Edward J. O'Loughlin

Molecular weight, polydispersity, and spectroscopic properties of aquatic humic substances

The number- and weight-averaged molecular weights of a number of aquatic fulvic acids, a commercial humic acid, and unfractionated organic matter from four natural water samples were measured by high-pressure size exclusion chromatography (HPSEC). Molecular weights determined in this manner compared favorably with those values reported in the literature. Both recent literature values and our data indicate that these substances are smaller and less polydisperse than previously believed. Moreover, the molecular weights of the organic matter from three of the four natural water samples compared favorably to the fulvic acid samples extracted from similar environments

XAFS investigation of the interactions of UVI with secondary mineralization products from the bioreduction of FeIII oxides.

Biogenic Fe(II) phases (magnetite, green rust, siderite, vivianite, etc.) provide a reservoir of reducing capacity in many subsurface environments that may contribute to the reduction of contaminants such as U(VI). We have examined the uptake and reduction of U(VI) in the presence of biogenic green rust (BioGR), magnetite (BioMAG), and siderite (BioSID) formed during the reduction of Fe(III) oxides by Shewanella putrefaciens CN32. Within 48 h, total solution-phase U(VI) concentrations decreased from 500 microM to 1.5 microM, 392 microM, and 472 microM in the U-BioGR, U-BioMAG, and U-BioSID systems, respectively. Analysis of the samples by U L(III) extended X-ray absorption fine structure spectroscopy (EXAFS) indicated that despite a stoichiometric excess of Fe(II), no more than 6% of U(VI) was reduced to U(IV) in the U-BioSID system, and no more than 22% of U(VI) was reduced in the U-BioMAG system. For comparison, in the U-BioGR system, >99% of U(VI) was reduced to U(IV). Uptake of U(VI) by BioGR and BioMAG was accompanied by formation of nanoparticulate uraninite. The U EXAFS data for the U-BioSID system were consistent with partial U(VI)/U(IV) substitution for Fe(II) in the surface layer of siderite particles and adsorption of U(IV).

Sulfur-mediated electron shuttling during bacterial iron reduction

How bacteria manage to breathe on rust In the absence of oxygen, anaerobic bacteria turn to other chemical compounds during respiration. This can be helpful in detoxifying heavy-metal pollution. Flynn et al. (see the Perspective by Friedrich and Finster) found that alkaline conditions prevent a detoxifying bug—Shewanella oneidensis—from using enzymes to reduce rust-like minerals. Instead, the bacteria reduce elemental sulfur compounds, generating hydrogen sulfide that reduces the iron indirectly. This interplay between anoxic biogeochemical cycles may explain why some anaerobic bacteria contain the genetic machinery necessary to reduce multiple compounds besides oxygen. Science, this issue p. 1039; see also p. 974 Bacterial respiration of ferric iron involves sulfur intermediates in alkaline conditions [Also see Perspective by Friedrich and Finster] Microbial reduction of ferric iron [Fe(III)] is an important biogeochemical process in anoxic aquifers. Depending on groundwater pH, dissimilatory metal-reducing bacteria can also respire alternative electron acceptors to survive, including elemental sulfur (S0). To understand the interplay of Fe/S cycling under alkaline conditions, we combined thermodynamic geochemical modeling with bioreactor experiments using Shewanella oneidensis MR-1. Under these conditions, S. oneidensis can enzymatically reduce S0 but not goethite (α-FeOOH). The HS– produced subsequently reduces goethite abiotically. Because of the prevalence of alkaline conditions in many aquifers, Fe(III) reduction may thus proceed via S0-mediated electron-shuttling pathways.

Reduction of halogenated ethanes by green rust.

Green rusts, mixed Fe(II)/Fe(III) hydroxide minerals present in many suboxic environments, have been shown to reduce a number of organic and inorganic contaminants. The reduction of halogenated ethanes was examined in aqueous suspensions of green rust, both alone and with the addition of Ag(I) (AgGR) and Cu(II) (CuGR). Hexachloroethane (HCA), pentachloroethane (PCA), 1,1,1,2-tetrachloroethane (1,1,1,2-TeCA), 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA), 1,1,1-trichloroethane (1,1,1-TCA), 1,1,2-trichloroethane (1,1,2-TCA), 1,1-dichloroethane (1,1-DCA), and 1,2-dibromoethane were reduced in the presence of green rust alone, AgGR, or CuGR; only 1,2-dichloroethane and chloroethane were nonreactive. The reduction was generally more rapid for more highly substituted ethanes than for ethanes having fewer halogen groups (HCA > PCA > 1,1,1,2-TeCA > 1,1,1-TCA > 1,1,2,2-TeCA > 1,1,2-TCA > 1,1-DCA), and isomers with the more asymmetric distributions of halogen groups were more rapidly reduced than the isomer with greater symmetry (e.g., 1,1,1-TCA > 1,1,2-TCA). The addition of Ag(I) or Cu(II) to green rust suspensions resulted in a substantial increase in the rate of halogenated ethane reduction as well as significant differences in the product distributions with respect to green rust alone.

Green Rust Formation from the Bioreduction of γ –FeOOH (Lepidocrocite): Comparison of Several Shewanella Species

Green rusts are mixed ferrous/ferric hydroxides that typically form under weakly acidic to alkaline conditions in suboxic environments. The recent identification of green rusts as products of the reduction of Fe(III) oxides and oxyhydroxides by Shewanella putrefaciens, a dissimilatory iron-reducing bacterium (DIRB), suggests that green rusts may play a role in the redox cycling of Fe in many aquatic and terrestrial environments. We examined the potential for green rust formation resulting from the bioreduction of lepidocrocite(γ -FeOOH) by a series of Shewanella species (S. alga BrY, S. amazonensis SB2B, S. baltica OS155, S. denitrificans OS217T, S. loihica PV-4, S. oneidensis MR-1, S. putrefaciens ATCC 8071, S. putrefaciens CN32, S. saccharophilia, and Shewanella sp. ANA-3). All Shewanella species, with the exception of S. denitrificans OS217T, were able to couple the oxidation of formate to the reduction of Fe(III) in lepidocrocite; however there were significant differences among species with respect to the rate and extent of Fe(II) production. Despite these differences, green rust was the only Fe(II)-bearing solid phase formed under our experimental conditions, as indicated by X-ray diffraction, Mössbauer spectroscopy, and scanning electron microscopy. The formation of green rust by Shewanella species isolated from a wide range of habitats and possessing varied metabolic capabilities suggests that under favorable conditions biogenic green rusts may be formed by a diverse array of DIRB.

Binding of HgII to High-Affinity Sites on Bacteria Inhibits Reduction to Hg0 by Mixed FeII/III Phases

Magnetite and green rust have been shown to reduce aqueous Hg(II) to Hg(0). In this study, we tested the ability of magnetite and green rust to reduce Hg(II) sorbed to 2 g · L(-1) of biomass (Bacillus subtilis), at high (50 μM) and low (5 μM) Hg loadings and at pH 6.5 and 5.0. At high Hg:biomass loading, where Hg(II) binding to biomass is predominantly through carboxyl functional groups, Hg L(III)-edge X-ray absorption spectroscopy showed reduction of Hg(II) to Hg(0) by magnetite. Reduction occurred within 2 h and 2 d at pH 6.5 and 5.0, respectively. At low Hg:biomass loading, where Hg(II) binds to biomass via sulfhydryl functional groups, Hg(II) was not reduced by magnetite at pH 6.5 or 5.0 after 2 months of reaction. Green rust, which is generally a stronger reductant than magnetite, reduced about 20% of the total Hg(II) bound to biomass via sulfhydryl groups to Hg(0) in 2 d. These results suggest that Hg(II) binding to carboxyl groups does not significantly inhibit the reduction of Hg(II) by magnetite. However, the binding of Hg(II) to biomass via sulfhydryl groups severely inhibits the ability of mixed Fe(II/III) phases like magnetite and green rust to reduce Hg(II) to Hg(0). The mobility of heavy metal contaminants in aquatic and terrestrial environments is greatly influenced by their speciation, especially their oxidation state. In the case of Hg, reduction of Hg(II) to Hg(0) can increase Hg mobility because of the volatility of Hg(0). Since Hg is typically present in aquatic and terrestrial systems at low concentrations, binding of Hg(II) to high-affinity sites on bacteria could have important implications for the potential reduction of Hg(II) to Hg(0) and the overall mobility of Hg in biostimulated subsurface environments.

Uranium Transformations in Static Microcosms

Elucidation of complex biogeochemical processes and their effects on speciation of U in the subsurface is critical for developing remediation strategies with an understanding of stability. We have developed static microcosms that are similar to bioreduction process studies in situ under laminar flow conditions or in sediment pores. Uranium L(3)-edge X-ray absorption near-edge spectroscopy analysis with depth in the microcosms indicated that transformation of U(VI) to U(IV) occurred by at least two distinct processes. Extended X-ray absorption fine structure (EXAFS) analysis indicated that initial U(VI) species associated with C- and P-containing ligands were transformed to U(IV) in the form of uraninite and U associated with Fe-bound ligands. Microbial community analysis identified putative Fe(III) and sulfate reducers at two different depths in the microcosms. The slow reduction of U(VI) to U(IV) may contribute the stability of U(IV) within microcosms at 11 months after a decrease in bioreducing conditions due to limited electron donors.

The complete genome sequence for putative H₂- and S-oxidizer Candidatus Sulfuricurvum sp., assembled de novo from an aquifer-derived metagenome.

We reconstructed the complete 2.4 Mb-long genome of a previously uncultivated epsilonproteobacterium, Candidatus Sulfuricurvum sp. RIFRC-1, via assembly of short-read shotgun metagenomic data using a complexity reduction approach. Genome-based comparisons indicate the bacterium is a novel species within the Sulfuricurvum genus, which contains one cultivated representative, S. kujiense. Divergence between the species appears due in part to extensive genomic rearrangements, gene loss and chromosomal versus plasmid encoding of certain (respiratory) genes by RIFRC-1. Deoxyribonucleic acid for the genome was obtained from terrestrial aquifer sediment, in which RIFRC-1 comprised ∼ 47% of the bacterial community. Genomic evidence suggests RIFRC-1 is a chemolithoautotrophic diazotroph capable of deriving energy for growth by microaerobic or nitrate-/nitric oxide-dependent oxidation of S°, sulfide or sulfite or H₂oxidation. Carbon may be fixed via the reductive tricarboxylic acid cycle. Consistent with these physiological attributes, the local aquifer was microoxic with small concentrations of available nitrate, small but elevated concentrations of reduced sulfur and NH(4)(+) /NH₃-limited. Additionally, various mechanisms for heavy metal and metalloid tolerance and virulence point to a lifestyle well-adapted for metal(loid)-rich environments and a shared evolutionary past with pathogenic Epsilonproteobacteria. Results expand upon recent findings highlighting the potential importance of sulfur and hydrogen metabolism in the terrestrial subsurface.

Orenia metallireducens sp. nov. Strain Z6, a Novel Metal-Reducing Member of the Phylum Firmicutes from the Deep Subsurface

ABSTRACT A novel halophilic and metal-reducing bacterium, Orenia metallireducens strain Z6, was isolated from briny groundwater extracted from a 2.02 km-deep borehole in the Illinois Basin, IL. This organism shared 96% 16S rRNA gene similarity with Orenia marismortui but demonstrated physiological properties previously unknown for this genus. In addition to exhibiting a fermentative metabolism typical of the genus Orenia, strain Z6 reduces various metal oxides [Fe(III), Mn(IV), Co(III), and Cr(VI)], using H2 as the electron donor. Strain Z6 actively reduced ferrihydrite over broad ranges of pH (6 to 9.6), salinity (0.4 to 3.5 M NaCl), and temperature (20 to 60°C). At pH 6.5, strain Z6 also reduced more crystalline iron oxides, such as lepidocrocite (γ-FeOOH), goethite (α-FeOOH), and hematite (α-Fe2O3). Analysis of X-ray absorption fine structure (XAFS) following Fe(III) reduction by strain Z6 revealed spectra from ferrous secondary mineral phases consistent with the precipitation of vivianite [Fe3(PO4)2] and siderite (FeCO3). The draft genome assembled for strain Z6 is 3.47 Mb in size and contains 3,269 protein-coding genes. Unlike the well-understood iron-reducing Shewanella and Geobacter species, this organism lacks the c-type cytochromes for typical Fe(III) reduction. Strain Z6 represents the first bacterial species in the genus Orenia (order Halanaerobiales) reported to reduce ferric iron minerals and other metal oxides. This microbe expands both the phylogenetic and physiological scopes of iron-reducing microorganisms known to inhabit the deep subsurface and suggests new mechanisms for microbial iron reduction. These distinctions from other Orenia spp. support the designation of strain Z6 as a new species, Orenia metallireducens sp. nov. IMPORTANCE A novel iron-reducing species, Orenia metallireducens sp. nov., strain Z6, was isolated from groundwater collected from a geological formation located 2.02 km below land surface in the Illinois Basin, USA. Phylogenetic, physiologic, and genomic analyses of strain Z6 found it to have unique properties for iron reducers, including (i) active microbial iron-reducing capacity under broad ranges of temperatures (20 to 60°C), pHs (6 to 9.6), and salinities (0.4 to 3.5 M NaCl), (ii) lack of c-type cytochromes typically affiliated with iron reduction in Geobacter and Shewanella species, and (iii) being the only member of the Halanaerobiales capable of reducing crystalline goethite and hematite. This study expands the scope of phylogenetic affiliations, metabolic capacities, and catalytic mechanisms for iron-reducing microbes.

Iron phase transformations resulting from the respiration of Shewanella putrefaciens on a mixed mineral phase

The initial Fe(III) minerals and the secondary mineralization products of Shewanella putrefaciens CN32 grown in the presence of dissolved phosphate and a commercial Fe(III) oxide, nominally nanoparticulate lepidocrocite, were determined using XRD and XAFS. The starting material was transformed by the bacteria from a reddish brown, rust colour mineral to a dark green phase over 90 days. Acid extraction of the bioreduced solids with 0.75 M HCl recovered 83% of the total iron as Fe(II), leaving a solid, acid-resistant phase. The latter was identified as nanoparticulate hematite by EXAFS. Subsequently, the starting Fe(III) phase was determined to be a mixture of 60% lepidocrocite, 26% ferrihydrite, and 14% hematite, using linear combination EXAFS analysis. For the acid-extractable phase, XANES and EXAFS indicated a predominantly Fe(II) valence state and a spectrum consistent with a mixture of brucite-type minerals(e.g., green rust or ferrous hydroxide) and siderite. The observed transformations suggest that in this mixed-mineral system, lepidocrocite and ferrihydrite are readily reducible to green rust and siderite, whereas hematite is less amenable to bacterial reduction. This study also demonstrates the utility of XAFS spectroscopy in the quantitative characterization of dissimilatory metal transformations, particularly in complex systems such as nanoparticulate minerals in hydrated mineral-bacteria assemblages.