Elsevier

Geochimica et Cosmochimica Acta

Volume 131, 15 April 2014, Pages 115-127
Geochimica et Cosmochimica Acta

The product of microbial uranium reduction includes multiple species with U(IV)–phosphate coordination

https://doi.org/10.1016/j.gca.2014.01.005Get rights and content

Abstract

Until recently, the reduction of U(VI) to U(IV) during bioremediation was assumed to produce solely the sparingly soluble mineral uraninite, UO2(s). However, results from several laboratories reveal other species of U(IV) characterized by the absence of an EXAFS U–U pair correlation (referred to here as noncrystalline U(IV)). Because it lacks the crystalline structure of uraninite, this species is likely to be more labile and susceptible to reoxidation. In the case of single species cultures, analyses of U extended X-ray fine structure (EXAFS) spectra have previously suggested U(IV) coordination to carboxyl, phosphoryl or carbonate groups. In spite of this evidence, little is understood about the species that make up noncrystalline U(IV), their structural chemistry and the nature of the U(IV)–ligand interactions. Here, we use infrared spectroscopy (IR), uranium LIII-edge X-ray absorption spectroscopy (XAS), and phosphorus K-edge XAS analyses to constrain the binding environments of phosphate and uranium associated with Shewanella oneidensis MR-1 bacterial cells. Systems tested as a function of pH included: cells under metal-reducing conditions without uranium, cells under reducing conditions that produced primarily uraninite, and cells under reducing conditions that produced primarily biomass-associated noncrystalline U(IV). P X-ray absorption near-edge structure (XANES) results provided clear and direct evidence of U(IV) coordination to phosphate. Infrared (IR) spectroscopy revealed a pronounced perturbation of phosphate functional groups in the presence of uranium. Analysis of these data provides evidence that U(IV) is coordinated to a range of phosphate species, including monomers and polymerized networks. U EXAFS analyses and a chemical extraction measurements support these conclusions. The results of this study provide new insights into the binding mechanisms of biomass-associated U(IV) species which in turn sheds light on the mechanisms of biological U(VI) reduction.

Introduction

Uranium contamination in the subsurface remains problematic in areas of active or historic uranium mining, milling or processing. One promising strategy for the remediation of uranium aims at transforming the soluble and mobile hexavalent form of uranium, U(VI), to the reduced and relatively immobile tetravalent form, U(IV) (O’Loughlin et al., 2003, Jeon et al., 2005, Wall and Krumholz, 2006, Burgos et al., 2008, Sheng et al., 2011, Zhang et al., 2011). Until recently, reduction of U(VI) to U(IV) was only found to produce the sparingly soluble mineral uraninite, UO2(s) (Lovley et al., 1991, Lovley and Phillips, 1992, Lovley, 1993, Burns, 1999, O’Loughlin et al., 2003, Wall and Krumholz, 2006, Burgos et al., 2008). However, recent research reveals that non-uraninite species of U(IV), i.e., those lacking the 3.85 Å U–U pair correlation characteristic of UO2 observed using X-ray absorption spectroscopy (XAS), can form as the product of U(VI) reduction by Gram-negative and Gram-positive bacteria (Bernier-Latmani et al., 2010, Fletcher et al., 2010, Boyanov et al., 2011, Cologgi et al., 2011, Ray et al., 2011, Sivaswamy et al., 2011), by biogenic Fe(II)-bearing minerals (Veeramani et al., 2011, Veeramani et al., 2013, Latta et al., 2012), and in biostimulated or naturally reduced sediments (Campbell et al., 2011, Sharp et al., 2011). It is unknown if these U(IV) species occurs as amorphous solids or coordination polymers, as complexes sorbed to biomass functional groups, or as a mixture of the above. Because of this ambiguity, we will henceforth refer to this species as noncrystalline U(IV). Its lack of crystalline structure and its susceptibility to complexation by bicarbonate (Alessi et al., 2012), makes it more labile and prone to reoxidation than U(IV) bound in uraninite (Alessi et al., 2013, Cerrato et al., 2013). For this reason, geochemical models that assume uraninite is the sole product of U(VI) reduction may be critically flawed.

The microbial reduction of U(VI) to U(IV) produces mixtures of nanoparticulate uraninite and noncrystalline U(IV) species associated with bacterial biomass (e.g., Senko et al., 2007, Bernier-Latmani et al., 2010, Boyanov et al., 2011). The presence and relative abundances of U(VI), UO2(s), and noncrystalline U(IV) species are typically estimated using U LIII-edge EXAFS data. The presence of a U–U pair correlation in these data at 3.85 Å is indicative (in our system) of the presence of uraninite (O’Loughlin et al., 2003, Schofield et al., 2008, Boyanov et al., 2011). In XAS spectra obtained from laboratory pure cultures (Bernier-Latmani et al., 2010) and natural sediments biostimulated with an electron donor (Sharp et al., 2011), there is permissive evidence that noncrystalline U(IV) is at least in part associated with phosphate groups on microbial biomass. There is also evidence that the presence of inorganic phosphate in the aqueous medium during microbial U(VI) reduction significantly increases the fraction of noncrystalline U(IV) produced (Bernier-Latmani et al., 2010, Boyanov et al., 2011). Boyanov et al. (2011) tested the reduction of carbonate-complexed U(VI) by a variety of Gram-positive and Gram-negative bacteria in the presence and absence of solution phosphate (290 μM as KH2PO4). The authors found that, regardless of bacterial species, a noncrystalline U(IV)–phosphate species associated with the solid phase was formed in the presence of solution phosphate. In the absence of phosphate, nano-uraninite was produced by Shewanella oneidensis MR-1 and Anaeromyxobacter dehalogenans 2CP-C, while a mixture of nanoparticulate uraninite and U(IV)–carbonate complexes was formed by Desulfitobacterium spp. A recent systematic study of the influence of solution chemistry on the nature of U(VI) reduction products shows that the presence of orthophosphate in the medium is not a prerequisite for noncrystalline U(IV) formation, thus indirectly suggesting that phosphate presumed to bind U(IV) may be of biological origin (Stylo et al., 2013). Previous results indicate that the formation of noncrystalline U(IV) versus uraninite depended strongly on the chemical environment in which S. oneidensis was carrying out U(VI) reduction (Bernier-Latmani et al., 2010). Hence, it is possible to modulate the formation of uraninite versus noncrystalline U(IV) based on solution composition.

Shell-by shell fitting of U LIII-edge EXAFS data has suggested the association of noncrystalline U(IV) with phosphoryl moieties on biomass or mineral surfaces (e.g., Senko et al., 2007, Bernier-Latmani et al., 2010, Chakraborty et al., 2010, Veeramani et al., 2011). However, the inability to unambiguously distinguish between U–P and U–C coordination pairs in the fitting and the potential speciation complexity (i.e., the presence of several species) of what is referred to as noncrystalline U(IV) call for a more rigorous evaluation of this species. Bargar et al. (2013) recently proposed that noncrystalline U(IV) species found in biostimulated sediments may be comprised of a spectrum of species ranging from true monomers to phosphate coordination polymers to which U(IV) is bound. To our knowledge no experimental evidence of these U(IV)–P associations in biomass is extant outside of inferences from EXAFS fits (see above). Crucially, the degree of polymerization of phosphate, if any, cannot be determined using U EXAFS. To address these questions, it is necessary to use spectroscopic techniques such as FTIR or P XAS that directly probe the coordination environment around phosphate. Premised on these studies and the hypothesis that a large fraction of noncrystalline U(IV) is bound to microbial phosphate functional groups, we investigated the coordination environment of noncrystalline U(IV) species associated with S. oneidensis MR-1 bacterial cells. To provide a holistic structural picture of the U(IV) complexes, we used techniques capable of directly characterizing the local structure of both the functional groups (FTIR and P XAS) and U(IV) (U LIII-edge EXAFS). Anoxic cultures containing biogenic uraninite, noncrystalline U(IV), or no uranium were produced as a function of pH and compared to standard materials using four complementary techniques. U LIII edge EXAFS and a bicarbonate chemical extraction method (Alessi et al., 2012) were used to verify the reduction of U(VI) and the production of noncrystalline U(IV) or biogenic uraninite. Phosphorus K-edge XAS analyses provided unambiguous evidence that noncrystalline U(IV) species are associated with organic P functional groups. Infrared spectroscopy (IR) allowed for more in-depth probing of the types of functional groups with which noncrystalline U(IV) is associated. Shell-by-shell fitting of the U EXAFS, constrained by the FTIR and P XAS results, offer further glimpses into the nature of U(IV) coordination. Our results show conclusively for the first time that noncrystalline U(IV) is coordinated to biomass phosphorus functional groups, as inorganic U(IV) phosphate species, and likely in the framework of phosphate coordination polymers.

Section snippets

Media and cultures

S. oneidensis MR-1 was cultured, grown in Luria Bertani (LB) medium, and processed as described previously (Bernier-Latmani et al., 2010). All reagents used in the study were of analytical grade or higher, and ultrapure water (resistivity 18.2 MΩ cm) was used in preparing all solutions. All components of the growth media were sterilized by autoclaving prior to use.

Uranium reduction

Bacterial uranium reduction experiments were conducted in an anoxic chamber (Coy Laboratory Products, Grass Lake, Michigan)

Uranium reduction products, and their quantification with bicarbonate extraction

To identify the conditions under which disordered noncrystalline U(IV) species, and nanoparticulate uraninite are formed by microbial reduction, U(VI) reduction was conducted in two chemical media – WLP or BP – to favor the formation of biomass-associated noncrystalline U(IV) species or uraninite, respectively. Notably, U(VI) reduction in either of these reduction media does not lead to the production of samples containing either 100% uraninite or 100% noncrystalline U(IV). The contribution of

Conclusions

It is now established that, in addition to crystalline uraninite, noncrystalline U(IV) species may form as the product of microbial U(VI) reduction in aquifers at uranium contaminated field sites (Campbell et al., 2011, Bargar et al., 2013). Due to the relatively higher lability of noncrystalline U(IV) (Alessi et al., 2012), unraveling its structure is critical to providing some understanding of conditions promoting its formation. Previous studies have established that the ratio of uraninite to

Acknowledgments

Work carried out at EPFL was funded by Swiss NSF grants No. 200021-113784 and 200020-126821, SNSF International Co-operation grant No. IZK0Z2-12355, SNSF International Short Visits grant No. IZK0Z2-133214 and the SLAC Science Focus Area (work package 10094) funded by the USDOE Office of Biological and Environmental Research, Subsurface Biogeochemical Research program. DSA was partially supported by a Marie Curie International Incoming Fellowship from the European Commission, grant

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  • Cited by (0)

    1

    Current address: Department of Earth and Atmospheric Sciences, University of Alberta, Edmonton, AB T5J 4B5, Canada.

    2

    Current address: Center for Advanced Radiation Sources, University of Chicago, Chicago, IL 60637, USA.

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