Dean A. Moore

Role of outer‐membrane cytochromes MtrC and OmcA in the biomineralization of ferrihydrite by Shewanella oneidensis MR‐1

In an effort to improve the understanding of electron transfer mechanisms at the microbe-mineral interface, Shewanella oneidensis MR-1 mutants with in-frame deletions of outer-membrane cytochromes (OMCs), MtrC and OmcA, were characterized for the ability to reduce ferrihydrite (FH) using a suite of microscopic, spectroscopic, and biochemical techniques. Analysis of purified recombinant proteins demonstrated that both cytochromes undergo rapid electron exchange with FH in vitro with MtrC displaying faster transfer rates than OmcA. Immunomicroscopy with cytochrome-specific antibodies revealed that MtrC co-localizes with iron solids on the cell surface while OmcA exhibits a more diffuse distribution over the cell surface. After 3-day incubation of MR-1 with FH, pronounced reductive transformation mineral products were visible by electron microscopy. Upon further incubation, the predominant phases identified were ferrous phosphates including vivianite [Fe(3)(PO(4))(2)x8H(2)O] and a switzerite-like phase [Mn(3),Fe(3)(PO(4))(2)x7H(2)O] that were heavily colonized by MR-1 cells with surface-exposed outer-membrane cytochromes. In the absence of both MtrC and OmcA, the cells ability to reduce FH was significantly hindered and no mineral transformation products were detected. Collectively, these results highlight the importance of the outer-membrane cytochromes in the reductive transformation of FH and support a role for direct electron transfer from the OMCs at the cell surface to the mineral.

Redox Reactions of Reduced Flavin Mononucleotide (FMN), Riboflavin (RBF), and Anthraquinone-2,6-disulfonate (AQDS) with Ferrihydrite and Lepidocrocite

Flavins are secreted by the dissimilatory iron-reducing bacterium Shewanella and can function as endogenous electron transfer mediators. To assess the potential importance of flavins in Fe(III) bioreduction, we investigated the redox reaction kinetics of reduced flavin mononucleotide (i.e., FMNH(2)) and reduced riboflavin (i.e., RBFH(2)) with ferrihydrite and lepidocrocite. The organic reductants rapidly reduced and dissolved ferrihydrite and lepidocrocite in the pH range 4-8. The rate constant k for 2-line ferrihydrite reductive dissolution by FMNH(2) was 87.5 ± 3.5 M(-1)·s(-1) at pH 7.0 in batch reactors, and k was similar for RBFH(2). For lepidocrocite, k was 500 ± 61 M(-1)·s(-1) for FMNH(2) and 236 ± 22 M(-1)·s(-1) for RBFH(2). The surface area normalized initial reaction rates (r(a)) were between 0.08 and 77 μmol·m(-2)·s(-1) for various conditions in stopped-flow experiments. Initial rates (r(o)) were first-order with respect to iron(III) oxide concentration, and r(a) increased with decreasing pH. Poorly crystalline 2-line ferrihydrite yielded the highest r(a), followed by more crystalline 6-line ferrihydrite and crystalline lepidocrocite. Compared to a previous whole-cell study with Shewanella oneidensis strain MR-1, our findings suggest that the reduction of electron transfer mediators by the Mtr (i.e., metal-reducing) pathway coupled to lactate oxidation is rate limiting, rather than heterogeneous electron transfer to the iron(III) oxide.

Thermodynamic model for the solubility of thorium dioxide in the Na+-Cl--OH--H2O system at 23°C and 90°C

Data are extremely limited on the effects of temperature on crystallinity and the resulting changes in solubility products of thermally transformed thorium oxide phases. Such data are required to reliably predict thorium behavior in high-level waste repositories where higher than ambient temperatures are expected. Solubility studies were conducted as a function of pH and time and at 0.1 M NaCl for 1) ThO2(am) at 23°C, 2) ThO2 (am→c), a thermally transformed amorphous thorium hydrous oxide at 90°C, and 3) ThO2(c) at 23°C and 90°C. Results show that when ThO2(am) is heated to 90°C, it transforms to a relatively insoluble and crystalline solid [ThO2(am→c)]. At a fixed pH, the observed solubility of ThO2(am) at 23°C is more than 11 orders of magnitude greater than those for ThO2(c) at 23°C or of ThO2(am→c) and ThO2(c) at 90°C. Solubility data were interpreted using the Pitzer ion-interaction model. The log of the solubility product for the thorium dioxide dissolution reaction [ThO2(s) + 2 H2O ↔ Th4+ + 4 OH-] was determined to be -44.9 for ThO2(am) at 23°C, ≥-56.9 for ThO2(c) at 23°C, and -51.4 for ThO2(c) at 90°C. At 90°C, a relatively less crystalline phase, ThO2(am→c), showed slightly higher solubility (log Ksp = -49.2) than crystalline ThO2(c).

Solubility and Solubility Product at 22°C of UO2(c) Precipitated from Aqueous U(IV) Solutions

Solubility studies on UO2(c), precipitated at 90°C from low-pH U(IV) solutions, were conducted under rigidly controlled redox conditions maintained by EuCl2 as a function of pH and from the oversaturation direction. Samples were equilibrated for 24 days at 90°C and then for 1 day at 22°C. X-ray diffraction (XRD) analyses of the solid phases, along with the observed solubility behavior, identified UO2(c) as the dominant phase at pH≳1.2 and UO2(am) as the dominant phase at pH≳1.2. The UV-Vis-NIR spectra of the aqueous phases showed that aqueous uranium was present in the tetravalent state. Our ability to effectively maintain uranium in the tetravalent state during experiments and the recent availability of reliable values of Pitzer ion-interactionparameters for this system have helped to set reliable upper limits for the log Ko value of ≤ −60.2 + 0.24 for the UO2(c) solubility [UO2(c) + 2H2O ⇌ U4+ + 4OH−] and of >−11.6 for the formation of U(OH)4(aq) [U4++ 4H2O ⇌ U(OH)4(aq) + 4H+]

Reductive dissolution of PuO2(am): The effect of Fe(II) and hydroquinone

PuO2(am) solubility was investigated as a function of time, for pH from 0.5 to 11, and in the presence of 0.001 M FeCl2 or 0.00052 M hydroquinone to determine the effect of environmentally important reducing agents on PuO2(am) solubilization under geological conditions. Equilibrium was reached in <4 days. The observed PuO2(am) solubilities were many orders of magnitude higher than the Pu(IV) concentrations predicted from thermodynamic data. Spectroscopic, solvent extraction, and thermodynamic analyses of data showed that Pu(III) was the dominant aqueous oxidation state. The experimental pH, pe, and Pu(III) concentrations from both the Fe(II) and hydroquinone systems provided a log K0 value of 15.5 ± 0.7 for [PuO2(am) + 4H+ + e− ⇌ Pu3+ + 2H2O]. The data show that reduction reactions involving Fe(II) and hydroquinone are relatively rapid and that reductive dissolution of PuO2(am), hitherto ignored, may play an important role in controlling Pu behavior under reducing environmental conditions.

Competitive reduction of pertechnetate (99TcO4-) by dissimilatory metal reducing bacteria and biogenic Fe(II).

The fate of pertechnetate ((99)Tc(VII)O(4)(-)) during bioreduction was investigated in the presence of 2-line ferrihydrite (Fh) and various dissimilatory metal reducing bacteria (DMRB) (Geobacter, Anaeromyxobacter, Shewanella) in comparison with TcO(4)(-) bioreduction in the absence of Fh. In the presence of Fh, Tc was present primarily as a fine-grained Tc(IV)/Fe precipitate that was distinct from the Tc(IV)O(2)·nH(2)O solids produced by direct biological Tc(VII) reduction. Aqueous Tc concentrations (<0.2 μm) in the bioreduced Fh suspensions (1.7 to 3.2 × 10(-9) mol L(-1)) were over 1 order of magnitude lower than when TcO(4)(-) was biologically reduced in the absence of Fh (4.0 × 10(-8) to 1.0 × 10(-7) mol L(-1)). EXAFS analyses of the bioreduced Fh-Tc products were consistent with variable chain length Tc-O octahedra bonded to Fe-O octahedra associated with the surface of the residual or secondary Fe(III) oxide. In contrast, biogenic TcO(2)·nH(2)O had significantly more Tc-Tc second neighbors and a distinct long-range order consistent with small particle polymers of TcO(2). In Fe-rich subsurface sediments, the reduction of Tc(VII) by Fe(II) may predominate over direct microbial pathways, potentially leading to lower concentrations of aqueous (99)Tc(IV).

Heterogeneous Reduction of PuO2 with Fe(II): Importance of the Fe(III) Reaction Product

Heterogeneous reduction of actinides in higher, more soluble oxidation states to lower, more insoluble oxidation states by reductants such as Fe(II) has been the subject of intensive study for more than two decades. However, Fe(II)-induced reduction of sparingly soluble Pu(IV) to the more soluble lower oxidation state Pu(III) has been much less studied, even though such reactions can potentially increase the mobility of Pu in the subsurface. Thermodynamic calculations are presented that show how differences in the free energy of various possible solid-phase Fe(III) reaction products can greatly influence aqueous Pu(III) concentrations resulting from reduction of PuO₂(am) by Fe(II). We present the first experimental evidence that reduction of PuO₂(am) to Pu(III) by Fe(II) was enhanced when the Fe(III) mineral goethite was spiked into the reaction. The effect of goethite on reduction of Pu(IV) was demonstrated by measuring the time dependence of total aqueous Pu concentration, its oxidation state, and system pe/pH. We also re-evaluated established protocols for determining Pu(III) {[Pu(III) + Pu(IV)] - Pu(IV)} by using thenoyltrifluoroacetone (TTA) in toluene extractions; the study showed that it is important to eliminate dissolved oxygen from the TTA solutions for accurate determinations. More broadly, this study highlights the importance of the Fe(III) reaction product in actinide reduction rate and extent by Fe(II).

Microbial reductive transformation of phyllosilicate Fe(III) and U(VI) in fluvial subsurface sediments.

The microbial reduction of Fe(III) and U(VI) was investigated in shallow aquifer sediments collected from subsurface flood deposits near the Hanford Reach of the Columbia River in Washington State. Increases in 0.5 N HCl-extractable Fe(II) were observed in incubated sediments and (57)Fe Mössbauer spectroscopy revealed that Fe(III) associated with phyllosilicates and pyroxene was reduced to Fe(II). Aqueous uranium(VI) concentrations decreased in subsurface sediments incubated in sulfate-containing synthetic groundwater with the rate and extent being greater in sediment amended with organic carbon. X-ray absorption spectroscopy of bioreduced sediments indicated that 67-77% of the U signal was U(VI), probably as an adsorbed species associated with a new or modified reactive mineral phase. Phylotypes within the Deltaproteobacteria were more common in Hanford sediments incubated with U(VI) than without, and in U(VI)-free incubations, members of the Clostridiales were dominant with sulfate-reducing phylotypes more common in the sulfate-amended sediments. These results demonstrate the potential for anaerobic reduction of phyllosilicate Fe(III) and sulfate in Hanford unconfined aquifer sediments and biotransformations involving reduction and adsorption leading to decreased aqueous U concentrations.

Optimization of a portable microanalytical system to reduce electrode fouling from proteins associated with biomonitoring of lead (Pb) in saliva

There is a need to develop reliable portable analytical systems for on-site and real-time biomonitoring of lead (Pb) from both occupational and environmental exposures. Saliva is an appealing matrix since it is easily obtainable, and therefore a potential substitute for blood due to existing reasonably good correlation between Pb levels in blood and saliva. The microanalytical system is based on flow-injection/stripping voltammetry with a wall-jet (flow-onto) microelectrochemical cell. Samples that contain as little as 1% saliva can cause electrode fouling, resulting in significantly reduced responsiveness and irreproducible quantitations. In addition, incomplete Pb release from salivary protein can also yield a lower Pb response than expected. This paper evaluates the extent of in vitro Pb-protein binding and the optimal pretreatment for releasing Pb from the saliva samples. Even in 50% by volume of rat saliva, the electrode fouling was not observed, due to the appropriate sample pretreatment and the constant flow of the sample and acidic carrier that prevented passivation by the protein. The system offered a linear response over a low Pb range of 1-10ppb, low detection limit of 1ppb, excellent reproducibility, and reliability. It also yielded the same Pb concentrations in unknown samples as did the ICP-MS. These encouraging results suggest that the microanalytical system represents an important analytical advancement for real-time non-invasive biomonitoring of Pb.

The Solubility of Th(IV) and U(IV) Hydrous Oxides in Concentrated Nahco 3 and Na 2 Co 3 Solutions

The solubility of Th(IV) and U(IV) hydrous oxide was studied in the aqueous HCO 3 − -CO 3 2− -OH − -H 2 O system extending to high concentration. The solubility of the Th(IV) and U(IV) hydrous oxides increases dramatically in both high bicarbonate and carbonate solutions and decreases with the increase in hydroxide at a fixed carbonate concentration. In general, the observed solubility of Th(IV) hydrous oxide at a given total carbonate concentration was three to four orders of magnitude higher than the solubility of U(IV) hydrous oxide. In the studies of the U(IV) system, extreme caution was used to ensure that the dissolved U was present as U(IV). Oxidation state analyses and systematic trends in the U(IV) solubility data similar to those for Th(IV), which is not redox sensitive, indicated that the dissolved U was present as U(IV).