Eric E. Roden

Enzymatic iron and uranium reduction by sulfate-reducing bacteria

Abstract The potential for sulfate-reducing bacteria (SRB) to enzymatically reduce Fe(III) and U(VI) was investigated. Five species of Desulfovibrio as well as Desulfobacterium autotrophicum and Desulfobulbus propionicus reduced Fe(III) chelated with nitrilotriacetic acid as well as insoluble Fe(III) oxide. Fe(III) oxide reduction resulted in the accumulation of magnetite and siderite. Desulfobacter postgatei reduced the chelated Fe(III) but not Fe(III) oxide. Desulfobacter curvatus, Desulfomonile tiedjei, and Desulfotomaculum acetoxidans did not reduce Fe(III). Only Desulfovibrio species reduced U(VI). U(VI) reduction resulted in the precipitation of uraninite. None of the SRB that reduced Fe(III) or U(VI) appeared to conserve enough energy to support growth from this reaction. However, Desulfovibrio desulfuricans metabolized H2 down to lower concentrations with Fe(III) or U(VI) as the electron acceptor than with sulfate, suggesting that these metals may be preferred electron acceptors at the low H2 concentrations present in most marine sediments. Molybdate did not inhibit Fe(III) reduction by D. desulfuricans. This indicates that the inability of molybdate to inhibit Fe(III) reduction in marine sediments does not rule out the possibility that SRB are important catalysts for Fe(III) reduction. The results demonstrate that although SRB were previously considered to reduce Fe(III) and U(VI) indirectly through the production of sulfide, they may also directly reduce Fe(III) and U(VI) through enzymatic mechanisms. These findings, as well as our recent discovery that the So-reducing microorganism Desulfuromonas acetoxidans can reduce Fe(III), demonstrate that there are close links between the microbial sulfur, iron, and uranium cycles in anaerobic marine sediments.

Influence of Biogenic Fe(II) on Bacterial Crystalline Fe(III) Oxide Reduction

Bacterial crystalline Fe(III) oxide reduction has the potential to significantly influence the biogeochemistry of anaerobic sedimentary environments where crystalline Fe(III) oxides are abundant relative to poorly crystalline (amorphous) phases. A review of published data on solid-phase Fe(III) abundance and speciation indicates that crystalline Fe(III) oxides are frequently 2- to S 10-fold more abundant than amorphous Fe(III) oxides in shallow subsurface sediments not yet subjected to microbial Fe(III) oxide reduction activity. Incubation experiments with coastal plain aquifer sediments demonstrated that crystalline Fe(III) oxide reduction can contribute substantially to Fe(II) production in the presence of added electron donors and nutrients. Controls on crystalline Fe(III) oxide reduction are therefore an important consideration in relation to the biogeochemical impacts of bacterial Fe(III) oxide reduction in subsurface environments. In this paper, the influence of biogenic Fe(II) on bacterial reduction of crystalline Fe(III) oxides is reviewed and analyzed in light of new experiments conducted with the acetate-oxidizing, Fe(III)-reducing bacterium (FeRB) Geobacter metallireducens . Previous experiments with Shewanella algae strain BrY indicated that adsorption and/or surface precipitation of Fe(II) on Fe(III) oxide and FeRB cell surfaces is primarily responsible for cessation of goethite ( f -FeOOH) reduction activity after only a relatively small fraction (generally < 10%) of the oxide is reduced. Similar conclusions are drawn from analogous studies with G. metallireducens . Although accumulation of aqueous Fe(II) has the potential to impose thermodynamic constraints on the extent of crystalline Fe(III) oxide reduction, our data on bacterial goethite reduction suggest that this phenomenon cannot universally explain the low microbial reducibility of this mineral. Experiments examining the influence of exogenous Fe(II) (20 mM FeCl 2 ) on soluble Fe(III)-citrate reduction by G. metallireducens and S. algae showed that high concentrations of Fe(II) did not inhibit Fe(III)-citrate reduction by freshly grown cells, which indicates that surface-bound Fe(II) does not inhibit Fe(III) reduction through a classical end-product enzyme inhibition mechanism. However, prolonged exposure of G. metallireducens and S. algae cells to high concentrations of soluble Fe(II) did cause inhibition of soluble Fe(III) reduction. These findings, together with recent documentation of the formation of Fe(II) surface precipitates on FeRB in Fe(III)-citrate medium, provide further evidence for the impact of Fe(II) sorption by FeRB on enzymatic Fe(III) reduction. Two different, but not mutually exclusive, mechanisms whereby accumulation of Fe(II) coatings on Fe(III) oxide and FeRB surfaces may lead to inhibition of enzymatic Fe(III) oxide reduction activity (in the absence of soluble electron shuttles and/or Fe(III) chelators) are identified and discussed in relation to recent experimental work and theoretical considerations.

SUCCESSIONAL CHANGES IN BACTERIAL ASSEMBLAGE STRUCTURE DURING EPILITHIC BIOFILM DEVELOPMENT

Although bacteria are often treated as one entity in ecological studies, bacterial assemblages are composed of individual species populations. Bacterial assemblages can have their own richness and structure, analogous to communities of plants and animals, although few studies have attempted to describe spatial or temporal patterns in their structure. In this study, we examined successional changes in the structure of bacterial assemblages using denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction amplified 16S rDNA fragments. Bacterial biofilm assemblages developing on glass slides in a mesocosm and a small lake showed an initial increase in richness over the first week, followed by a slight decrease and a subsequent increase after two to three months. Functional changes in the bacterial community were examined using most probable number estimates and revealed decreases in the abundance of glucose- and cellulose-degraders during biofilm development, whereas benzoate-degraders became more abundant in the lake biofilms. The banding patterns observed on DGGE gels were used to derive rank-abundance profiles for each stage of biofilm development. These profiles resembled those observed for communities of macroorganisms and could usually be described by geometric series models. These models suggested greater equitability in bacterial community structure as the biofilms developed. A comparison of two successional series of biofilms separated by 30 d revealed that neither successional stage nor time of sampling was the major factor influencing bacterial assemblage structure. Our results allowed us to suggest a general model for the development of bacterial biofilm assemblages that emphasizes the interaction of species and resource diversity. This model suggests that, at least in biofilms, bacterial assemblages may not be structured by the resource competition or niche-driven patterns typical of communities of macroorganisms.

Microbial and surface chemistry controls on reduction of synthetic Fe(III) oxide minerals by the dissimilatory iron‐reducing bacterium Shewanella alga

The role of Fe(II) biosorption and the effect of medium components on the rate and long‐term extent of Fe(III) oxide reduction (FeRed) by a dissimilatory Fe(III)‐reducing bacterium (Shewanella alga strain BrY) were examined in batch culture experiments. Introduction of fresh S. alga cells into month‐old cultures in which Fe(III) reduction had ceased resulted in further reduction of synthetic amorphous Fe(III) oxide, hematite, and two forms of goethite (Gt). Fresh S. alga cells were also able to reduce a substantial amount of synthetic Gt that had been partly or completely saturated with sorbed Fe(II). Cells that had been precoated with Fe(II) showed a reduced rate and capacity for FeRed. These results indicated that biosorption of Fe(II) had a major impact on FeRed. S. alga cells were shown to have an Fe(II) sorption capacity of ∼0.1 mmol g−1, compared with ∼0.25 mmol g−1 determined for the synthetic Gt. Sorption experiments with component mixtures indicated that direct interaction between cells and oxide...

Bacterial reductive dissolution of crystalline Fe(III) oxide in continuous-flow column reactors

ABSTRACT Bacterial reductive dissolution of synthetic crystalline Fe(III) oxide-coated sand was studied in continuous-flow column reactors in comparison with parallel batch cultures. The cumulative amount of aqueous Fe(II) exported from the columns over a 6-month incubation period corresponded to (95.0 ± 3.7)% (n = 3) of their original Fe(III) content. Wet-chemical analysis revealed that only (6.5 ± 3.2)% of the initial Fe(III) content remained in the columns at the end of the experiment. The near-quantitative removal of Fe was visibly evidenced by extensive bleaching of color from the sand in the columns. In contrast to the column reactors, Fe(II) production quickly reached an asymptote in batch cultures, and only (13.0 ± 2.2)% (n = 3) of the Fe(III) oxide content was reduced. Sustained bacterial-cell growth occurred in the column reactors, leading to the production and export of a quantity of cells 100-fold greater than that added during inoculation. Indirect estimates of cell growth, based on the quantity of Fe(III) reduced, suggest that only an approximate doubling of initial cell abundance was likely to have occurred in the batch cultures. Our results indicate that removal of biogenic Fe(II) via aqueous-phase transport in the column reactors decreased the passivating influence of surface-bound Fe(II) on oxide reduction activity, thereby allowing a dramatic increase in the extent of Fe(III) oxide reduction and associated bacterial growth. These findings have important implications for understanding the fate of organic and inorganic contaminants whose geochemical behavior is linked to Fe(III) oxide reduction.

Suboxic Deposition of Ferric Iron by Bacteria in Opposing Gradients of Fe(II) and Oxygen at Circumneutral pH

ABSTRACT The influence of lithotrophic Fe(II)-oxidizing bacteria on patterns of ferric oxide deposition in opposing gradients of Fe(II) and O2 was examined at submillimeter resolution by use of an O2 microelectrode and diffusion microprobes for iron. In cultures inoculated with lithotrophic Fe(II)-oxidizing bacteria, the majority of Fe(III) deposition occurred below the depth of O2 penetration. In contrast, Fe(III) deposition in abiotic control cultures occurred entirely within the aerobic zone. The diffusion microprobes revealed the formation of soluble or colloidal Fe(III) compounds during biological Fe(II) oxidation. The presence of mobile Fe(III) in diffusion probes from live cultures was verified by washing the probes in anoxic water, which removed ca. 70% of the Fe(III) content of probes from live cultures but did not alter the Fe(III) content of probes from abiotic controls. Measurements of the amount of Fe(III) oxide deposited in the medium versus the probes indicated that ca. 90% of the Fe(III) deposited in live cultures was formed biologically. Our findings show that bacterial Fe(II) oxidation is likely to generate reactive Fe(III) compounds that can be immediately available for use as electron acceptors for anaerobic respiration and that biological Fe(II) oxidation may thereby promote rapid microscale Fe redox cycling at aerobic-anaerobic interfaces.

Potential for Microscale Bacterial Fe Redox Cycling at the Aerobic-Anaerobic Interface

Recent studies of bacterial Fe(II) oxidation at circumneutral pH by a newly-isolated lithotrophic β-Proteobacterium (strain TW2) are reviewed in relation to a conceptual model that accounts for the influence of biogenic Fe(III)-binding ligands on patterns of Fe(II) oxidation and Fe(III) oxide deposition in opposing gradients of Fe(II) and O2. The conceptual model envisions complexation of Fe(III) by biogenic ligands as mechanism which alters the locus of Fe(III) oxide deposition relative to Fe(II) oxidation so as to delay/retard cell encrustation with Fe(III) oxides. Experiments examining the potential for bacterial Fe redox cycling in microcosms containing ferrihydrite-coated sand and a coculture of a lithotrophic Fe(II)-oxidizing bacterium (strain TW2) and a dissimilatory Fe(III)-reducing bacterium (Shewanella algae strain BrY) are described and interpreted in relation to an extended version of the conceptual model in which Fe(III)-binding ligands promote rapid microscale Fe redox cycling. The coculture systems showed minimal Fe(III) oxide accumulation at the sand-water interface, despite intensive O2 input from the atmosphere and measurable dissolved O2 to a depth of 2 mm below the sand-water interface. In contrast, a distinct layer of oxide precipitates formed in systems containing Fe(III)-reducing bacteria alone. Voltammetric microelectrode measurements revealed much lower concentrations of dissolved Fe(II) in the coculture systems. Examination of materials from the cocultures by fluorescence in situ hybridization indicated close physical juxtapositioning of Fe(II)-oxidizing and Fe(III)reducing bacteria in the upper few mm of sand. Together these results indicate that Fe(II)-oxidizing bacteria have the potential to enhance the coupling of Fe(II) oxidation and Fe(III) reduction at redox interfaces, thereby promoting rapid microscale cycling of Fe.

Microbial Iron Redox Cycling in a Circumneutral-pH Groundwater Seep

ABSTRACT The potential for microbially mediated redox cycling of iron (Fe) in a circumneutral-pH groundwater seep in north central Alabama was studied. Incubation of freshly collected seep material under anoxic conditions with acetate-lactate or H2 as an electron donor revealed the potential for rapid Fe(III) oxide reduction (ca. 700 to 2,000 μmol liter−1 day−1). Fe(III) reduction at lower but significant rates took place in unamended controls (ca. 300 μmol liter−1 day−1). Culture-based enumerations (most probable numbers [MPNs]) revealed significant numbers (102 to 106 cells ml−1) of organic carbon- and H2-oxidizing dissimilatory Fe(III)-reducing microorganisms. Three isolates with the ability to reduce Fe(III) oxides by dissimilatory or fermentative metabolism were obtained (Geobacter sp. strain IST-3, Shewanella sp. strain IST-21, and Bacillus sp. strain IST-38). MPN analysis also revealed the presence of microaerophilic Fe(II)-oxidizing microorganisms (103 to 105 cells ml−1). A 16S rRNA gene library from the iron seep was dominated by representatives of the Betaproteobacteria including Gallionella, Leptothrix, and Comamonas species. Aerobic Fe(II)-oxidizing Comamonas sp. strain IST-3 was isolated. The 16S rRNA gene sequence of this organism is 100% similar to the type strain of the betaproteobacterium Comamonas testosteroni (M11224). Testing of the type strain showed no Fe(II) oxidation. Collectively our results suggest that active microbial Fe redox cycling occurred within this habitat and support previous conceptual models for how microbial Fe oxidation and reduction can be coupled in surface and subsurface sedimentary environments.

Repeated anaerobic microbial redox cycling of iron.

ABSTRACT Some nitrate- and Fe(III)-reducing microorganisms are capable of oxidizing Fe(II) with nitrate as the electron acceptor. This enzymatic pathway may facilitate the development of anaerobic microbial communities that take advantage of the energy available during Fe-N redox oscillations. We examined this phenomenon in synthetic Fe(III) oxide (nanocrystalline goethite) suspensions inoculated with microflora from freshwater river floodplain sediments. Nitrate and acetate were added at alternate intervals in order to induce repeated cycles of microbial Fe(III) reduction and nitrate-dependent Fe(II) oxidation. Addition of nitrate to reduced, acetate-depleted suspensions resulted in rapid Fe(II) oxidation and accumulation of ammonium. High-resolution transmission electron microscopic analysis of material from Fe redox cycling reactors showed amorphous coatings on the goethite nanocrystals that were not observed in reactors operated under strictly nitrate- or Fe(III)-reducing conditions. Microbial communities associated with N and Fe redox metabolism were assessed using a combination of most-probable-number enumerations and 16S rRNA gene analysis. The nitrate-reducing and Fe(III)-reducing cultures were dominated by denitrifying Betaproteobacteria (e.g., Dechloromonas) and Fe(III)-reducing Deltaproteobacteria (Geobacter), respectively; these same taxa were dominant in the Fe cycling cultures. The combined chemical and microbiological data suggest that both Geobacter and various Betaproteobacteria participated in nitrate-dependent Fe(II) oxidation in the cycling cultures. Microbially driven Fe-N redox cycling may have important consequences for both the fate of N and the abundance and reactivity of Fe(III) oxides in sediments.

Characterization of a Neutrophilic, Chemolithoautotrophic Fe(II)-Oxidizing β -Proteobacterium from Freshwater Wetland Sediments

A neutrophilic Fe(II)-oxidizing bacterium (FeOB) isolated from Fe-rich freshwater wetland sediments has been phylogenetically and physiologically characterized. The 16S rRNA gene sequence of this organism (designated strain TW2) places it among the Rhodocyclus group within the β-proteobacteria. The closest known relative to strain TW2 is the heterotrophic perchlorate reducer Dechlorosoma suillum, with 94% 16S rRNA gene identity. TW2 grows chemolithoautotrophically with Fe(II) as an electron donor and O2 as an electron acceptor. Inorganic carbon fixation during growth on Fe(II) was demonstrated via H14CO3 − fixation experiments. The organism can also grow organotrophically with acetate as the sole carbon and energy source, and can utilize acetate as an auxiliary source of fixed carbon which enhances cell yield (2–3-fold) during lithotrophic growth on Fe(II). No other electron donors and no electron acceptors other than O2 were utilized. The organisms ability to grow with Fe(II) and acetate, along with its limitations with respect to electron acceptor utilization, suggests a specific adaptation to microaerobic niches in redox interfacial environments. The unique metabolism of strain TW2, together with the 16S rRNA sequence data, suggests that this organism represents a novel taxonomic group at the genus level.