Edward J. O’Loughlin

Reduction of AgI, AuIII, CuII, and HgII by FeII/FeIII hydroxysulfate green rust

Green rusts are mixed Fe{sup II}/Fe{sup III} hydroxides that are found in many suboxic environments where they are believed to play a central role in the biogeochemical cycling of iron. X-ray absorption fine structure analysis of hydroxysulfate green rust suspensions spiked with aqueous solutions of AgCH{sub 3}COO, AuCl{sub n}(OH){sub 4-n}, CuCl{sub 2}, or HgCl{sub 2} showed that Ag{sup I}, Au{sup III}, Cu{sup II}, and Hg{sup II} were readily reduced to Ag{sup 0}, Au{sup 0}, Cu{sup 0}, and Hg{sup 0}. Imaging of the resulting solids from the Ag{sup I}-, Au{sup III}-, and Cu{sup II}-amended green rust suspensions by transmission electron microscopy indicated the formation of submicron-sized particles of Ag{sup 0}, Au{sup 0}, and Cu{sup 0}. The facile reduction of Ag{sup I}, Au{sup III}, Cu{sup II}, and Hg{sup II} to Ag{sup 0}, Au{sup 0}, Cu{sup 0}, and Hg{sup 0}, respectively, by green rust suggests 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.

Solution and Microbial Controls on the Formation of Reduced U(IV) Species

Reduction of U(VI) to U(IV) as the result of direct or indirect microbial activity is currently being explored for in situ remediation of subsurface U plumes, under the assumption that U(IV) solubility is controlled by the low-solubility mineral uraninite (U(IV)-dioxide). However, recent characterizations of U in sediments from biostimulated field sites, as well as laboratory U(VI) bioreduction studies, report on the formation of U(IV) species that lack the U═O(2)═U coordination of uraninite, suggesting that phases other than uraninite may be controlling U(IV) solubility in environments with complexing surfaces and ligands. To determine the controls on the formation of such nonuraninite U(IV) species, the current work studied the reduction of carbonate-complexed U(VI) by (1) five Gram-positive Desulfitobacterium strains, (2) the Gram-negative bacteria Anaeromyxobacter dehalogenans 2CP-C and Shewanella putrefaciens CN32, and (3) chemically reduced 9,10-anthrahydroquinone-2,6-disulfonate (AH(2)QDS, a soluble reductant). Further, the effects of 0.3 mM dissolved phosphate on U(IV) species formation were explored. Extended X-ray absorption fine structure (EXAFS) spectroscopy analysis demonstrated that the addition of phosphate causes the formation of a nonuraninite, phosphate-complexed U(IV) species, independent of the biological or abiotic mode of U(VI) reduction. In phosphate-free medium, U(VI) reduction by Desulfitobacterium spp. and by AH(2)QDS resulted in nonuraninite, carbonate-complexed U(IV) species, whereas reduction by Anaeromyxobacter or Shewanella yielded nanoparticulate uraninite. These findings suggest that the Gram-positive Desulfitobacterium strains and the Gram-negative Anaeromyxobacter and Shewanella species use distinct mechanisms to reduce U(VI).

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 Mössbauer 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.

Bioreduction of Hydrogen Uranyl Phosphate: Mechanisms and U(IV) Products

The mobility of uranium (U) in subsurface environments is controlled by interrelated adsorption, redox, and precipitation reactions. Previous work demonstrated the formation of nanometer-sized hydrogen uranyl phosphate (abbreviated as HUP) crystals on the cell walls of Bacillus subtilis, a non-U(VI)-reducing, Gram-positive bacterium. The current study examined the reduction of this biogenic, cell-associated HUP mineral by three dissimilatory metal-reducing bacteria, Anaeromyxobacter dehalogenans strain K, Geobacter sulfurreducens strain PCA, and Shewanella putrefaciens strain CN-32, and compared it to the bioreduction of abiotically formed and freely suspended HUP of larger particle size. Uranium speciation in the solid phase was followed over a 10- to 20-day reaction period by X-ray absorption fine structure spectroscopy (XANES and EXAFS) and showed varying extents of U(VI) reduction to U(IV). The reduction extent of the same mass of HUP to U(IV) was consistently greater with the biogenic than with the abiotic material under the same experimental conditions. A greater extent of HUP reduction was observed in the presence of bicarbonate in solution, whereas a decreased extent of HUP reduction was observed with the addition of dissolved phosphate. These results indicate that the extent of U(VI) reduction is controlled by dissolution of the HUP phase, suggesting that the metal-reducing bacteria transfer electrons to the dissolved or bacterially adsorbed U(VI) species formed after HUP dissolution, rather than to solid-phase U(VI) in the HUP mineral. Interestingly, the bioreduced U(IV) atoms were not immediately coordinated to other U(IV) atoms (as in uraninite, UO2) but were similar in structure to the phosphate-complexed U(IV) species found in ningyoite [CaU(PO4)2·H2O]. This indicates a strong control by phosphate on the speciation of bioreduced U(IV), expressed as inhibition of the typical formation of uraninite under phosphate-free conditions.

The transport behavior of As, Cu, Pb, and Zn during electrokinetic remediation of a contaminated soil using electrolyte conditioning

Electrokinetic remediation (also known as electrokinetics) is a promising technology for removing metals from fine-grained soils. However, few studies have been conducted regarding the transport behavior of multi-metals during electrokinetics. We investigated the transport of As, Cu, Pb, and Zn from soils during electrokinetics, the metal fractionation before and after electrokinetics, the relationships between metal transport and fractionation, and the effects of electrolyte conditioning. The main transport mechanisms of the metals were electroosmosis and electromigration during the first two weeks and electromigration during the following weeks. The direction of electroosmotic flow was from the anode to the cathode, and the metals in the dissolved and reducible-oxides fractions were transported to the anode or cathode by electromigration according to the chemical speciation of the metal ions in the pore water. Moreover, a portion of the metals that were initially in the residual fraction transitioned to the reducible and soluble fractions during electrokinetic treatment. However, this alteration was slow and resulted in decreasing metal removal rates as the electrokinetic treatment progressed. In addition, the use of NaOH, H3PO4, and Na2SO4 as electrolytes resulted in conditions that favored the precipitation of metal hydroxides, phosphates, and sulfates in the soil. These results demonstrated that metal removal was affected by the initial metal fractionation, metal speciation in the pore solution, and the physical-chemical parameters of the electrolytes, such as pH and electrolyte composition. Therefore, the treatment time, use of chemicals, and energy consumption could be reduced by optimizing pretreatment and by choosing appropriate electrolytes for the target metals.

Influence of Chloride and Fe(II) Content on the Reduction of Hg(II) by Magnetite

Abiotic reduction of inorganic mercury by natural organic matter and native soils is well-known, and recently there is evidence that reduced iron (Fe) species, such as magnetite, green rust, and Fe sulfides, can also reduce Hg(II). Here, we evaluated the reduction of Hg(II) by magnetites with varying Fe(II) content in both the absence and presence of chloride. Specifically, we evaluated whether magnetite stoichiometry (x = Fe(II)/Fe(III)) influences the rate of Hg(II) reduction and formation of products. In the absence of chloride, reduction of Hg(II) to Hg(0) is observed over a range of magnetite stoichiometries (0.29 < x < 0.50) in purged headspace reactors and unpurged low headspace reactors, as evidenced by Hg recovery in a volatile product trap solution and Hg L(III)-edge X-ray absorption near edge spectroscopy (XANES). In the presence of chloride, however, XANES spectra indicate the formation of a metastable Hg(I) calomel species (Hg2Cl2) from the reduction of Hg(II). Interestingly, Hg(I) species are only observed for the more oxidized magnetite particles that contain lower Fe(II) content (x < 0.42). For the more reduced magnetite particles (x ≥ 0.42), Hg(II) is reduced to Hg(0) even in the presence of high chloride concentrations. As previously observed for nitroaromatic compounds and uranium, magnetite stoichiometry appears to influence the rate of Hg(II) reduction (both in the presence and absence of chloride) confirming that it is important to consider magnetite stoichiometry when assessing the fate of contaminants in Fe-rich subsurface environments.

Geochemical and microbiological processes contributing to the transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in contaminated aquifer material

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a potential human carcinogen, and its contamination of subsurface environments is a significant threat to public health. This study investigated abiotic and biological degradation of RDX in contaminated aquifer material. Anoxic batch systems were started with and without pre-aeration of aquifer material to distinguish initial biological RDX reduction from abiotic RDX reduction. Aerating the sediment eliminated chemical reductants in the native aquifer sediment, primarily Fe(II) sorbed to mineral surfaces. RDX (50 μM) was completely reduced and transformed to ring cleavage products when excess concentrations (2mM) of acetate or lactate were provided as the electron donor for aerated sediment. RDX was reduced concurrently with Fe(III) when acetate was provided, while RDX, Fe(III), and sulfate were reduced simultaneously with lactate amendment. Betaproteobacteria were the dominant microorganisms associated with RDX and Fe(III)/sulfate reduction. In particular, Rhodoferax spp. increased from 21% to 35% and from 28% to 60% after biostimulation by acetate and lactate, respectively. Rarefaction analyses demonstrated that microbial diversity decreased in electron-donor-amended systems with active RDX degradation. Although significant amounts of Fe(III) and/or sulfate were reduced after biostimulation, solid-phase reactive minerals such as magnetite or ferrous sulfides were not observed, suggesting that RDX reduction in the aquifer sediment is due to Fe(II) adsorbed to solid surfaces as a result of Fe(III)-reducing microbial activity. These results suggest that both biotic and abiotic processes play an important role in RDX reduction under in situ conditions.

Geochemical characteristics and microbial community composition in toxic metal-rich sediments contaminated with Au–Ag mine tailings

The effects of extreme geochemical conditions on microbial community composition were investigated for two distinct sets of sediment samples collected near weathered mine tailings. One set (SCH) showed extraordinary geochemical characteristics: As (6.7-11.5%), Pb (1.5-2.1%), Zn (0.1-0.2%), and pH (3.1-3.5). The other set (SCL) had As (0.3-1.2%), Pb (0.02-0.22%), and Zn (0.01-0.02%) at pH 2.5-3.1. The bacterial communities in SCL were clearly different from those in SCH, suggesting that extreme geochemical conditions affected microbial community distribution even on a small spatial scale. The clones identified in SCL were closely related to acidophilic bacteria in the taxa Acidobacterium (18%), Acidomicrobineae (14%), and Leptospirillum (10%). Most clones in SCH were closely related to Methylobacterium (79%) and Ralstonia (19%), both well-known metal-resistant bacteria. Although total As was extremely high, over 95% was in the form of scorodite (FeAsO4·2H2O). Acid-extractable As was only ∼118 and ∼14 mg kg(-1) in SCH and SCL, respectively, below the level known to be toxic to bacteria. Meanwhile, acid-extractable Pb and Zn in SCH were above toxic concentrations. Because As was present in an oxidized, stable form, release of Pb and/or Zn (or a combination of toxic metals in the sediment) from the sediment likely accounts for the differences in microbial community structure. The results also suggest that care should be taken when investigating mine tailings, because large differences in chemical/biological properties can occur over small spatial scales.

Impact of Organic Carbon Electron Donors on Microbial Community Development under Iron- and Sulfate-Reducing Conditions.

Although iron- and sulfate-reducing bacteria in subsurface environments have crucial roles in biogeochemical cycling of C, Fe, and S, how specific electron donors impact the compositional structure and activity of native iron- and/or sulfate-reducing communities is largely unknown. To understand this better, we created bicarbonate-buffered batch systems in duplicate with three different electron donors (acetate, lactate, or glucose) paired with ferrihydrite and sulfate as the electron acceptors and inoculated them with subsurface sediment as the microbial inoculum. Sulfate and ferrihydrite reduction occurred simultaneously and were faster with lactate than with acetate. 16S rRNA-based sequence analysis of the communities over time revealed that Desulfotomaculum was the major driver for sulfate reduction coupled with propionate oxidation in lactate-amended incubations. The reduction of sulfate resulted in sulfide production and subsequent abiotic reduction of ferrihydrite. In contrast, glucose promoted faster reduction of ferrihydrite, but without reduction of sulfate. Interestingly, the glucose-amended incubations led to two different biogeochemical trajectories among replicate bottles that resulted in distinct coloration (white and brown). The two outcomes in geochemical evolution might be due to the stochastic evolution of the microbial communities or subtle differences in the initial composition of the fermenting microbial community and its development via the use of different glucose fermentation pathways available within the community. Synchrotron-based x-ray analysis indicated that siderite and amorphous Fe(II) were formed in the replicate bottles with glucose, while ferrous sulfide and vivianite were formed with lactate or acetate. These data sets reveal that use of different C utilization pathways projects significant changes in microbial community composition over time that uniquely impact both the geochemistry and mineralogy of subsurface environments.

Tepidibacillus decaturensis sp. nov., a microaerophilic, moderately thermophilic iron-reducing bacterium isolated from 1.7 km depth groundwater.

A Gram-stain-negative, microaerophilic rod-shaped organism designated as strain Z9T was isolated from groundwater of 1.7 km depth from the Mt. Simon Sandstone of the Illinois Basin, Illinois, USA. Cells of strain Z9T were rod shaped with dimensions of 0.3×(1-10) µm and stained Gram-negative. Strain Z9T grew within the temperature range 20-60 °C (optimum at 30-40 °C), between pH 5 and 8 (optimum 5.2-5.8) and under salt concentrations of 1-5 % (w/v) NaCl (optimum 2.5 % NaCl). In addition to growth by fermentation and nitrate reduction, this strain was able to reduce Fe(III), Mn(IV), Co(III) and Cr(VI) when H2 or organic carbon was available as the electron donor, but did not actively reduce oxidized sulfur compounds (e.g. sulfate, thiosulfate or S0). The G+C content of the DNA from strain Z9T was 36.1 mol%. Phylogenetic analysis of the 16S rRNA gene from strain Z9T showed that it belongs to the class Bacilli and shares 97 % sequence similarity with the only currently characterized member of the genus Tepidibacillus, T.fermentans. Based on the physiological distinctness and phylogenetic information, strain Z9T represents a novel species within the genus Tepidibacillus, for which the name Tepidibacillus decaturensis sp. nov. is proposed. The type strain is Z9T (=ATCC BAA-2644T=DSM 103037T).