Reducing Bacterium Geobacter Sulfurreducens Research Articles

Microbial Reduction of Antimony(V)-Bearing Ferrihydrite by Geobacter sulfurreducens.

The reduction of Sb(V)-bearing ferrihydrite by Geobacter sulfurreducens was studied to determine the fate of the metalloid in Fe-rich systems undergoing redox transformations. Sb(V) added at a range of concentrations adsorbed readily to ferrihydrite, and the loadings had a pronounced impact on the rate and extent of Fe(III) reduction and the products formed. Magnetite dominated at low (0.5 and 1 mol%) Sb(V) concentrations, with crystallite sizes decreasing at higher Sb loadings: 37-, 25-, and 17-nm particles for no-Sb, 0.5% Sb, and 1% Sb samples, respectively. In contrast, goethite was the dominant end product for samples with higher antimony loadings (2 and 5 mol%), with increased goethite grain size in the 5% Sb sample. Inductively coupled mass spectrometry (ICP-MS) analysis confirmed that Sb was not released to solution during the bioreduction process, and X-ray photoelectron spectroscopy (XPS) analyses showed that no Sb(III) was formed throughout the experiments, confirming that the Fe(III)-reducing bacterium Geobacter sulfurreducens cannot reduce Sb(V) enzymatically or via biogenic Fe(II). These findings suggest that Fe (bio)minerals have a potential role in limiting antimony pollution in the environment, even when undergoing redox transformations. IMPORTANCE Antimony is an emerging contaminant that shares chemical characteristics with arsenic. Metal-reducing bacteria (such as Geobacter sulfurreducens) can cause the mobilization of arsenic from Fe(III) minerals under anaerobic conditions, causing widespread contamination of aquifers worldwide. This research explores whether metal-reducing bacteria can drive the mobilization of antimony under similar conditions. In this study, we show that G. sulfurreducens cannot reduce Sb(V) directly or cause Sb release during the bioreduction of the Fe(III) mineral ferrihydrite [although the sorbed Sb(V) did alter the Fe(II) mineral end products formed]. Overall, this study highlights the tight associations between Fe and Sb in environmental systems, suggesting that the microbial reduction of Fe(III)/Sb mineral assemblages may not lead to Sb release (in stark contrast to the mobilization of As in iron-rich systems) and offers potential Fe-based remediation options for Sb-contaminated environments.

Microbial Biosynthesis of Thiol Compounds: Implications for Speciation, Cellular Uptake, and Methylation of Hg(II).

Cellular uptake of inorganic divalent mercury (Hg(II)) is a key step in microbial formation of neurotoxic methylmercury (MeHg), but the mechanisms remain largely unidentified. We show that the iron reducing bacterium Geobacter sulfurreducens produces and exports appreciable amounts of low molecular mass thiol (LMM-RSH) compounds reaching concentrations of about 100 nM in the assay medium. These compounds largely control the chemical speciation and bioavailability of Hg(II) by the formation of Hg(LMM-RS)2 complexes (primarily with cysteine) in assays without added thiols. By characterizing these effects, we show that the thermodynamic stability of Hg(II)-complexes is a principal controlling factor for Hg(II) methylation by this bacterium such that less stable complexes with mixed ligation involving LMM-RSH, OH-, and Cl- are methylated at higher rates than the more stable Hg(LMM-RS)2 complexes. The Hg(II) methylation rate across different Hg(LMM-RS)2 compounds is also influenced by the chemical structure of the complexes. In contrast to the current perception of microbial uptake of Hg, our results adhere to generalized theories for metal biouptake based on metal complexation with cell surface ligands and refine the mechanistic understanding of Hg(II) availability for microbial methylation.

Scale-up of the production of highly reactive biogenic magnetite nanoparticles using Geobacter sulfurreducens.

Although there are numerous examples of large-scale commercial microbial synthesis routes for organic bioproducts, few studies have addressed the obvious potential for microbial systems to produce inorganic functional biomaterials at scale. Here we address this by focusing on the production of nanoscale biomagnetite particles by the Fe(III)-reducing bacterium Geobacter sulfurreducens, which was scaled up successfully from laboratory- to pilot plant-scale production, while maintaining the surface reactivity and magnetic properties which make this material well suited to commercial exploitation. At the largest scale tested, the bacterium was grown in a 50 l bioreactor, harvested and then inoculated into a buffer solution containing Fe(III)-oxyhydroxide and an electron donor and mediator, which promoted the formation of magnetite in under 24 h. This procedure was capable of producing up to 120 g of biomagnetite. The particle size distribution was maintained between 10 and 15 nm during scale-up of this second step from 10 ml to 10 l, with conserved magnetic properties and surface reactivity; the latter demonstrated by the reduction of Cr(VI). The process presented provides an environmentally benign route to magnetite production and serves as an alternative to harsher synthetic techniques, with the clear potential to be used to produce kilogram to tonne quantities.

Redox cycling of Fe(II) and Fe(III) in magnetite by Fe-metabolizing bacteria.

Microorganisms are a primary control on the redox-induced cycling of iron in the environment. Despite the ability of bacteria to grow using both Fe(II) and Fe(III) bound in solid-phase iron minerals, it is currently unknown whether changing environmental conditions enable the sharing of electrons in mixed-valent iron oxides between bacteria with different metabolisms. We show through magnetic and spectroscopic measurements that the phototrophic Fe(II)-oxidizing bacterium Rhodopseudomonas palustris TIE-1 oxidizes magnetite (Fe3O4) nanoparticles using light energy. This process is reversible in co-cultures by the anaerobic Fe(III)-reducing bacterium Geobacter sulfurreducens. These results demonstrate that Fe ions bound in the highly crystalline mineral magnetite are bioavailable as electron sinks and electron sources under varying environmental conditions, effectively rendering magnetite a naturally occurring battery.

Biogenic nano-magnetite and nano-zero valent iron treatment of alkaline Cr(VI) leachate and chromite ore processing residue

Highly reactive nano-scale biogenic magnetite (BnM), synthesized by the Fe(III)-reducing bacterium Geobacter sulfurreducens, was tested for the potential to remediate alkaline Cr(VI) contaminated waters associated with chromite ore processing residue (COPR). The performance of this biomaterial, targeting aqueous Cr(VI) removal, was compared to a synthetic alternative, nano-scale zero valent iron (nZVI). Samples of highly contaminated alkaline groundwater and COPR solid waste were obtained from a contaminated site in Glasgow, UK. During batch reactivity tests, Cr(VI) removal from groundwater was inhibited by ∼25% (BnM) and ∼50% (nZVI) when compared to the treatment of less chemically complex model pH 12 Cr(VI) solutions. In both the model Cr(VI) solutions and contaminated groundwater experiments the surface of the nanoparticles became passivated, preventing complete coupling of their available electrons to Cr(VI) reduction. To investigate this process, the surfaces of the reacted samples were analyzed by TEM-EDX, XAS and XPS, confirming Cr(VI) reduction to the less soluble Cr(III) on the nanoparticle surface. In groundwater reacted samples the presence of Ca, Si and S was also noted on the surface of the nanoparticles, and is likely responsible for earlier onset of passivation. Treatment of the solid COPR material in contact with water, by addition of increasing weight % of the nanoparticles, resulted in a decrease in aqueous Cr(VI) concentrations to below detection limits, via the addition of ⩾5% w/w BnM or ⩾1% w/w nZVI. XANES analysis of the Cr K edge, showed that the % Cr(VI) in the COPR dropped from 26% to a minimum of 4–7% by the addition of 5% w/w BnM or 2% w/w nZVI, with higher additions unable to reduce the remaining Cr(VI). The treated materials exhibited minimal re-mobilization of soluble Cr(VI) by re-equilibration with atmospheric oxygen, with the bulk of the Cr remaining in the solid fraction. Both nanoparticles exhibited a considerable capacity for the remediation of COPR related Cr(VI) contamination, with the synthetic nZVI demonstrating greater reactivity than the BnM. However, the biosynthesized BnM was also capable of significant Cr(VI) reduction and demonstrated a greater efficiency for the coupling of its electrons towards Cr(VI) reduction than the nZVI.

Redox interactions between Cr(VI) and Fe(II) in bioreduced biotite and chlorite.

Contamination of the environment with Cr as chromate (Cr(VI)) from industrial activities is of significant concern as Cr(VI) is a known carcinogen, and is mobile in the subsurface. The capacity of Fe(II)-containing phyllosilicates including biotite and chlorite to alter the speciation, and thus the mobility, of redox-sensitive contaminants including Cr(VI) is of great interest since these minerals are common in soils and sediments. Here, the capacity of bacteria, ubiquitous in the surface and near-surface environment, to reduce Fe(III) in phyllosilicate minerals and, thus, alter their redox reactivity was investigated in two-step anaerobic batch experiments. The model Fe(III)-reducing bacterium Geobacter sulfurreducens was used to reduce Fe(III) in the minerals, leading to a significant transformation of structural Fe(III) to Fe(II) of 0.16 mmol/g (∼ 40%) in biotite and 0.15 mmol/g (∼ 20%) in chlorite. The unaltered minerals could not remove Cr(VI) from solution despite containing a larger excess of Fe(II) than would be required to reduce all the added Cr(VI), unless they were supplied in a very high concentration (a 1:10 solid to solution ratio). By contrast, even at very low concentrations, the addition of bioreduced biotite and chlorite caused removal of Cr(VI) from solution, and surface and near surface X-ray absorption spectroscopy confirmed that this immobilization was through reductive transformation to Cr(III). We provide empirical evidence that the amount of Fe(II) generated by microbial Fe(III) reduction is sufficient to reduce the Cr(VI) removed and, in the absence of reduction by the unaltered minerals, suggest that only the microbially reduced fraction of the iron in the minerals is redox-active against the Cr(VI).

Biosynthesis of Zinc Substituted Magnetite Nanoparticles with Enhanced Magnetic Properties

The magnetic moments of magnetite nanoparticles are dramatically enhanced through the addition of zinc in a microbiologically driven synthesis procedure. The particles are produced through the reduction of Fe(III)‐compounds containing Zn(II) by the iron reducing bacterium Geobacter sulfurreducens. Results indicate a significant increase in the saturation magnetization by over 50% compared to magnetite at both room and low temperatures for relatively minor quantities of zinc substitution. A maximum saturation magnetization of nearly 100 emu g−1 of sample is measured at room temperature. Analysis of the cation site ordering reveals a complex dependence on the Zn content, with the combined effect of Zn substitution of Fe3+ ions on tetrahedral sites, together with Fe2+ cation oxidation, leading to the observed magnetization enhancement for low Zn doping levels. The improved magnetic properties give superior performance in MRI applications with an MRI contrast enhancement among the largest values reported, being more than 5 times larger than a commercial contrast agent (Feridex) measured under identical conditions. The synthesis technique applied here involves an environmentally benign route and offers the potential to tune the magnetic properties of magnetic nanoparticles, with increased overall magnetization desirable for many different commercial applications.

Harnessing the Extracellular Bacterial Production of Nanoscale Cobalt Ferrite with Exploitable Magnetic Properties

Nanoscale ferrimagnetic particles have a diverse range of uses from directed cancer therapy and drug delivery systems to magnetic recording media and transducers. Such applications require the production of monodisperse nanoparticles with well-controlled size, composition, and magnetic properties. To fabricate these materials purely using synthetic methods is costly in both environmental and economical terms. However, metal-reducing microorganisms offer an untapped resource to produce these materials. Here, the Fe(III)-reducing bacterium Geobacter sulfurreducens is used to synthesize magnetic iron oxide nanoparticles. A combination of electron microscopy, soft X-ray spectroscopy, and magnetometry techniques was employed to show that this method of biosynthesis results in high yields of crystalline nanoparticles with a narrow size distribution and magnetic properties equal to the best chemically synthesized materials. In particular, it is demonstrated here that cobalt ferrite (CoFe(2)O(4)) nanoparticles with low temperature coercivity approaching 8 kOe and an effective anisotropy constant of ∼10(6) erg cm(-3) can be manufactured through this biotechnological route. The dramatic enhancement in the magnetic properties of the nanoparticles by the introduction of high quantities of Co into the spinel structure represents a significant advance over previous biomineralization studies in this area using magnetotactic bacteria. The successful production of nanoparticulate ferrites achieved in this study at high yields could open up the way for the scaled-up industrial manufacture of nanoparticles using environmentally benign methodologies.

Time-resolved synchrotron powder X-ray diffraction study of magnetite formation by the Fe(III)-reducing bacterium Geobacter sulfurreducens

The Fe(III)-reducing bacterium Geobacter sulfurreducens produces nanometer-sized magnetite by transferring electrons from organic matter or hydrogen to ferrihydrite, resulting in the reductive transformation of the ferrihydrite to magnetite, and the generation of ATP for growth. Electron transfer can occur by direct contact between the cell surface and the mineral or via a soluble shuttle, for example a quinone-containing humic species. The minerals produced at different stages of ferrihydrite reduction during two experiments, one with and one without the humic analog anthraquinone-2, 6-disulphonate (AQDS), were measured using high-resolution synchrotron powder X-ray diffraction. Amorphous 2-line ferrihydrite converts to goethite, then to a mixture of goethite and magnetite, and finally to magnetite. Samples with and without AQDS showed the same general mineralogical trends, and the rate of reaction was faster in the presence of AQDS. In addition, two transient minerals structurally similar to goethite and magnetite were observed to form as intermediates between ferrihydrite and goethite and goethite and magnetite, but only in samples produced in the absence of the electron shuttle. These transient minerals were named proto-goethite and proto-magnetite. Proto-goethite has a shorter c-axis [4.467(20) angstrom] than crystalline goethite, a function of size (<2 nm) where quantum properties prevail. Proto-magnetite is identified by long tetrahedral (2.113 angstrom) and short octahedral (1.943 angstrom) Fe-O bonds compared to stoichiometric magnetite, possibly indicative of a coordination crossover caused by charge density [Fe(II)] migration to tetrahedral sites. Fe(II) in solution or sorbed to the mineral surface is considered to be the catalyst causing the mineral transformations. The Fe(II) is thought to form predominantly from the reductive dissolution of 2-line ferrihydrite by G. sulfurreducens.

On the use of cyclic voltammetry for the study of anodic electron transfer in microbial fuel cells

In this communication we discuss, by means of the metal reducing bacterium Geobacter sulfurreducens, a strategy to use cyclic voltammetry for the study of anodic bioelectrocatalytic electron transfer in microbial fuel cells.

Growth of Geobacter sulfurreducens on Poorly Crystalline Fe(III) Oxyhydroxide Coatings

Few studies have examined the molecular to micron-scale interactions between dissimilatory Fe(III)-reducing bacteria and poorly crystalline Fe(III) phases which are frequently the most bioavailable Fe(III) sources in the subsurface. Here we describe methods for analysing these interactions using a range of chemical and spectroscopic techniques. Glass slides were coated with a synthetic poorly crystalline Fe(III) phase and then incubated in the presence of the Fe(III)-reducing bacterium Geobacter sulfurreducens and a suitable growth medium. Growth on the Fe(III)-coating was observed via cell staining and environmental scanning electron microscopy while microbial Fe(III) reduction was quantified using a colorimetric assay. However, following microbial reduction, Fe(II) could not be detected on the slide surface using X-ray photoelectron spectroscopy. Fe(II)-coated control slides showed that the mineral surface was not re-oxidised during handling or analysis. Further experiments intended to demonstrate removal of Tc(VII) and Cr(VI) from solution via abiotic reduction mediated by biogenic Fe(II) on the slide surface resulted in far lower levels of Tc(VII) and Cr(VI) reduction than expected. These data may indicate that the electrons transferred from G. sulfurreducens to poorly crystalline Fe(III) involves the deeper mineral structure, so that Fe(II) phases are not detectable on the surface. The environmental implications of this hypothesis are discussed.

Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens.

A 9.6 kDa periplasmic c -type cytochrome, designated PpcA, was purified from the Fe(III)-reducing bacterium Geobacter sulfurreducens and characterized. The purified protein is basic (pI 9.5), contains three haems and has an N-terminal amino acid sequence closely related to those of the previously described trihaem c (7) cytochromes of Geobacter metallireducens and Desulfuromonas acetoxidans. The gene encoding PpcA was identified from the G. sulfurreducens genome using the N-terminal sequence, and encodes a protein of 71 amino acids (molecular mass 9.58 kDa) with 49% identity to the c (7) cytochrome of D. acetoxidans. In order to determine the physiological role of PpcA, a knockout mutant was prepared with a single-step recombination method. Acetate-dependent Fe(III) reduction was significantly inhibited in both growing cultures and cell suspensions of the mutant. When ppcA was expressed in trans, the full capacity for Fe(III) reduction with acetate was restored. The transfer of electrons from acetate to anthraquinone 2,6-disulphonate (AQDS; a humic acid analogue) and to U(VI) was also compromised in the mutant, but acetate-dependent reduction of fumarate was not altered. The rates of reduction of Fe(III), AQDS, U(VI) and fumarate were also the same in the wild type and ppcA mutant when hydrogen was supplied as the electron donor. When taken together with previous studies on other electron transport proteins in G. sulfurreducens, these results suggest that PpcA serves as an intermediary electron carrier from acetate to terminal Fe(III) reductases in the outer membrane, and is also involved in the transfer of electrons from acetate to U(VI) and humics.

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 TcO(2) 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 TcO(2) 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 microM 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.

Characterization of a membrane-bound NADH-dependent Fe 3+ reductase from the dissimilatory Fe 3+-reducing bacterium Geobacter sulfurreducens

Geobacter sulfurreducens produces a single, membrane-associated Fe 3+ reductase activity when grown on fumarate or Fe 3+. The activity was initially isolated by solubilization of membranes with the non-ionic detergent dodecyl-β- D-maltoside, and partially purified by a combination of ion exchange chromatography and preparative non-denaturing gel electrophoresis. Molecular mass of the reductase, as determined by gel filtration chromatography, was approximately 300 kDa. Cofactor analysis of the purified reductase demonstrates that it contains a hemoprotein and flavin adenine dinucleotide. Kinetic and inhibitor studies show that the reductase is specific for NADH as electron donor, and confirm that the reductase enzymatically reduces Fe 3+. The cytochrome associated with the complex undergoes a reoxidation upon addition of Fe 3+ compounds, indicating an ability to pass reducing equivalents to Fe 3+. This is the first description of a purified NADH-dependent Fe 3+ reductase from a microorganism capable of coupling Fe 3+ reduction to growth.