Identification of fluorescent U(V) and U(VI) microparticles in a multispecies biofilm by confocal laser scanning microscopy and fluorescence spectroscopy

Fluorescent uranium(V) and uranium(VI) particles were observed for the first time in vivo by a combined laser fluorescence spectroscopy and confocal laser scanning microscopy approach in a living multispecies biofilm grown on biotite plates. These particles ranged between 1 and 7 um in width and up to 20 microm in length and were located at the bottom and at the edges of biofilms colonies. Analysis of amplified 16S rRNA fragments and fluorescence in situ hybridization were used to characterize the biofilm communities. Laser fluorescence spectroscopy was used to identify these particles. The particles showed either a characteristic fluorescence spectrum in the wavelength range of 415-475 nm, indicative for uranium(V), or in the range of 480-560 nm, which is typical for uranium(VI). Particles of uranium(V) as well as uranium(VI) were simultaneously observed in the biofilms. These uranium particles were attributed for uranium(VI) to biologically mediated precipitation and for uranium(V) to redox processes taking place within the biofilm. The detection of uranium(V) in a multispecies biofilm was interpreted as a short-lived intermediate of the uranium(VI) to uranium(IV) redox reaction. Its presence clearly documents that the uranium(VI) reduction is not a two electron step but that only one electron is involved.     

Cryogenic laser induced U(VI) fluorescence studies of a U(VI) substituted natural calcite: implications to U(VI) speciation in contaminated Hanford sediments

Time-resolved laser-induced fluorescence spectroscopy (TRLFS) and imaging spectromicroscopy (TRLFISM) were used to examine the chemical speciation of uranyl in contaminated subsurface sediments from the U.S. Department of Energy (U.S. DOE) Hanford Site, Washington. Spectroscopic measurements for contaminant U(VI) were compared to those from a natural, uranyl-bearing calcite (NUC) that had been found via X-ray absorption spectroscopy (XAS) to include uranyl in the same coordination environment as calcium. Spectral deconvolution of TRLFS measurements on the NUC revealed the unexpected presence of two distinct chemical environments consistent with published spectra of U(VI)-substituted synthetic calcite and aragonite. Apparently, some U(VI) substitution sites in calcite distorted to exhibit a local, more energetically favorable aragonite structure. TRLFS measurements of the Hanford sediments NP4-1 and NP1-6 were similar to the NUC in terms of peak positions and intensity, despite a small CaCO3 content (1.0 to 3.2 mass %). Spectral deconvolution of the sediments revealed the presence of U(VI) in calcite and aragonite structural environments. A third, unidentified U(VI) species was also present in the NP1-6 sediment. TRLFISM measurements at multiple locations in the different sediments displayed only minor variation, indicating a uniform speciation pattern. Collectively, the measurements implied that waste U(VI), long-resident beneath the sampled disposal pond (32 y), had coprecipitated within carbonates. These findings have major implications for the solubility and fate of contaminant U(VI).     

Cryogenic laser induced fluorescence characterization of U(VI) in Hanford Vadose Zone pore waters

Ambient and liquid helium temperature laser-induced time-resolved uranyl fluorescence spectroscopy was applied to study the speciation of aqueous uranyl solutions containing carbonate and phosphate and two porewater samples obtained by ultracentrifugation of U(VI)-contaminated sediments. The significantly enhanced fluorescence signal intensity and spectral resolution found at liquid helium temperature allowed, for the first time, direct fluorescence spectroscopic observation of the higher aqueous uranyl complexes with carbonate: UO2(CO3)2(2-), UO2(CO3)3(4-), and (UO2)2(OH)3CO3-. The porewater samples were nonfluorescent at room temperature. However, at liquid helium temperature, both porewater samples displayed strong, well-resolved fluorescence spectra. Comparisons of the spectroscopic characteristics of the porewaters with those of the standard uranyl-carbonate complexes confirmed that U(VI) in the porewaters existed primarily as UO2(CO3)3(4-) along with a small amount of other minor components, such as dicalcium-urano-tricarbonate complex, Ca2UO2(CO3)3, consistent with thermodynamic calculation. The U(VI)-carbonate complex is apparently the mobile species responsible for the subsurface migration of U(VI), even though the majority of the in-ground U(VI) inventory at the site from which the samples were obtained exists as intragrain U(VI)-silicate precipitates.     

Inhibition of a U(VI)- and sulfate-reducing consortia by U(VI)

The stimulation of microbial U(VI) reduction is currently being investigated as a means to reduce uranium's mobility in groundwater, but little is known about the concentration at which U(VI) might inhibit microbial activity, or the effect of U(VI) on bacterial community structure. We investigated these questions with an ethanol-fed U(VI)- and sulfate-reducing enrichment developed from sediment from the site of an ongoing field biostimulation experiment at Area 3 of the Oak Ridge Field Research Center (FRC). Sets of triplicate enrichments were spiked with increasing concentrations of U(VI) (from 49 microm to 9.2 mM). As the U(VI) concentration increased to 224 microM, the culture's production of acetate from ethanol slowed, and at or above 1.6 mM U(VI) little acetate was produced over the time frame of the experiment. An uncoupling inhibition model was applied to the data, and the inhibition coefficient for U(VI), Ku, was found to be approximately 100 microM U(VI), or 24 mg/L, indicating the inhibitory effect is relevant at highly contaminated sites. Microbial community structure at the conclusion of the experiment was analyzed with terminal restriction fragment length polymorphism (T-RFLP) analysis. T-RFs associated with Desulfovibrio-like organisms decreased in relative abundance with increasing U(VI) concentration, whereas Clostridia-like T-RFs increased.     

Structural characterization of U(VI) in apatite by X-ray absorption spectroscopy

X-ray absorption spectroscopy was used to determine the local structure of U(VI) within synthetic fluorapatite at a concentration of 2.3 wt %. Extended X-ray absorption fine structure indicates that U(VI) substitutes into the Ca1 site. To accommodate this substitution the apatite structure significantly distorts such that the Ca1 site approximates octahedral coordination, with six uniform U-0 distances of 2.06A. An X-ray adsorption edge structure, with two inflection points, and optical emission spectra are consistent with 6d orbital crystal field splitting. These results indicate that significant amounts of U(VI) can be accommodated in the apatite structure but with an unexpected coordination, which may bear on the ultimate development of apatite-hosted nuclear-waste forms.     

U(VI) sorption and reduction kinetics on the magnetite (111) surface

Sorption of contaminants onto mineral surfaces is an important process that can restrict their transport in the environment. In the current study, uranium (U) uptake on magnetite (111) was measured as a function of time and solution composition (pH, [CO(3)](T), [Ca]) under continuous batch-flow conditions. We observed, in real-time and in situ, adsorption and reduction of U(VI) and subsequent growth of UO(2) nanoprecipitates using atomic force microscopy (AFM) and newly developed batch-flow U L(III)-edge grazing-incidence X-ray absorption spectroscopy near-edge structure (GI-XANES) spectroscopy. U(VI) reduction occurred with and without CO(3) present, and coincided with nucleation and growth of UO(2) particles. When Ca and CO(3) were both present no U(VI) reduction occurred and the U surface loading was lower. In situ batch-flow AFM data indicated that UO(2) particles achieved a maximum height of 4-5 nm after about 8 h of exposure, however, aggregates continued to grow laterally after 8 h reaching up to about 300 nm in diameter. The combination of techniques indicated that U uptake is divided into three-stages; (1) initial adsorption of U(VI), (2) reduction of U(VI) to UO(2) nanoprecipitates at surface-specific sites after 2-3 h of exposure, and (3) completion of U(VI) reduction after ~6-8 h. U(VI) reduction also corresponded to detectable increases in Fe released to solution and surface topography changes. Redox reactions are proposed that explicitly couple the reduction of U(VI) to enhanced release of Fe(II) from magnetite. Although counterintuitive, the proposed reaction stoichiometry was shown to be largely consistent with the experimental results. In addition to providing molecular-scale details about U sorption on magnetite, this work also presents novel advances for collecting surface sensitive molecular-scale information in real-time under batch-flow conditions.     

Identification of simultaneous U(VI) sorption complexes and U(IV) nanoprecipitates on the magnetite (111) surface

Sequestration of uranium (U) by magnetite is a potentially important sink for U in natural and contaminated environments. However, molecular-scale controls that favor U(VI) uptake including both adsorption of U(VI) and reduction to U(IV) by magnetite remain poorly understood, in particular, the role of U(VI)-CO(3)-Ca complexes in inhibiting U(VI) reduction. To investigate U uptake pathways on magnetite as a function of U(VI) aqueous speciation, we performed batch sorption experiments on (111) surfaces of natural single crystals under a range of solution conditions (pH 5 and 10; 0.1 mM U(VI); 1 mM NaNO(3); and with or without 0.5 mM CO(3) and 0.1 mM Ca) and characterized surface-associated U using grazing incidence extended X-ray absorption fine structure spectroscopy (GI-EXAFS), grazing incidence X-ray diffraction (GI-XRD), and scanning electron microscopy (SEM). In the absence of both carbonate ([CO(3)](T), denoted here as CO(3)) and calcium (Ca), or in the presence of CO(3) only, coexisting adsorption of U(VI) surface species and reduction to U(IV) occurs at both pH 5 and 10. In the presence of both Ca and CO(3), only U(VI) adsorption (VI) occurs. When U reduction occurs, nanoparticulate UO(2) forms only within and adjacent to surface microtopographic features such as crystal boundaries and cracks. This result suggests that U reduction is limited to defect-rich surface regions. Further, at both pH 5 and 10 in the presence of both CO(3) and Ca, U(VI)-CO(3)-Ca ternary surface species develop and U reduction is inhibited. These findings extend the range of conditions under which U(VI)-CO(3)-Ca complexes inhibit U reduction.     

Investigation of bacterial spore structure by high resolution solid-state nuclear magnetic resonance spectroscopy and transmission electron microscopy

High resolution solid-state nuclear magnetic resonance spectroscopy (NMR) in combination with transmission electron microscopy (TEM) of spores of Bacillus cereus, an outer coatless mutant B. subtilis 322, an inner coatless mutant B. subtilis 325 and of germinated spores of B. subtilis CMCC 604 were carried out. Structural differences in the coats, mainly protein of spores were reflected by NMR spectra which indicated also differences in molecular mobility of carbohydrates which was partially attributed to the cortex. Dipicolinic acid (DPA) of spores of B. cereus displayed a high degree of solid state order and may be crystalline. Heat activation was studied on spores of B. subtilis 357 lux + and revealed a structural change when analysed by TEM but this was not associated with increases in molecular mobility since no effects were measured by NMR.     

U(VI)-kaolinite surface complexation in absence and presence of humic acid studied by TRLFS

Time-resolved laser-induced fluorescence spectroscopy (TRLFS) was applied to study the U(VI) surface complexes on kaolinite in the presence and absence of humic acid (HA). Two uranyl surface species with fluorescence lifetimes of 5.9 +/- 1.4 and 42.5 +/- 3.4 micros and 4.4 +/- 1.2 and 30.9 +/- 7.2 micros were identified in the binary (U(VI)-kaolinite) and ternary system (U(VI)-HA-kaolinite), respectively. The fluorescence spectra of adsorbed uranyl surface species are described with six and five fluorescence emission bands in the binary and ternary system, respectively. The positions of peak maxima are shifted significantly to higher wavelengths compared to the free uranyl ion in perchlorate medium. HA has no influence on positions of the fluorescence emission bands. In the binary system, both surface species can be attributed to adsorbed bidentate mononuclear surface complexes, which differ in the number of water molecules in their coordination environment. In the ternary system, U(VI) prefers direct binding on kaolinite rather than via HA, but it is sorbed as a uranyl-humate complex. Consequently, the hydration shell of the U(VI) surface complexes is displaced with complexed HA, which is simultaneously distributed between kaolinite particles. Aluminol binding sites are assumed to control the sorption of U(VI) onto kaolinite.     

Influence of magnetite stoichiometry on U(VI) reduction

Hexavalent uranium (U(VI)) can be reduced enzymatically by various microbes and abiotically by Fe(2+)-bearing minerals, including magnetite, of interest because of its formation from Fe(3+) (oxy)hydroxides via dissimilatory iron reduction. Magnetite is also a corrosion product of iron metal in suboxic and anoxic conditions and is likely to form during corrosion of steel waste containers holding uranium-containing spent nuclear fuel. Previous work indicated discrepancies in the extent of U(VI) reduction by magnetite. Here, we demonstrate that the stoichiometry (the bulk Fe(2+)/Fe(3+) ratio, x) of magnetite can, in part, explain the observed discrepancies. In our studies, magnetite stoichiometry significantly influenced the extent of U(VI) reduction by magnetite. Stoichiometric and partially oxidized magnetites with x ≥ 0.38 reduced U(VI) to U(IV) in UO(2) (uraninite) nanoparticles, whereas with more oxidized magnetites (x < 0.38) and maghemite (x = 0), sorbed U(VI) was the dominant phase observed. Furthermore, as with our chemically synthesized magnetites (x ≥ 0.38), nanoparticulate UO(2) was formed from reduction of U(VI) in a heat-killed suspension of biogenic magnetite (x = 0.43). X-ray absorption and M?ssbauer spectroscopy results indicate that reduction of U(VI) to U(IV) is coupled to oxidation of Fe(2+) in magnetite. The addition of aqueous Fe(2+) to suspensions of oxidized magnetite resulted in reduction of U(VI) to UO(2), consistent with our previous finding that Fe(2+) taken up from solution increased the magnetite stoichiometry. Our results suggest that magnetite stoichiometry and the ability of aqueous Fe(2+) to recharge magnetite are important factors in reduction of U(VI) in the subsurface.     

Role of surface alteration in determining the mobility of U(VI) in the presence of citrate: implications for extraction of U(VI) from soils

In the present study, the adsorption of U(VI) by a natural iron-rich sand in the presence of citrate was studied over a range of citrate concentrations and pH values. Adsorption of U(VI) on the iron-rich sand decreased in the presence of increasing concentrations of citrate. Adsorption of citrate to the sand was weak under most conditions studied. Several explanations for the adsorption behavior of U(VI) and citrate were investigated, including aqueous complexation of U(VI) by citrate, competition of U(VI) and citrate for adsorption sites, and extraction of Fe and Al from the sorbent surface by citrate (surface alteration). Although aqueous complexation of U(VI) by citrate may still play a significant role, both competitive adsorption and aqueous complexation proved to be inadequate explanations of the adsorption behavior. Both physical surface alteration (i.e., loss of surface area) and chemical surface alteration (i.e., change in the chemical composition of the sand surface) were investigated, with chemical surface alteration controlling the bulk of U(VI) adsorption. Considering these results, remediation schemes that involve organic complexing agents should address the possibility of surface alteration affecting radionuclide adsorption and mobility.     

Europium uptake and partitioning in oat (Avena sativa) roots as studied by laser-induced fluorescence spectroscopy and confocal microscopy profiling technique

The uptake of Eu3+ by elongating oat roots was studied by fluorescence spectroscopy, fluorescence lifetime measurement, and a laser excitation time-resolved confocal fluorescence profiling technique. The results of this work indicated that initial uptake of Eu3+ was highest within the undifferentiated cells of the root tip just behind the root cap, a region of maximal cell growth and differentiation and with incomplete formation of the Casparian strip around the central vascular cylinder. Distribution of assimilated Eu3+ within the root's differentiation and elongation zone was nonuniform. Higher concentrations of Eu3+ were observed within the vascular cylinder, specifically in the phloem and developing xylem parenchyma. Elevated levels of the metal were also observed in the root hairs of the mature root zone. Fluorescence spectroscopic characteristics of the assimilated Eu3+ suggested that the Eu3+ exists as inner-sphere mononuclear complexes inside the root. This work also demonstrated the effectiveness of a time-resolved Eu3+ fluorescence spectroscopy and confocal fluorescence profiling techniques for the in vivo, real-time study of metal [Eu3+] accumulation by a functioning intact plant root. This approach can prove valuable for basic and applied studies in plant nutrition and environmental uptake of actinide radionuclides.     

Sorption and desorption of perchlorate and U(VI) by strong-base anion-exchange resins

This study investigated the sorption affinity and capacity of six strong-base anion-exchange (SBA) resins for both uranium [U(VI)] and perchlorate (ClO4-) in simulated groundwater containing varying concentrations of sulfate (SO4(2-)). Additionally, desorption of U(VI) from spent resins was studied to separate U(VI) from resins with sorbed ClO4- for waste segregation and minimization. Results indicate that all SBA resins investigated in this study strongly sorb U(VI). The gel-type polyacrylic resin (Purolite A850) showed the highest sorption affinity and capacityfor U(VI) butwasthe least effective in sorbing ClO4-. The presence of SO4(2-) had little impact on the sorption of U(VI) but significantly affected the sorption of ClO4-, particularly on monofunctional SBA resins. A dilute acid wash was found to be effective in desorbing U(VI) but ineffective in desorbing ClO4- from bifunctional resins (Purolite A530E and WBR109). A single wash removed approximately 75% of sorbed U(VI) but only approximately 0.1% of sorbed ClO4- from the bifunctional resins. On the other hand, only 21.4% of sorbed U(VI) but approximately 34% of sorbed ClO4- was desorbed from the Purolite A850 resin. This study concludes that bifunctional resins could be used effectively to treatwater contaminated with ClO4- and traces of U(VI), and dilute acid washes could minimize hazardous wastes by separating sorbed U(VI) from ClO4- prior to the regeneration of the spent resin loaded with ClO4-.     

Uptake of Cm(III) and Eu(III) by calcium silicate hydrates: a solution chemistry and time-resolved laser fluorescence spectroscopy study

The interaction of the two chemical homologues [Cm(III) and Eu(III)] with calcium silicate hydrates (CSH phases) at pH 13.3 has been investigated in batch-type sorption studies using Eu(III) and complemented with time-resolved laser fluorescence spectroscopy (TRLFS) using Cm(III). The sorption data for Eu(III) reveal fast sorption kinetics and a strong uptake by CSH phases with distribution ratios of (6 +/- 3) x 10(5) L kg(-1). Three different Cm(III) species have been identified: A nonfluorescing species, which was identified as a curium hydroxide (surface) precipitate, and two fluorescing Cm(III)/CSH-sorbed species. The fluorescing sorbed species have characteristic emission spectra with main peak maxima at 618.9 and 620.9 nm and fluorescence emission lifetimes of 289 +/- 11 and 1482 +/- 200 micros, respectively. From the fluorescence lifetimes, it was calculated that the two fluorescing Cm(III) species have one or two and no water molecules left in their first coordination sphere, suggesting that these species are incorporated into the CSH structure. A structural model for Cm(III) and Eu(III) incorporation into CSH phases is proposed based on the substitution for Ca at two different types of sites in the CSH structure.     

U(VI) adsorption to heterogeneous subsurface media: application of a surface complexation model

The pH-dependent adsorption of U(VI) onto three heterogeneous, subsurface media from the Department of Energy (DOE) Oak Ridge Reservation, Savannah River Site, and Hanford Reservation was investigated. The three materials contained significant quantities of iron and manganese oxides with nearly identical extractable iron oxide contents (25.3-25.8 g/kg). A model independently developed for the adsorption of U(VI) to synthetic ferrihydrite (Waite, T. D.; Davis, J. A.; Payne, T. E.; Waychunas, G. A.; Xu, N. Geochim. Cosmochim. Acta 1994, 58, 5465-5478) was able to predict the major features of the pH-dependent U(VI) adsorption to the materials under the assumption that all the dithionite-citrate-bicarbonate extractable iron oxide was present as ferrihydrite. Further experiments with the Oak Ridge soil as a function of carbonate and U(VI) concentration indicated that the model could predict pH-dependent U(VI) adsorption to within a root mean square error of 0.163-0.408, even under conditions outside of those for which the model was developed. These results indicate that this model could be used as a first approximation in predicting U(VI) adsorption and transport in the subsurface. U(VI) adsorption also decreased at pH >10, even in the absence of carbonate, which is of potential importance to U(VI) mobility in extreme environments present in the subsurface at some DOE facilities. The pH-dependent adsorption of U(VI) was fundamentally different in systems with a constant CO2 partial pressure as compared to a constant total carbonate concentration. Experiments at constant CO2 partial pressure may not be representative of the conditions present in the subsurface, and a constant carbonate concentration does not always result in decreased U(VI) adsorption at higher pH values.     

Elucidation of protein aggregation in frozen cod and haddock by transmission electron microscopy/immunocytochemistry,light microscopy and atomic force microscopy

Polyclonal antibodies raised to both native cod myosin and actin as well as to aggregated proteins obtained from frozen cod stored for 11 months at ?10 °C were used to investigate disposition of muscle proteins in frozen cod and haddock fillets by transmission electron microscopy. Specimens from cod and haddock fillets, stored at ?10 °C, treated with anti‐aggregate antibody as the primary antibody, showed significantly more gold particles, especially around the protein aggregates and muscle fibres compared with fish stored at ?30 °C. Samples that were treated with anti‐myosin or anti‐actin antibody showed opposite results. Similar binding properties were observed in ELISA experiments involving the reaction of actin and myosin to both native and aggregate antibodies; thus immunological tests can be used for monitoring aggregate and texture changes in frozen stored fish. In addition, atomic force microscopy images obtained from cod muscle also indicated structural changes in frozen cod muscle proteins. The mica surface was covered with a continuous layer of muscle proteins comprising mainly small globular particles and a few large particles for the control cod sample stored at ?30 °C for 11 months. In contrast, cod fillets stored at ?10 °C showed a thin layer of proteins with small holes and an increased number of large particles denoting aggregates. Formation of ice crystals between the muscle fibres of frozen cod and haddock muscle was monitored without thawing by light microscopy at ?20 °C. The micrographs showed a greater proportion of large ice crystals and extensive protein fibre changes in fillets stored at ?10 °C compared with the control at ?30 °C. Copyright © 2004 Society of Chemical Industry     

Non-destructive evaluation of ATP content and plate count on pork meat surface by fluorescence spectroscopy

The potential of fluorescence spectroscopy was investigated for the non-destructive evaluation of ATP content and plate count on pork meat surface stored aerobically at 15 °C during three days. Excitation (Ex) Emission (Em) Matrix of fluorescence intensity was obtained and fluorescence from tryptophan (Ex = 295 nm and Em = 335 nm) and NADPH (Ex = 335 nm and Em = 450 nm) was detected. Because tryptophan and NADPH fluorescence changed along with the growth of microorganisms, microbial spoilage on meat could be detected from fluorescence. By applying PLSR (Partial Least Squares Regression) analysis, ATP content and plate count were predicted with good determination coefficient (0.94–0.97 in calibration and 0.84–0.88 in validation).