Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by Deinococcus radiodurans R1

Deinococcus radiodurans is an exceptionally radiation-resistant microorganism capable of surviving acute exposures to ionizing radiation doses of 15,000 Gy and previously described as having a strictly aerobic respiratory metabolism. Under strict anaerobic conditions, D. radiodurans R1 reduced Fe(III)-nitrilotriacetic acid coupled to the oxidation of lactate to CO(2) and acetate but was unable to link this process to growth. D. radiodurans reduced the humic acid analog anthraquinone-2,6-disulfonate (AQDS) to its dihydroquinone form, AH(2)DS, which subsequently transferred electrons to the Fe(III) oxides hydrous ferric oxide and goethite via a previously described electron shuttle mechanism. D. radiodurans reduced the solid-phase Fe(III) oxides in the presence of either 0.1 mM AQDS or leonardite humic acids (2 mg ml(-1)) but not in their absence. D. radiodurans also reduced U(VI) and Tc(VII) in the presence of AQDS. In contrast, Cr(VI) was directly reduced in anaerobic cultures with lactate although the rate of reduction was higher in the presence of AQDS. The results are the first evidence that D. radiodurans can reduce Fe(III) coupled to the oxidation of lactate or other organic compounds. Also, D. radiodurans, in combination with humic acids or synthetic electron shuttle agents, can reduce U and Tc and thus has potential applications for remediation of metal- and radionuclide-contaminated sites where ionizing radiation or other DNA-damaging agents may restrict the activity of more sensitive organisms.     

Alkaline extracellular reduction: isolation and characterization of an alkaliphilic and halotolerant bacterium, Bacillus pseudofirmus MC02

Aims: To isolate an alkaliphilic bacterium and to investigate its ability of extracellular reduction. Methods and Results: An alkaliphilic and halotolerant humus‐reducing anaerobe, Bacillus pseudofirmus MC02, was successfully isolated from a pH 10·0 microbial fuel cell. To examine its ability of extracellular reduction, AQDS (anthraquinone‐2, 6‐disulfonae), humic acids (HA) and Fe(III) oxides were chosen as representative electron acceptors. All the experiments were conducted in a pH 9·5 carbonate buffer. The results are as follows: (i) Sucrose, lactate, glucose and glycerol were the favourable electron donors for AQDS reduction by the strain MC02; (ii) The strain had the ability of reducing HA in the presence of sucrose; (iii) It could effectively reduce Fe(III) oxides coupled with sucrose fermentation when AQDS was added as electron shuttle and its Fe(III) reducing capacity ranked as: lepidocrocite (γ‐FeOOH) > goethite (α‐FeOOH) > haematite(α‐Fe₂O₃); (iv) The strain could decolourize azo dye Orange I. Conclusions: Bacillus pseudofirmus MC02 was capable of extracellular reduction in AQDS, HA and Fe(III) oxides, and it can be used for decolourizing azo dye (Orange I) in alkaline conditions. Significance and Impact of the Study: This is the first report of an alkaliphlic strain of B. pseudofirmus capable of extracellular reduction in AQDS, HA, Fe(III) oxides and decolourization of Orange I. This study could provide valuable information on alkaline biotransformation in the printing and dyeing wastewater and saline‐alkali soil.     

Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates

The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp. (WS01, WS18, and ES5) and a known Cellulomonas strain ( Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions. In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI). In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) [Fe(III)-NTA] from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM. All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls. This is the first report of Cellulomonas sp. reducing Fe(III) and U(VI). Further, this is the first report of Cellulomonas spp. coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI).     

Microbially Mediated Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5- Triazine by Extracellular Electron Shuttling Compounds

The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of Geobacter metallireducens and G. sulfurreducens with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of G. metallireducens alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.     

Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria

The dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO₂ at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO₂ and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single Geobacter sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.     

Reduction of Fe(III), Mn(IV), and Toxic Metals at 100°C by Pyrobaculum islandicum

It has recently been noted that a diversity of hyperthermophilic microorganisms have the ability to reduce Fe(III) with hydrogen as the electron donor, but the reduction of Fe(III) or other metals by these organisms has not been previously examined in detail. When Pyrobaculum islandicum was grown at 100°C in a medium with hydrogen as the electron donor and Fe(III)-citrate as the electron acceptor, the increase in cell numbers of P. islandicum per mole of Fe(III) reduced was found to be ca. 10-fold higher than previously reported. Poorly crystalline Fe(III) oxide could also serve as the electron acceptor for growth on hydrogen. The stoichiometry of hydrogen uptake and Fe(III) oxide reduction was consistent with the oxidation of 1 mol of hydrogen resulting in the reduction of 2 mol of Fe(III). The poorly crystalline Fe(III) oxide was reduced to extracellular magnetite. P. islandicum could not effectively reduce the crystalline Fe(III) oxide minerals goethite and hematite. In addition to using hydrogen as an electron donor for Fe(III) reduction, P. islandicum grew via Fe(III) reduction in media in which peptone and yeast extract served as potential electron donors. The closely related species P. aerophilum grew via Fe(III) reduction in a similar complex medium. Cell suspensions of P. islandicum reduced the following metals with hydrogen as the electron donor: U(VI), Tc(VII), Cr(VI), Co(III), and Mn(IV). The reduction of these metals was dependent upon the presence of cells and hydrogen. The metalloids arsenate and selenate were not reduced. U(VI) was reduced to the insoluble U(IV) mineral uraninite, which was extracellular. Tc(VII) was reduced to insoluble Tc(IV) or Tc(V). Cr(VI) was reduced to the less toxic, less soluble Cr(III). Co(III) was reduced to Co(II). Mn(IV) was reduced to Mn(II) with the formation of manganese carbonate. These results demonstrate that biological reduction may contribute to the speciation of metals in hydrothermal environments and could account for such phenomena as magnetite accumulation and the formation of uranium deposits at ca. 100°C. Reduction of toxic metals with hyperthermophilic microorganisms or their enzymes might be applied to the remediation of metal-contaminated waters or waste streams.     

Electron Transfer by Desulfobulbus propionicus to Fe(III) and Graphite Electrodes

Desulfobulbus propionicus was able to grow with Fe(III), the humic acids analog anthraquinone-2,6-disulfonate (AQDS), or a graphite electrode as an electron acceptor. These results provide an explanation for the enrichment of Desulfobulbaceae species on the surface of electrodes harvesting electricity from anaerobic marine sediments and further expand the diversity of microorganisms known to have the ability to use both sulfate and Fe(III) as an electron acceptor.     

Fe(III) Reduction and U(VI) Immobilization by Paenibacillus sp. Strain 300A,Isolated from Hanford 300A Subsurface Sediments

A facultative iron-reducing [Fe(III)-reducing] Paenibacillus sp. strain was isolated from Hanford 300A subsurface sediment biofilms that was capable of reducing soluble Fe(III) complexes [Fe(III)-nitrilotriacetic acid and Fe(III)-citrate] but unable to reduce poorly crystalline ferrihydrite (Fh). However, Paenibacillus sp. 300A was capable of reducing Fh in the presence of low concentrations (2 μM) of either of the electron transfer mediators (ETMs) flavin mononucleotide (FMN) or anthraquinone-2,6-disulfonate (AQDS). Maximum initial Fh reduction rates were observed at catalytic concentrations (<10 μM) of either FMN or AQDS. Higher FMN concentrations inhibited Fh reduction, while increased AQDS concentrations did not. We also found that Paenibacillus sp. 300A could reduce Fh in the presence of natural ETMs from Hanford 300A subsurface sediments. In the absence of ETMs, Paenibacillus sp. 300A was capable of immobilizing U(VI) through both reduction and adsorption. The relative contributions of adsorption and microbial reduction to U(VI) removal from the aqueous phase were ∼7:3 in PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)] and ∼1:4 in bicarbonate buffer. Our study demonstrated that Paenibacillus sp. 300A catalyzes Fe(III) reduction and U(VI) immobilization and that these reactions benefit from externally added or naturally existing ETMs in 300A subsurface sediments.     

Extracellular Electron Transfer to Fe(III) Oxides by the Hyperthermophilic Archaeon Geoglobus ahangari via a Direct Contact Mechanism

The microbial reduction of Fe(III) plays an important role in the geochemistry of hydrothermal systems, yet it is poorly understood at the mechanistic level. Here we show that the obligate Fe(III)-reducing archaeon Geoglobus ahangari uses a direct-contact mechanism for the reduction of Fe(III) oxides to magnetite at 85°C. Alleviating the need to directly contact the mineral with the addition of a chelator or the electron shuttle anthraquinone-2,6-disulfonate (AQDS) stimulated Fe(III) reduction. In contrast, entrapment of the oxides within alginate beads to prevent cell contact with the electron acceptor prevented Fe(III) reduction and cell growth unless AQDS was provided. Furthermore, filtered culture supernatant fluids had no effect on Fe(III) reduction, ruling out the secretion of an endogenous mediator too large to permeate the alginate beads. Consistent with a direct contact mechanism, electron micrographs showed cells in intimate association with the Fe(III) mineral particles, which once dissolved revealed abundant curled appendages. The cells also produced several heme-containing proteins. Some of them were detected among proteins sheared from the cell''s outer surface and were required for the reduction of insoluble Fe(III) oxides but not for the reduction of the soluble electron acceptor Fe(III) citrate. The results thus support a mechanism in which the cells directly attach and transfer electrons to the Fe(III) oxides using redox-active proteins exposed on the cell surface. This strategy confers on G. ahangari a competitive advantage for accessing and reducing Fe(III) oxides under the extreme physical and chemical conditions of hot ecosystems.     

Microbially mediated biodegradation of hexahydro-1,3,5-trinitro-1,3,5- triazine by extracellular electron shuttling compounds

The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of Geobacter metallireducens and G. sulfurreducens with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of G. metallireducens alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.     

Reduction kinetics of Fe(III), Co(III), U(VI), Cr(VI), and Tc(VII) in cultures of dissimilatory metal-reducing bacteria

The reduction kinetics of Fe(III)citrate, Fe(III)NTA, Co(III)EDTA-, U(VI)O(2) (2+), Cr(VI)O(4) (2-), and Tc(VII)O(4) (-) were studied in cultures of dissimilatory metal reducing bacteria (DMRB): Shewanella alga strain BrY, Shewanella putrefaciens strain CN32, Shewanella oneidensis strain MR-1, and Geobacter metallireducens strain GS-15. Reduction rates were metal specific with the following rate trend: Fe(III)citrate > or = Fe(III)NTA > Co(III)EDTA- > UO(2)(2+) > CrO(4)(2-) > TcO(4)(-), except for CrO(4) (2-) when H(2) was used as electron donor. The metal reduction rates were also electron donor dependent with faster rates observed for H(2) than lactate- for all Shewanella species despite higher initial lactate (10 mM) than H2 (0.48 mM). The bioreduction of CrO(4) (2-) was anomalously slower compared to the other metals with H(2) as an electron donor relative to lactate and reduction ceased before all the CrO(4)(2-) had been reduced. Transmission electron microscopic (TEM) and energy-dispersive spectroscopic (EDS) analyses performed on selected solids at experiment termination found precipitates of reduced U and Tc in association with the outer cell membrane and in the periplasm of the bacteria. The kinetic rates of metal reduction were correlated with the precipitation of reduced metal phases and their causal relationship discussed. The experimental rate data were well described by a Monod kinetic expression with respect to the electron acceptor for all metals except CrO(4)(2-), for which the Monod model had to be modified to account for incomplete reduction. However, the Monod models became statistically over-parameterized, resulting in large uncertainties of their parameters. A first-order approximation to the Monod model also effectively described the experimental results, but the rate coefficients exhibited far less uncertainty. The more precise rate coefficients of the first-order model provided a better means than the Monod parameters, to quantitatively compare the reduction rates between metals, electron donors, and DMRB species.     

Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH₂QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bifunctional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH₂QDS. Using stopped-flow kinetic measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for ¹³C,¹⁵N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and ¹H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens.     

Hexavalent chromium reduction by Cellulomonas sp. strain ES6: the influence of carbon source, iron minerals, and electron shuttling compounds

The reduction of hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III), can be an important aspect of remediation processes at contaminated sites. Cellulomonas species are found at several Cr(VI) contaminated and uncontaminated locations at the Department of Energy site in Hanford, Washington. Members of this genus have demonstrated the ability to effectively reduce Cr(VI) to Cr(III) fermentatively and therefore play a potential role in Cr(VI) remediation at this site. Batch studies were conducted with Cellulomonas sp. strain ES6 to assess the influence of various carbon sources, iron minerals, and electron shuttling compounds on Cr(VI) reduction rates as these chemical species are likely to be present in, or added to, the environment during in situ bioremediation. Results indicated that the type of carbon source as well as the type of electron shuttle present influenced Cr(VI) reduction rates. Molasses stimulated Cr(VI) reduction more effectively than pure sucrose, presumably due to presence of more easily utilizable sugars, electron shuttling compounds or compounds with direct Cr(VI) reduction capabilities. Cr(VI) reduction rates increased with increasing concentration of anthraquinone-2,6-disulfonate (AQDS) regardless of the carbon source. The presence of iron minerals and their concentrations did not significantly influence Cr(VI) reduction rates. However, strain ES6 or AQDS could directly reduce surface-associated Fe(III) to Fe(II), which was capable of reducing Cr(VI) at a near instantaneous rate. These results suggest the rate limiting step in these systems was the transfer of electrons from strain ES6 to the intermediate or terminal electron acceptor whether that was Cr(VI), Fe(III), or AQDS.     

Reduction of Fe(III) oxide by methanogens in the presence and absence of extracellular quinones

Five methanogens (Methanosarcina barkeri MS, Methanosphaera cuniculi 1R7, Methanobacterium palustre F, Methanococcus voltaei A3 and Methanolobus vulcani PL-12/M) were investigated for their ability to reduce Fe(III) oxide and the soluble quinone anthraquinone-2,6-disulphonate (AQDS). Two species (M. barkeri and M. voltaei) reduced significant amounts of Fe(III) oxide using hydrogen as the electron donor, and 0.1 mM AQDS greatly accelerated Fe(III) reduction by these organisms. Although Fe(III) appeared to inhibit growth and methanogenesis of some strains, hydrogen partial pressures under donor-limited conditions were much lower (<0.5 Pa) in the presence of Fe(III) than in normal media (1-10 Pa) for all species except for M. vulcani. These results demonstrate that electrons were transferred to Fe(III) by hydrogen-utilizing methanogens even when growth and methanogenesis were inhibited. All species except the obligate methylotroph M. vulcani were able to reduce AQDS when their growth substrates were present as electron donors, and rates were highest when organisms used hydrogen as the electron donor. Purified soil humic acids could also be reduced by the AQDS-reducing methanogens. The ability of methanogens to interact with extracellular quinones, humic acids and Fe(III) oxides raises the possibility that this functional group of organ-isms contributes to Fe(III) and humic acid reduction under certain conditions in the environment and provides an alternative explanation for the inhibition of methanogenesis in some Fe(III)-containing ecosystems.     

Characterization of Fe(III) Reduction by Chlororespiring Anaeromxyobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [¹⁴C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.     

Effect of iron(III), humic acids and anthraquinone-2,6-disulfonate on biodegradation of cyclic nitramines by Clostridium sp. EDB2

AIMS: To determine the biodegradation of cyclic nitramines by an anaerobic marine bacterium, Clostridium sp. EDB2, in the presence of Fe(III), humic acids (HA) and anthraquinone-2,6-disulfonate (AQDS). METHODS AND RESULTS: An obligate anaerobic bacterium, Clostridium sp. EDB2, degraded RDX and HMX, and produced similar product distribution including nitrite, methylenedinitramine, nitrous oxide, ammonium, formaldehyde, formic acid and carbon dioxide. Carbon (C) and nitrogen (N) mass balance for RDX products were 87% and 82%, respectively, and for HMX were 88% and 74%, respectively. Bacterial growth and biodegradation of RDX and HMX were stimulated in the presence of Fe(III), HA and AQDS suggesting that strain EDB2 utilized Fe(III), HA and AQDS as redox mediators to transfer electrons to cyclic nitramines. CONCLUSIONS: Strain EDB2 demonstrated a multidimensional approach to degrade RDX and HMX: first, direct degradation of the chemicals; second, indirect degradation by reducing Fe(III) to produce reactive-Fe(II); third, indirect degradation by reducing HA and AQDS which act as electron shuttles to transfer electrons to the cyclic nitramines. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study could be helpful in determining the fate of cyclic nitramine energetic chemicals in the environments rich in Fe(III) and HA.     

Periplasmic Cytochrome c3 of Desulfovibrio vulgaris Is Directly Involved in H2-Mediated Metal but Not Sulfate Reduction

Kinetic parameters and the role of cytochrome c₃ in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (K_m = 220 μM), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H₂ and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H₂ and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H₂, lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate- or pyruvate-reduced, but not H₂-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H₂ was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H₂ was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c₃ is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.     

Shewanella oneidensis MR-1 Restores Menaquinone Synthesis to a Menaquinone-Negative Mutant

The mechanisms underlying the use of insoluble electron acceptors by metal-reducing bacteria, such as Shewanella oneidensis MR-1, are currently under intensive study. Current models for shuttling electrons across the outer membrane (OM) of MR-1 include roles for OM cytochromes and the possible excretion of a redox shuttle. While MR-1 is able to release a substance that restores the ability of a menaquinone (MK)-negative mutant, CMA-1, to reduce the humic acid analog anthraquinone-2,6-disulfonate (AQDS), cross-feeding experiments conducted here showed that the substance released by MR-1 restores the growth of CMA-1 on several soluble electron acceptors. Various strains derived from MR-1 also release this substance; these include mutants lacking the OM cytochromes OmcA and OmcB and the OM protein MtrB. Even though strains lacking OmcB and MtrB cannot reduce Fe(III) or AQDS, they still release a substance that restores the ability of CMA-1 to use MK-dependent electron acceptors, including AQDS and Fe(III). Quinone analysis showed that this released substance restores MK synthesis in CMA-1. This ability to restore MK synthesis in CMA-1 explains the cross-feeding results and challenges the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration.     

Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate

A thermophilic bacterium that can use O2, NO3-, Fe(III), and S0 as terminal electron acceptors for growth was isolated from groundwater sampled at a 3.2-km depth in a South African gold mine. This organism, designated SA-01, clustered most closely with members of the genus Thermus, as determined by 16S rRNA gene (rDNA) sequence analysis. The 16S rDNA sequence of SA-01 was >98% similar to that of Thermus strain NMX2 A.1, which was previously isolated by other investigators from a thermal spring in New Mexico. Strain NMX2 A.1 was also able to reduce Fe(III) and other electron acceptors. Neither SA-01 nor NMX2 A.1 grew fermentatively, i.e., addition of an external electron acceptor was required for anaerobic growth. Thermus strain SA-01 reduced soluble Fe(III) complexed with citrate or nitrilotriacetic acid (NTA); however, it could reduce only relatively small quantities (0.5 mM) of hydrous ferric oxide except when the humic acid analog 2,6-anthraquinone disulfonate was added as an electron shuttle, in which case 10 mM Fe(III) was reduced. Fe(III)-NTA was reduced quantitatively to Fe(II); reduction of Fe(III)-NTA was coupled to the oxidation of lactate and supported growth through three consecutive transfers. Suspensions of Thermus strain SA-01 cells also reduced Mn(IV), Co(III)-EDTA, Cr(VI), and U(VI). Mn(IV)-oxide was reduced in the presence of either lactate or H2. Both strains were also able to mineralize NTA to CO2 and to couple its oxidation to Fe(III) reduction and growth. The optimum temperature for growth and Fe(III) reduction by Thermus strains SA-01 and NMX2 A.1 is approximately 65 degrees C; their optimum pH is 6.5 to 7.0. This is the first report of a Thermus sp. being able to couple the oxidation of organic compounds to the reduction of Fe, Mn, or S.     

Immobilization of Radionuclides and Heavy Metals through Anaerobic Bio-Oxidation of Fe(II)

Adsorption of heavy metals and radionuclides (HMR) onto iron and manganese oxides has long been recognized as an important reaction for the immobilization of these compounds. However, in environments containing elevated concentrations of these HMR the adsorptive capacity of the iron and manganese oxides may well be exceeded, and the HMR can migrate as soluble compounds in aqueous systems. Here we demonstrate the potential of a bioremediative strategy for HMR stabilization in reducing environments based on the recently described anaerobic nitrate-dependent Fe(II) oxidation by Dechlorosoma species. Bio-oxidation of 10 mM Fe(II) and precipitation of Fe(III) oxides by these organisms resulted in rapid adsorption and removal of 55 μM uranium and 81 μM cobalt from solution. The adsorptive capacity of the biogenic Fe(III) oxides was lower than that of abiotically produced Fe(III) oxides (100 μM for both metals), which may have been a result of steric hindrance by the microbial cells on the iron oxide surfaces. The binding capacity of the biogenic oxides for different heavy metals was indirectly correlated to the atomic radius of the bound element. X-ray absorption spectroscopy indicated that the uranium was bound to the biogenically produced Fe(III) oxides as U(VI) and that the U(VI) formed bidentate and tridentate inner-sphere complexes with the Fe(III) oxide surfaces. Dechlorosoma suillum oxidation was specific for Fe(II), and the organism did not enzymatically oxidize U(IV) or Co(II). Small amounts (less than 2.5 μM) of Cr(III) were reoxidized by D. suillum; however, this appeared to be inversely dependent on the initial concentration of the Cr(III). The results of this study demonstrate the potential of this novel approach for stabilization and immobilization of HMR in the environment.