contaminant of global concern, as bioaccumulation of methylmercury poses significant risk to aquatic ecosystems and human health. Controlling the transport of mercury in the environment is challenging due to deposition of airborne mercury at locations far from point sources. Mobility of mercury is strongly dependent on its chemical form, with the elemental mercury being volatile and hence mobile in the environment, while oxidized forms are much less mobile (though more toxic). This study, led by Dr. Mishra along with follow researchers of molecular environmental science group at Argonne National Laboratory, has provided improved understanding of the role of bacteria in controlling the chemical form of mercury in subsurface environments. Using X-ray absorption spectroscopy experiments at the Advanced Photon Source to study the sorption of oxidized HgII to Bacillus subtilis, a gram positive soil bacterium, they determined that HgII sorbs to bacterial cells via high and low affinity sulfhydryl and carboxyl binding groups on the cell surfaces. Additionally, they found that HgII that is sorbed to cells via high affinity sulfhydryl groups remains unavailable for reduction by magnetite, a reactive iron-containing mineral often found in sediments, even after two months of reaction time. This is in sharp contrast to their observation of complete reduction of HgII to Hg0 within two hours when HgII is sorbed to cells via the lower affinity carboxyl groups. Since binding of HgII to high-affinity sulfhydryl groups on bacteria could have important implications for the overall mobility of Hg in subsurface environments, these results identify a mechanism by which mercury might be immobilized in the environment.

Bhoopesh Mishra, Edward J. O'Loughlin, Maxim I. Boyanov, and Kenneth M. Kemner, “Binding of Hg(II) to High-Affinity Sites on Bacteria Inhibits Reduction to Hg(0) by Mixed Fe(II/III) Phases” (2011) Environmental Science & Technology, 45, pp 9597–9603 DOI: 10.1021/es201820c.

Unexpected Uranium Solids Formed by Iron-Bearing Soil

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The redox state of soils and sediments exists on a continuum from oxidized to reduced, which can affect the mobility of propagating uranium plumes. Under oxidized conditions, uranium (U) is rather soluble in the U6+ valence state, whereas under reducing conditions U can be immobilized as the less-soluble U4+ valence state. Reductive immobilization of U in sediments is often considered the result of direct biological action of microorganisms; however, chemical reactions in model systems with minerals containing iron in the Fe2+ redox state have also been shown to effectively reduce U6+. Dr. Latta along with follow researchers at the University of Iowa and at Argonne National Laboratory have found that a complex mixture of Fe2+-bearing minerals in a naturally reduced soil is capable of reducing and immobilizing uranium. Using Mössbauer spectroscopy at the University of Iowa and synchrotron x-ray absorption spectroscopy at the Advanced Photon Source at Argonne National Laboratory, we found that uranium was abiotically reduced by Fe2+ found within clay minerals and also by a less-common, transient, and highly reactive Fe2+-mineral called green rust. Furthermore, we observed that the reduced U4+ atoms formed a product different from the uraninite mineral (UO2) commonly observed in laboratory studies, providing evidence for the diversity in chemical speciation of reduced U in natural systems. This study, conducted as part of the Argonne Subsurface Science Focus Area project, provides detailed information necessary for the understanding of toxic and radioactive contaminant mobility, contributes to the long-term stewardship of U.S. Department of Energy legacy sites, and helps to protect the public from exposure to environmental hazards.

Reference: Latta, D. E.; Boyanov, M. I.; Kemner, K. M.; O’Loughlin, E. J.; Scherer, M. M., Abiotic Reduction of Uranium by Fe(II) in Soil. Appl. Geochem. 2012, 27 (8), 1512-1524.

Common Rust Material is a Rechargeable Magnet for Uranium

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The common iron rust mineral, magnetite (Fe3O4), named for its magnetic properties, is expected to play an important role in the mobility of uranium (U) at contaminated U.S. Department of Energy sites and in spent nuclear waste repositories. Magnetite forms when iron metal rusts and when certain species of microorganisms transform iron oxides present in aquifer sediments. In research led by Dr. Latta during his thesis work at The University of Iowa and other members of the Argonne National Laboratory subsurface science focus area found that the amount of Fe(II) present in magnetite controls whether soluble and oxidized U(VI) is reduced to the much less soluble U(IV) mineral, uraninite (UO2). Using the Advanced Photon Source we found that when the magnetite was partially oxidized, U(VI) adsorbed to the surface of magnetite particles, but U in solution remained above the U.S. EPA defined concentration limit for uranium. When magnetite had an Fe(II) content near the stoichiometric value (Fe(II):Fe(III) = 1:2), U(VI) was reduced to the highly-insoluble mineral uraninite. Furthermore, we found that addition of Fe(II) to solutions containing oxidized magnetite recharged the reactivity of the magnetite. The recharged magnetite was capable of reducing U(VI) to U(IV) again. The results of our research suggest that the rechargeable properties of magnetite could be harnessed to help remediate subsurface U contamination.

“Influences on Magnetite Stoichiometry on U(VI) Reduction,” D. E. Latta, C. A. Gorski, M. I. Boyanov, E. J. O’Loughlin, K. M. Kemner, M. M. Scherer, Environ. Sci. Technol. 46(2) 778-86, 2012.

How Subsurface Bacteria Breathe Affects Uranium Mobility and Dispersal

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In situ remediation of subsurface uranium plumes by stimulating indigenous microorganisms is a promising tool for contaminant cleanup in groundwater. Two different types of bacteria, gram-positive and gram-negative, differ in how they are thought to transfer electrons to outside acceptors; that is, in how they breathe. The effect of these differences on the formation of reduced U(IV) products has implications for the mobility of uranium as a contaminant and therefore implications for its remediation. Researchers have thought that the reduced U(IV) always formed uraninite (UO2), the most stable and insoluble U mineral. However, studies by the Molecular Environmental Science group at the Biosciences Division involving x-ray spectroscopy measurements at the Advanced Photon Source suggest that bioreduced U(IV) in natural sediments remain as single U(IV) atoms for extended periods, either complexed to bacterial or mineral surfaces or incorporated in less-stable U(IV) minerals. In a controlled laboratory study, the reduction of carbonate-complexed U(VI) by active gram-positive (G+) and gram-negative (G-) bacteria was explored and compared to reduction by a soluble reductant in the same medium. Both types of bacteria reduced dissolved uranium U(VI) to less soluble U(IV). When phosphate was not present in the solution, G- bacteria created uraninite; in contrast, G+ bacteria created a mononuclear, complexed form of U(IV), which may be less stable, and more easily dissolved and dispersed. These findings also suggest that G+ and G- strains use distinct mechanisms to transfer electrons and reduce U(VI). Conversely, by determining the coordination environment of reduced U(IV) atoms the chemical conditions of their formation and the mechanism of electron transfer to U(VI) can be inferred. In the presence of phosphate, EXAFS shows U(VI) reduction to a non-uraninite, phosphate-complexed U(IV) species, independent of microbial activity. These results highlight previously unappreciated controls of phosphate and electron transfer mechanism on reduced U(IV) speciation.

“Solution and Microbial Controls on the Formation of Reduced U(IV) Species,” M.I.Boyanov, K.E.Fletcher, M.-J.Kwon, X.Rui, E.J. O'Loughlin, F.E. Loffler, K.M.Kemner, Environ. Sci. Technol. 45, 8336 (2011)

Figure for Formation of green rust during bioreduction of Fe(III) oxides linked to presence of oxyanions, natural organic matter, and bacterial cell density
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Formation of green rust during bioreduction of Fe(III) oxides linked to presence of oxyanions (e.g., phosphate), natural organic matter, and bacterial cell density.

Microbial reduction of Fe(III) oxides results in the production of Fe(II) and may lead to the subsequent formation of Fe(II)-bearing secondary mineralization products including magnetite, siderite, vivianite, chukanovite (ferrous hydroxy carbonate), and green rust; however, the factors controlling the formation of specific Fe(II) phases are often not well defined. This study examined the effects of organic (e.g., aliphatic acids, humic substances, and bacterial exudates) and inorganic (e.g., arsenate, phosphate, silicate) ligands commonly found in soils as well as the number and type of DIRB on the rates of bioreduction of the iron oxide lepidocrocite. Faster Fe(II) production rates were accompanied by the formation of magnetite and ferrous hydroxy carbonate, while green rust formed in the presence of ligands that promoted slower Fe(II) production rates. The results are consistent with a conceptual model whereby competitive sorption of more strongly bound anions blocks access of bacterial cells and reduced electron-shuttling compounds to sites on the iron oxide surface, thereby limiting the rate of bioreduction. The differential formation of green rust versus magnetite has implications for the potential reduction of contaminants such as U(VI) and Hg(II) by Fe(II) species resulting from the bioreduction of Fe(III) oxides.

O’Loughlin, E. J., C. Gorski, M. M. Scherer, M. I. Boyanov, and K. M. Kemner. (2010). Effects of oxyanions, natural organic matter, and bacterial cell density on the bioreduction of lepidocrocite (γ–FeOOH) and secondary mineral formation. Environ. Sci. Technol. 44(12):4570-4576. DOI 10.1021/es100294w

Effectiveness of electron shuttles for enhancement of the bioreduction of Fe(III) oxides is linked to their reduction potential

Figure for Effectiveness of electron shuttles for enhancement of the bioreduction of Fe(III) oxides is linked to their reduction potential
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Because of the relative insolubility of Fe(III) oxides under conditions in typical aquatic and terrestrial environments (i.e., circumneutral pH), their use by Fe(III)-reducing microorganisms as terminal electron acceptors for anaerobic respiration requires different mechanisms for electron transfer relative to soluble terminal electron acceptors that are easily transported into the cell (e.g., molecular oxygen, nitrate, sulfate, etc.). The transfer of electrons from the cell to external electron acceptors like Fe(III) oxides can be facilitated by soluble electron shuttles, i.e., compounds that can be reversibly oxidized and reduced. This study examined the effects of a series of compounds representing major classes of natural and synthetic organic electron shuttles—including low-molecular-mass quinones, humic substances, phenazines, phenoxazines, phenothiazines, and indigo derivatives—on the bioreduction of lepidocrocite (γ-FeOOH) by the dissimilatory Fe(III)-reducing bacterium Shewanella putrefaciens CN32. Although S. putrefaciens CN32 was able to reduce lepidocrocite in the absence of exogenous electron shuttles, the addition of exogenous shuttles enhanced the bioreduction of lepidocrocite. In general, the rate of Fe(II) production correlated well with the reduction potentials of the electron shuttles, such that the more negative the reduction potential the faster the rate of Fe(II) production. The addition of humic acids or unfractionated natural organic matter at concentrations of 10 mg organic C L-1 resulted in, at best, a minimal enhancement of lepidocrocite bioreduction. This observation suggests that electron shuttling by humic substances is not likely to play a major role in Fe(III) bioreduction in oligotrophic environments such as subsurface sediments with low organic C contents.

O’Loughlin, E. J. (2008). Effects of electron transfer mediators on the bioreduction of lepidocrocite (γ-FeOOH) by Shewanella putrefaciens CN32. Environ. Sci. Technol. 42(18):6876-6882. DOI 10.1021/es800686d

Reduction of uranium(VI) by mixed Fe(II)/Fe(III) hydroxide (green rust) results in formation of uraninite nanoparticles

Figure for Reduction of uranium(VI) by mixed Fe(II)/Fe(III) hydroxide (green rust) results in formation of uraninite nanoparticles

We have reported the reduction of U(VI) to U(IV) by synthetic green rust, resulting in the formation of nanoparticulate uraninite (UO2). Analysis by U L3-edge XANES of aqueous green rust suspensions spiked with uranyl (UVI) showed that U(VI) was stoichiometrically reduced to U(IV) by green rust. The EXAFS data for the U(VI) reduced by green rust indicated the formation of a UO2 phase. The fits of a model based on the crystal structure of UO2 to the data for the uranium in the green rust samples indicate that the number of nearest-neighbor uranium atoms decreased from 12 for the UO2 structure to 5.4 for the uranium-green rust sample. With an assumed 4 near-neighbor uranium atoms per uranium atom on the surface of UO2, the best-fit value for the average number of uranium atoms indicated UO2 particles with an average diameter of 1.7 ± 0.6 nm. The formation of nanometer-scale particles of UO2, suggested by the modeling of the EXAFS data, was confirmed by high-resolution transmission electron microscopy, which showed discrete particles (~2-9 nm in diameter) of crystalline UO2. Our results clearly indicate that U(VI) (as soluble uranyl ion) is readily reduced by green rust to U(IV) in the form of relatively insoluble UO2 nanoparticles, suggesting that the presence of green rusts in the subsurface may have significant effects on the mobility of uranium, particularly under iron-reducing conditions.

O'Loughlin, E.J., S.D. Kelly, R.E. Cook, R. Csencsits and K.M. Kemner. 2003. Reduction of uranium(VI) by mixed Fe(II)/Fe(III) hydroxide (green rust): Formation of UO2 nanoparticles. Environ. Sci. Technol. 37: 721-727. DOI 10.1021/es0208409

Hg(II) reduction by Fe(II)—An overlooked component of mercury biogeochemical cycling?

Figure for Hg(II) reduction by Fe(II)—An overlooked component of mercury biogeochemical cycling?
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Green rusts are mixed Fe(II)/Fe(III) hydroxides that are found in many suboxic environments where they are believed to play a central role in the biogeochemical cycling of iron. Recent research has shown that green rusts are capable of reducing a number of organic and inorganic contaminants, suggesting that green rusts may be highly reactive reductants in suboxic environments. We show that green rust is an effective reductant for the reduction of Ag(I), Au(III), Cu(II), and Hg(II) to Ag(0), Au(0), Cu(0), and Hg(0). These results suggest that the presence of green rusts in suboxic soils and sediments can have a significant impact on the biogeochemistry of silver, gold, copper, and mercury, particularly with respect to their mobility. Indeed, the reduction of Hg(II) to Hg(0) by green rust indicates the potential for abiotic Hg(II) reduction in Fe(II)-rich environments and suggests that Hg(II) reduction by Fe(II) may represent an overlooked component of the biogeochemical cycling of mercury.

O'Loughlin, E.J., S.D. Kelly, K.M. Kemner, R. Cesncsits, and R.E. Cook. 2003. Reduction of AgI, AuIII, CuII, and HgII by FeII/FeIII hydroxysulfate green rust. Chemosphere 53: 437-446. DOI 10.1016/S0045-6535(03)00545-9

The role of c-type cytochromes in U(VI) reduction and accumulation in the extracellular EPS matrix

The ability of several dissimilatory Fe(III)-reducing bacteria to reduce U(VI) and produce less-soluble U(IV) species is now well established, but the mechanisms by which the enzymatic U(VI) reduction occurs are unclear. Reduction rates appear dependent on the presence or absence of certain c-type cytochromes in the cell membrane of Shewanella oneidensis MR-1. We examined both wild-type cultures and targeted gene deletion cultures lacking in certain cytochromes to determine the uranium distribution within the cells and in the intercellular space. TEM imaging showed distinct regions of uranium accumulation: A) in the periplasmic space, and B) along 100 nm thin and several micron long strings in the intercellular space, presumably associated with the extracellular polymeric substances (EPS) exuded by cells. Using synchrotron X-ray fluorescence imaging with 150 nm resolution we were able to correlate the regions of high uranium accumulation to high Fe and P content in the fibroid structures (B) but not in extracellular regions void of the structures (C), implicating heme-containing outer membrane proteins in the reduction of U(VI) in the extracellular EPS-UO2 matrix.

“c-Type Cytochrome-Dependent Formation of U(IV) Nanoparticles by Shewanella oneidensis”, M. Marshall, A. Beliaev, A. Dohnalkova, D. Kennedy, L. Shi, Z.Wang, M. Boyanov, B. Lai, K. Kemner, J. McLean, S. Reed, D. Culley, V. Bailey, C. Simonson, D. Saffarini, M. Romine, J. Zachara, J. Fredrickson , PLoS Biology 4(8), 1324-1333 (2006)


Elemental and Redox Analysis of Single Bacterial Cells by X-ray Microbeam Analysis

We have used hard x-ray fluorescence measurements, with 150 nm spatial resolution, to obtain elemental maps and quantitative chemical analyses of single planktonic and adhered cells of Pseudomonas fluorescens str. NCIMB 11764. Differences between the planktonic and adhered cells with regard to morphology, elemental composition, and their sensitivity to Cr(VI).  The ability to obtain this information at spatial scales of 150 nm on living cells (i.e. without the need for high vacuum) opens new vistas with regard to studying microorganisms under conditions relevant to their activities in natural systems. 

K.M. Kemner, et. al., Science 306, pg. 686, 2004


Reduction of U(VI) by surface sorbed Fe(II): investigation by acid-base titration and Fe/U edge XAFS spectroscopy

The reduction of aqueous U(VI) by Fe(II) is a potentially important pathway for this contaminant’s immobilization in subsurface environments. The reaction is kinetically inhibited in homogeneous solution, but occurs rapidly in the presence of surfaces. The reasons for this are unclear—possibilities include changes in the redox potential due to surface complexation, specific chemical conditions near the charged surface, or facilitated connection between redox centers through the crystal lattice. To obtain more insight we are studying the adsorption mechanisms of U(VI) and Fe(II) to an environmentally relevant surface ligand and the conditions under which reduction of U(VI) by Fe(II) is favorable. Uranium LIII edge XAFS shows only adsorbed U(VI) at pH 7.5, whereas complete reduction to U(IV) nanoparticles is observed at pH 8.4. In the absence of uranium, iron K-edge XAFS shows monomerically adsorbed Fe(II) at pH 7.5 and oligomeric Fe(II) clusters at pH 8.4. Based on these results we propose that the control on the U(VI)-Fe(II) redox reaction is the ability of a two-electron transfer to occur during a single U(VI) complexation reaction. This mechanism can also explain the commonly observed higher U(VI) reduction rate by Fe(II) in the presence of oxide surfaces.

“Adsorption of Fe(II) and U(VI) to carboxyl-functionalized microspheres: The influence of speciation on uranyl reduction studied by titration and XAFS”, M.I.Boyanov, E.J. O'Loughlin, E.E.Roden, J.B.Fein, K.M.Kemner, Geochim.Cosmochim.Acta 71, 1898-1912 (2007)


Formation of mixed valence Fe minerals inside of Shewanella putrefaciens CN32 cells and their role in bacterial respiration

The formation of intra-cellular precipitates in living orgranisms is unusual and far less common than the abundant external mineral formation during dissimilatory Fe(III) reduction (DIR). The functional role of internal precipitates is largely unknown, although a respiratory role is hypothesized. We studied the valence state of internal Fe precipitates formed during DIR inside Shewanella putrefaciens CN32 cells. The spatial distribution of elemental content within single bacterial cells was determined using synchrotron-based X-ray fluorescence microscopy with 150 nm resolution, followed by micro-XANES scans of different regions on the cell and from the extracellular precipitates. Results show that the Fe valence state of external microprecipitates is consistent with magnetite, whereas Fe associated with the cells is more reduced. Within the cell, Fe is most reduced in regions free from internal precipitates and more oxidized where the precipitates are formed. These results suggest a respiratory role of the internal precipitates. This study also demonstrates the chemical complexity of microbial environments and the need for spatially resolved spectroscopic methods to accurately investigate the processes.

“Mixed valence cytoplasmic iron granules are linked to anaerobic respiration,” S. Glasauer, S. Langley, M. Boyanov, B. Lai, K. M. Kemner, T. J. Beveridge, Appl. Environ. Microb. 73(3), 993-996 (2007)


XRF mapping of single bacterial cells of Shewanella Oneidensis and E. coli (shown below)

We have used x-ray fluorescence microscopy to investigate the spatial distribution of 3d elements in single Shewanella oneidensis cells grown with oxygen and fumarate as electron acceptors. Measurements were made on subsamples of cells taken at varying times during a 5 day growth period. Cells analyzed were either in a surface-adhered or planktonic state. The zone plate used in these microscopy experiments produced a focused beam with a cross section (and hence spatial resolution) of 0.15-0.30 micron. The samples (both planktonic and biofilm) were all grown in a consistent manner in a defined minimal salts medium.

Results from x-ray fluorescence imaging experiments indicate that the distribution of P, S, Cl, Ca, Fe, Ni, Cu, and Zn can define the location of the microbe. Additionally, quantitative elemental analysis of individual microbes identified significant changes in concentration of 3d transition elements depending on the age of the culture and the type of electron acceptor presented to the microbes.


Extended Aqueous Calcium-Uranyl-Carbonate Complex 

Current research on bioremediation of uranium-contaminated groundwater focuses on supplying indigenous metal-reducing bacteria with the appropriate metabolic requirements to induce microbiological reduction of soluble uranium(VI) to poorly soluble uranium(IV). Recent studies of uranium(VI) bioreduction in the presence of environmentally relevant levels of calcium revealed limited and slowed uranium(VI) reduction and the formation of a Ca-UO2-CO3 complex. However, the stoichiometry of the complex is poorly defined and may be complicated by the presence of a Na-UO2-CO3 complex. Such a complex might exist even at high calcium concentrations, as some UO2-CO3 complexes will still be present. The number of calcium and/or sodium atoms coordinated to a uranyl carbonate complex will determine the net charge of the complex. Such a change in aqueous speciation of uranium(VI) in calcareous groundwater may affect the fate and transport properties of uranium. In this paper, we present the results from X-ray absorption fine structure (XAFS) measurements of a series of solutions containing 50 mM uranium(VI) and 30 mM sodium bicarbonate, with various calcium concentrations of 0 to 5 mM. Use of the data series reduces the uncertainty in the number of calcium atoms bound to the UO2-CO3 complex to approximately 0.6 and enables spectroscopic identification of the Na-UO2-CO3 complex. At nearly neutral pH values, the numbers of sodium and calcium atoms bound to the uranyl triscarbonate species are found to depend on the calcium concentration, as predicted by speciation calculations.

Caption: Schematic of calcium-uranyl-carbonate species.  The uranyl (large light blue) at the center.  Surrounding the centers are oxygen (small red), carbon (small grey) atoms, and calcium (larger brown) atoms.

Brooks, S. C.; Fredrickson, J. K.; Carroll, S. L.; Kennedy, D. W.; Zachara, J. M.; Plymale, A. E.; Kelly, S. D.; Kemner, K. M.; Fendorf, S. Inhibition of bacterial U(VI) reduction by calcium. Environ. Sci. Technol. 2003, 37 (9), 1850-1858.

Kelly, S. D.; Kemner, K. M.; Brooks, S. C. X-ray absorption spectroscopy identifies calcium-uranyl-carbonate complexes at environmental concentrations. Geochimica Et Cosmochimica Acta 2007, 71 (4), 821-834.


Uranyl Incorporation in Natural Calcite

The Earth's crust contains some 4% by weight of the mineral calcite, making calcite one of the most common materials in the crust. While the mechanism was not understood, it has previously been shown that calcite (CaCO3) can incorporate hexavalent uranium (U(VI)) into its chemical composition. This leads to two important effects: first, uranium bound in a calcite could be used for geological dating; second, calcite that incorporates excess uranium -- perhaps from a contaminated site -- will keep that uranium out of the groundwater over the long term. By studying an ancient 298 million-year-old organic rich calcite (calcrete) we have for the first time shown the mineral's chemical composition around a stable uranyl -- the most common form of U(VI) -- contained therein. We believe that the uranyl environment may evolve over long time scales, becoming more calcite-like and even more stable. This is good news for those interested both in remediation and dating techniques alike.

Caption: Schematic of two calcites.  The left diagram shows calcium (small dark blue) at the center, and the right diagram has uranyl (large light blue) at the center.  Surrounding the centers are oxygen (small red) and carbon (small grey) atoms.

S.D.Kelly, M.G. Newville, L. Cheng, K.M. Kemner, S.R. Sutton, P. Fenter, N.C. Sturchio, C. SPotl, ES&T 2003:

S.D. Kelly, E.T. Rasbury, S. Chattopadhyay, A. J. Kropf, and K.M. Kemner, “Evidence of a Stable uranyl site in Ancient Organic-Rich Calcite”, Environ. Sci. Technol. 40, 2262-2268 2006.


Investigations of nanoparticles formed via biogeochemical interactions

Minerals commonly found in soils, bacteria and the extracellular material associated with them are thought to play a key role in determining an element's speciation and thus its mobility in the subsurface. In addition, the size of contaminant-associated particles has also been shown to be a factor that controls subsurface mobility via colloid transport. We have performed a number of x-ray absorption spectroscopy, x-ray microscopy, and electron microscopy studies of biogeochemical systems and have identified nanoparticle formation in a number of them. Such experiments include studies of the oxidation state and local environment of uranium exposed to (1) green rusts (GR) in an anaerobic environment, and (2) sulfate-reducing bacteria (SRB) (Desulfosporosinus sp.). An additional study includes trace metal analysis of biomineralization products created by SRBs (Desulfovibrio sp.) isolated from an abandoned Pb and Zn mine. The XANES spectra of the GR and Desulfosporosinus systems indicate that U(VI) is reduced when exposed to GR and the SRB. Additionally, EXAFS and electron microscopy studies verify that theUO2 moieties formed are nano-sized. XRF microprobe and electron microscopy studies of biomineralization products formed by Desulfovibrio sp. indicate the formation of micron-size particles consisting of randomly-packed nano-sized ZnS moieties that also contain appreciable quantities of As and Se.

Picture:  Biofilms of sulfate-reducing bacteria (blue) growing in dilute groundwater (~1 part per million dissolved zinc) associated with an abandoned lead-zinc deposit produce sphalerite (ZnS) particles that aggregate (light green) and form micrometer-diameter spheres (gold). Such biomineralization may assist in groundwater remediation and may play a role in the genesis of some ore deposits.[Scanning electron microscope image: J. F. Banfield, S. A. Welch, M. Diman, M. Labrenz]

M. Labrenz, G. K. Druschel, T. Thomsen-Ebert, B. Gilbert, S. A. Welch, K. M. Kemner, G. A. Logan, R. E. Summons, G. De Stasio, P. L. Bond, B. Lai, S.D. Kelly, J. F. Banfield, "Sphalerite (ZnS) deposits forming in natural biofilms of sulfate reducing bacteria," Science 290 1744-1747, 2000.



Adsorption of Cadmium and Uranium to B. subtilis Bacterial Cell Wall pH-Dependent XAFS Spectroscopy Study

Modeling the transport and fate of heavy metals in the environment is a central issue in environmental engineering and geochemistry. Interaction with diverse complexing media (minerals, biomass, etc.) must be considered under ambient conditions. Fluorescence XAFS was used to identify and quantify in-situ the adsorption channels of Cd to the isolated cell walls of a common groundwater bacterium, Bacillus subtilis. The results indicate that Cd binds predominantly to protonated phosphoryl ligands below pH 4.4, while at higher pH adsorption to carboxyl groups becomes increasingly important. At pH 7.8 we observe the activation of an additional binding site, which we tentatively ascribe to deprotonated phosphoryl ligands. This work is a collaboration with the groups of Prof. Bruce Bunker and Prof. Jeremy Fein from the University of Notre Dame, IN.

M.I.Boyanov, S.D.Kelly, K.M.Kemner, B.A.Bunker, J.B.Fein, D.A.Fowle. Geochim. et Cosmochim. Acta 67(18), 3299-3311 (2003):

S.D. Kelly, K. M. Kemner, J. B. Fein, D. A. Fowle, M. I. Boyanov, B. A. Bunker, N. Yee, "X-ray absorption fine-structure determination of pH dependent U-bacterial cell wall interactions", Geochem. Cosmo. Acta, 66(22) 3875-3891, Nov 2002.


Recent Collaborations

  • Dion Antonopoulos of Argonne National Laboratory, “Metagenomics-enabled understanding of the functions and activities of microbial communities”
  • Folker Meyer of Argonne National Laboratory, “Utilization of MG-RAST and bioinformatics approaches to analyze microbial community structure”
  • William Burgos of Penn State University, "Reactivity of iron-rich clays with uranium through redox transition zones"
  • Huifang Xu and Eric Roden of University of Wisconsin, Madison, "The role of nanopores on U(VI) sorption and redox behavior"  
  • Jeremy Fein, Bruce Bunker of University of Notre Dame, "X-ray absorption spectroscopy investigations of the absorption of metals to bacteria cell walls."  
  • Frank Loeffler of University of Tennessee, Rob Sanford of University of Illinois, Urbana/Champaign, Kurt Pennell of Tufts University, "MURMoT: Design and Application of Microbial Uranium Reduction Monitoring Tools"
  • Man Jae Kwon of KIST Gangnueng Environmental Research Group, "Investigations of redox transformations of iron and subsequent controls on elemental cycling"
  • Michelle Scherer of University of Iowa, "Electron and atom exchange between aqueous Fe(II) and structural Fe(III) in clays"
  • Alan Konopka and Vanessa Bailey of Pacific Northwest National Laboratory, "Micrometer-scale physical structure and microbial composition of soil macroaggregates"
  • John Zachara, Kevin Rosso, and Jim Fredrickson of Pacific Northwest National Laboratory - Role of microenvironments and transition zones in subsurface reactive transport
  • Terry Marsh of Michigan State University, "Metagenomics-enabled understanding of the functions and activities of microbial communities"
  • Mike McCormick of Hamilton College, "Electron shuttle analysis with EC-LC/MS"
  • Scott C. Brooks, Dave Watson, and Baohua Gu of Oak Ridge National Laboratory "Molecular scale transformations in field samples"


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