Phenazines and Other Redox-Active Antibiotics Promote Microbial Mineral Reduction

Natural products with important therapeutic properties are known to be produced by a variety of soil bacteria, yet the ecological function of these compounds is not well understood. Here we show that phenazines and other redox-active antibiotics can promote microbial mineral reduction. Pseudomonas chlororaphis PCL1391, a root isolate that produces phenazine-1-carboxamide (PCN), is able to reductively dissolve poorly crystalline iron and manganese oxides, whereas a strain carrying a mutation in one of the phenazine-biosynthetic genes (phzB) is not; the addition of purified PCN restores this ability to the mutant strain. The small amount of PCN produced relative to the large amount of ferric iron reduced in cultures of P. chlororaphis implies that PCN is recycled multiple times; moreover, poorly crystalline iron (hydr)oxide can be reduced abiotically by reduced PCN. This ability suggests that PCN functions as an electron shuttle rather than an iron chelator, a finding that is consistent with the observation that dissolved ferric iron is undetectable in culture fluids. Multiple phenazines and the glycopeptidic antibiotic bleomycin can also stimulate mineral reduction by the dissimilatory iron-reducing bacterium Shewanella oneidensis MR1. Because diverse bacterial strains that cannot grow on iron can reduce phenazines, and because thermodynamic calculations suggest that phenazines have lower redox potentials than those of poorly crystalline iron (hydr)oxides in a range of relevant environmental pH (5 to 9), we suggest that natural products like phenazines may promote microbial mineral reduction in the environment.     

Influence of environmental conditions on the production of phenazine-1-carboxamide by Pseudomonas chlororaphis PCL1391

Pseudomonas chlororaphis PCL1391 produces the secondary metabolite phenazine-1-carboxamide (PCN), which is an antifungal metabolite required for biocontrol activity of the strain. Identification of conditions involved in PCN production showed that some carbon sources and all amino acids tested promote PCN levels. Decreasing the pH from 7 to 6 or decreasing the growth temperature from 21 to 16 degrees C decreased PCN production dramatically. In contrast, growth at 1% oxygen as well as low magnesium concentrations increased PCN levels. Salt stress, low concentrations of ferric iron, phosphate, sulfate, and ammonium ions reduced PCN levels. Fusaric acid, a secondary metabolite produced by the soilborne Fusarium spp. fungi, also reduced PCN levels. Different nitrogen sources greatly influenced PCN levels. Analysis of autoinducer levels at conditions of high and low PCN production demonstrated that, under all tested conditions, PCN levels correlate with autoinducer levels, indicating that the regulation of PCN production by environmental factors takes place at or before autoinducer production. Moreover, the results show that autoinducer production not only is induced by a high optical density but also can be induced by certain environmental conditions. We discuss our findings in relation to the success of biocontrol in the field.     

Rapid Assay for Microbially Reducible Ferric Iron in Aquatic Sediments

The availability of ferric iron for microbial reduction as directly determined by the activity of iron-reducing organisms was compared with its availability as determined by a newly developed chemical assay for microbially reducible iron. The chemical assay was based on the reduction of poorly crystalline ferric iron by hydroxylamine under acidic conditions. There was a strong correlation between the extent to which hydroxylamine could reduce various synthetic ferric iron forms and the susceptibility of the iron to microbial reduction in an enrichment culture of iron-reducing organisms. When sediments that contained hydroxylamine-reducible ferric iron were incubated under anaerobic conditions, ferrous iron accumulated as the concentration of hydroxylamine-reducible ferric iron declined over time. Ferrous iron production stopped as soon as the hydroxylamine-reducible ferric iron was depleted. In anaerobic incubations of reduced sediments that did not contain hydroxylamine-reducible ferric iron, there was no microbial iron reduction, even though the sediments contained high concentrations of oxalate-extractable ferric iron. A correspondence between the presence of hydroxylamine-reducible ferric iron and the extent of ferric iron reduction in anaerobic incubations was observed in sediments from an aquifer and in fresh- and brackish-water sediments from the Potomac River estuary. The assay is a significant improvement over previously described procedures for the determination of hydroxylamine-reducible ferric iron because it provides a correction for the high concentrations of solid ferrous iron which may also be extracted from sediments with acid. This is a rapid, simple technique to determine whether ferric iron is available for microbial reduction.     

Preliminary characterization and biological reduction of putative biogenic iron oxides (BIOS) from the Tonga-Kermadec Arc, southwest Pacific Ocean

Sediment samples were obtained from areas of diffuse hydrothermal venting along the seabed in the Tonga sector of the Tonga‐Kermadec Arc, southwest Pacific Ocean. Sediments from Volcano 1 and Volcano 19 were analyzed by X‐ray diffraction (XRD) and found to be composed primarily of the iron oxyhydroxide mineral, two‐line ferrihydrite. XRD also suggested the possible presence of minor amounts of more ordered iron (hydr)oxides (including six‐line ferrihydrite, goethite/lepidocrocite and magnetite) in the biogenic iron oxides (BIOS) from Volcano 1; however, Mössbauer spectroscopy failed to detect any mineral phases more crystalline than two‐line ferrihydrite. The minerals were precipitated on the surfaces of abundant filamentous microbial structures. Morphologically, some of these structures were similar in appearance to the known iron‐oxidizing genus Mariprofundus spp., suggesting that the sediments are composed of biogenic iron oxides. At Volcano 19, an areally extensive, active vent field, the microbial cells appeared to be responsible for the formation of cohesive chimney‐like structures of iron oxyhydroxide, 2–3 m in height, whereas at Volcano 1, an older vent field, no chimney‐like structures were apparent. Iron reduction of the sediment material (i.e. BIOS) by Shewanella putrefaciens CN32 was measured, in vitro, as the ratio of [total Fe(II)]:[total Fe]. From this parameter, reduction rates were calculated for Volcano 1 BIOS (0.0521 day^?1), Volcano 19 BIOS (0.0473 day^?1), and hydrous ferric oxide, a synthetic two‐line ferrihydrite (0.0224 day^?1). Sediments from both BIOS sites were more easily reduced than synthetic ferrihydrite, which suggests that the decrease in effective surface area of the minerals within the sediments (due to the presence of the organic component) does not inhibit subsequent microbial reduction. These results indicate that natural, marine BIOS are easily reduced in the presence of dissimilatory iron‐reducing bacteria, and that the use of common synthetic iron minerals to model their reduction may lead to a significant underestimation of their biological reactivity.     

Organic matter mineralization with the reduction of ferric iron: A review

A review of the literature indicates that numerous microorganisms can reduce ferric iron during the metabolism of organic matter. In most cases, the reduction of ferric iron appears to be enzymatically catalyzed and, in some instances, may be coupled to an electron transport chain that could generate ATP. However, the physiology and biochemistry of ferric iron reduction are poorly understood. In pure culture, ferric iron‐reducing organisms metabolize fermentable substrates, such as glucose, primarily to typical fermentation products, and transfer only a minor portion of the electron equivalents in the fermentable substrates to ferric iron. However, fermentation products, especially hydrogen and acetate, may be important electron donors for ferric iron reduction in natural environments. The ability of some organisms to couple the oxidation of fermentation products to the reduction of ferric iron means that it is possible for a food chain of iron‐reducing organisms to completely mineralize nonrecalcitrant organic matter with ferric iron as the sole electron acceptor. The rate and extent of ferric iron reduction depend on the forms of ferric iron that are available. Most of the ferric iron in sediments is resistant to microbial reduction. Ferric iron‐reducing organisms can exclude sulfate reduction and methane production from the zone of ferric iron reduction in sediments by outcompeting sulfate‐reducing and methanogenic food chains for organic matter when ferric iron is available as amorphic ferric oxyhydroxide. There are few quantitative estimates of the rates of ferric iron reduction in natural environments, but there is evidence that ferric iron reduction can be an important pathway for organic matter decomposition in some environments. There is a strong need for further study on all aspects of microbial reduction of ferric iron.     

Dominance of sulfur-fueled iron oxide reduction in low-sulfate freshwater sediments

A central tenant in microbial biogeochemistry is that microbial metabolisms follow a predictable sequence of terminal electron acceptors based on the energetic yield for the reaction. It is thereby oftentimes assumed that microbial respiration of ferric iron outcompetes sulfate in all but high-sulfate systems, and thus sulfide has little influence on freshwater or terrestrial iron cycling. Observations of sulfate reduction in low-sulfate environments have been attributed to the presumed presence of highly crystalline iron oxides allowing sulfate reduction to be more energetically favored. Here we identified the iron-reducing processes under low-sulfate conditions within columns containing freshwater sediments amended with structurally diverse iron oxides and fermentation products that fuel anaerobic respiration. We show that despite low sulfate concentrations and regardless of iron oxide substrate (ferrihydrite, Al-ferrihydrite, goethite, hematite), sulfidization was a dominant pathway in iron reduction. This process was mediated by (re)cycling of sulfur upon reaction of sulfide and iron oxides to support continued sulfur-based respiration—a cryptic sulfur cycle involving generation and consumption of sulfur intermediates. Although canonical iron respiration was not observed in the sediments amended with the more crystalline iron oxides, iron respiration did become dominant in the presence of ferrihydrite once sulfate was consumed. Thus, despite more favorable energetics, ferrihydrite reduction did not precede sulfate reduction and instead an inverse redox zonation was observed. These findings indicate that sulfur (re)cycling is a dominant force in iron cycling even in low-sulfate systems and in a manner difficult to predict using the classical thermodynamic ladder.     

The production of antifungal metabolites by Pseudomonas chlororaphis grown on different nutrient sources

It was found that the antifungal activity of Pseudomonas chlororaphis SPB1217 is due to phenazine-1-carboxylic acid, phenazine-1-carboxamide, and two unidentified exometabolites. The carbon source used for the growth of this bacterial strain and iron ions present in the medium considerably influenced the proportion between the antifungal metabolites. The maximum production of phenazines was observed in the media enriched in amino acids and iron ions. The absence of correlation between the production of phenazines and antifungal activity indicates that phenazines are not the only antifungal metabolites of the strain. Organic acids as nutrient sources provide for more intense production of exometabolites and for a higher level of antifungal activity than do sugars.     

Degradation of acyl-homoserine lactone molecules by Acinetobacter sp. strain C1010

A bacterium C1010, isolated from the rhizospheres of cucumbers in fields in Korea, degraded the microbial quorum-sensing molecules, hexanoyl homoserine lactone (HHSL), and octadecanoyl homoserine lactone (OHSL). Morphological characteristics and 16S rRNA sequence analysis identified C1010 as Acinetobacter sp. strain C1010. This strain was able to degrade the acyl-homoserine lactones (AHLs) produced by the biocontrol bacterium, Pseudomonas chlororaphis O6, and a phytopathogenic bacterium, Burkholderia glumae. Co-cultivation studies showed that the inactivation of AHLs by C1010 inhibited production of phenazines by P. chlororaphis O6. In virulence tests, the C1010 strain attenuated soft rot symptom caused by Erwinia carotovora ssp. carotovora. We suggest Acinetobacter sp. strain C1010 could be a useful bacterium to manipulate biological functions that are regulated by AHLs in various Gram-negative bacteria.     

Nanosized Iron Oxide Colloids Strongly Enhance Microbial Iron Reduction

Microbial iron reduction is considered to be a significant subsurface process. The rate-limiting bioavailability of the insoluble iron oxyhydroxides, however, is a topic for debate. Surface area and mineral structure are recognized as crucial parameters for microbial reduction rates of bulk, macroaggregate iron minerals. However, a significant fraction of iron oxide minerals in the subsurface is supposed to be present as nanosized colloids. We therefore studied the role of colloidal iron oxides in microbial iron reduction. In batch growth experiments with Geobacter sulfurreducens, colloids of ferrihydrite (hydrodynamic diameter, 336 nm), hematite (123 nm), goethite (157 nm), and akaganeite (64 nm) were added as electron acceptors. The colloidal iron oxides were reduced up to 2 orders of magnitude more rapidly (up to 1,255 pmol h⁻¹ cell⁻¹) than bulk macroaggregates of the same iron phases (6 to 70 pmol h⁻¹ cell⁻¹). The increased reactivity was not only due to the large surface areas of the colloidal aggregates but also was due to a higher reactivity per unit surface. We hypothesize that this can be attributed to the high bioavailability of the nanosized aggregates and their colloidal suspension. Furthermore, a strong enhancement of reduction rates of bulk ferrihydrite was observed when nanosized ferrihydrite aggregates were added.Dissimilatory iron reduction is an important anaerobic respiration process in anoxic subsurface environments. However, the reactivity of ferric iron is mostly limited by the reduction kinetics of the poorly soluble, extracellular iron minerals. Electron transfer from microorganisms to iron oxides can occur via direct contact or by electron shuttling compounds (46). Transport of the electron shuttle between the redox partners is then assumed to occur via diffusion. For example, humic substances can serve as natural electron shuttles that can be reduced by microorganisms and subsequently chemically oxidized by the ferric oxide (18). Shewanella oneidensis excretes a flavin to stimulate hematite reduction, functioning in a similar manner (27). As another option, formation of conductive pili serving as nanowires was described as a possible way of transferring electrons to the oxide surface (15, 34). Nevertheless, direct attachment has been recognized as a major mode of accessing iron oxides as electron acceptors (12). Direct transfer between microbial outer membrane reductases and the ferric minerals, however, requires close contact of less than 14 Å between the terminal iron reductase on the cell surface and the iron oxide molecule at the mineral surface (19, 25), limiting the rates of electron transfer between cell and mineral.Several parameters have been discussed in this context as being decisive for the bioavailability and reactivity of iron oxides, such as, e.g., the mineral surface area (8, 41). Larger surface areas have been shown to be accompanied by higher initial reduction rates. Another parameter that might determine reactivity is the low solubility of ferric iron in water at neutral pH (20). Low solubility entails high crystallinity, which reduces reaction rates (4). Therefore, crystalline bulk iron phases such as goethite or hematite (9) are poorly reducible by microorganisms, in contrast to amorphous ferrihydrite (41). Naturally, well crystalline minerals have lower surface areas, and the effects of surface area and solubility cannot be distinguished sharply. Cell density, initial oxide and substrate concentrations, and ferrous iron adsorbed to the bulk mineral surface were also reported to control microbial reduction rates by exhibiting mutual saturation behavior in Michaelis-Menten-type kinetics (3, 22, 40).The latter studies also considered particle sizes, a parameter that has often been overlooked so far. All concepts mentioned above generally assumed a bulk state of the electron-accepting iron oxide. Indeed, iron oxides used in microbiological experiments appear mainly as coarse, flocculating macroaggregates, visible to the naked eye as sludge-like precipitates. In nature, however, nanosized iron oxides are abundant (32, 45) and play a vital role in many biogeochemical processes (2, 16, 28). Such nanoparticles may appear in stable colloidal suspension, even if aggregated as a stable cluster of multiple particles (13). Ferric oxide particles can appear in colloidal suspensions of different aggregate sizes and densities.Different particle aggregate sizes might influence the bioavailability of iron oxides in microbial reduction. Nanosized aggregates appearing in colloidal suspensions might be spatially more accessible for microorganisms than large aggregates flocculating as bulk phases. Therefore, the present study aims at assessing the reactivity and putative role of aggregate sizes of iron oxides in dissimilatory iron reduction. A set of ferrihydrite, hematite, goethite, and akaganeite colloids was compared to their respective noncolloidal bulk phases to evaluate this effect.     

Ferric Iron Reduction by Acidophilic Heterotrophic Bacteria

Fifty mesophilic and five moderately thermophilic strains of acidophilic heterotrophic bacteria were tested for the ability to reduce ferric iron in liquid and solid media under aerobic conditions; about 40% of the mesophiles (but none of the moderate thermophiles) displayed at least some capacity to reduce iron. Both rates and extents of ferric iron reduction were highly strain dependent. No acidophilic heterotroph reduced nitrate or sulfate, and (limited) reduction of manganese(IV) was noted in only one strain (Acidiphilium facilis), an acidophile which did not reduce iron. Insoluble forms of ferric iron, both amorphous and crystalline, were reduced, as well as soluble iron. There was evidence that, in at least some acidophilic heterotrophs, iron reduction was enzymically mediated and that ferric iron could act as a terminal electron acceptor. In anaerobically incubated cultures, bacterial biomass increased with increasing concentrations of ferric but not ferrous iron. Mixed cultures of Thiobacillus ferrooxidans or Leptospirillum ferrooxidans and an acidophilic heterotroph (SJH) produced sequences of iron cycling in ferrous iron-glucose media.     

Iron metabolism in anoxic environments at near neutral pH

Anaerobic dissimilatory ferric iron-reducing and ferrous iron-oxidizing bacteria gain energy through reduction or oxidation of iron minerals and presumably play an important role in catalyzing iron transformations in anoxic environments. Numerous ferric iron-reducing bacteria have been isolated from a great diversity of anoxic environments, including sediments, soils, deep terrestrial subsurfaces, and hot springs. In contrast, only few ferrous iron-oxidizing bacteria are known so far. At neutral pH, iron minerals are barely soluble, and the mechanisms of electron transfer to or from iron minerals are still only poorly understood. In natural habitats, humic substances may act as electron carriers for ferric iron-reducing bacteria. Also fermenting bacteria were shown to channel electrons to ferric iron via humic acids. Whether quinones or cytochromes released from cells act as electron transfer components in ferric iron reduction is still a matter of debate. Anaerobic ferrous iron-oxidizing phototrophic bacteria, on the other hand, appear to excrete complexing agents to prevent precipitation of ferric iron oxides at their cell surfaces. The present review evaluates recent findings on the physiology of ferric iron-reducing and ferrous iron-oxidizing bacteria with respect to their relevance to microbial iron transformations in nature.     

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.     

Methanogens rapidly transition from methane production to iron reduction

Methanogenesis, the microbial methane (CH₄) production, is traditionally thought to anchor the mineralization of organic matter as the ultimate respiratory process in deep sediments, despite the presence of oxidized mineral phases, such as iron oxides. This process is carried out by archaea that have also been shown to be capable of reducing iron in high levels of electron donors such as hydrogen. The current pure culture study demonstrates that methanogenic archaea (Methanosarcina barkeri) rapidly switch from methanogenesis to iron‐oxide reduction close to natural conditions, with nitrogen atmosphere, even when faced with substrate limitations. Intensive, biotic iron reduction was observed following the addition of poorly crystalline ferrihydrite and complex organic matter and was accompanied by inhibition of methane production. The reaction rate of this process was of the first order and was dependent only on the initial iron concentrations. Ferrous iron production did not accelerate significantly with the addition of 9,10‐anthraquinone‐2,6‐disulfonate (AQDS) but increased by 11–28% with the addition of phenazine‐1‐carboxylate (PCA), suggesting the possible role of methanophenazines in the electron transport. The coupling between ferrous iron and methane production has important global implications. The rapid transition from methanogenesis to reduction of iron–oxides close to the natural conditions in sediments may help to explain the globally‐distributed phenomena of increasing ferrous concentrations below the traditional iron reduction zone in the deep ‘methanogenic’ sediment horizon, with implications for metabolic networking in these subsurface ecosystems and in past geological settings.     

Sorption of Fe (hydr)oxides to the surface of Shewanella putrefaciens: cell-bound fine-grained minerals are not always formed de novo.

Shewanella putrefaciens, a gram-negative, facultative anaerobe, is active in the cycling of iron through its interaction with Fe (hydr)oxides in natural environments. Fine-grained Fe precipitates that are attached to the outer membranes of many gram-negative bacteria have most often been attributed to precipitation and growth of the mineral at the cell surface. Our study of the sorption of nonbiogenic Fe (hydr)oxides revealed, however, that large quantities of nanometer-scale ferrihydrite (hydrous ferric oxide), goethite (alpha-FeOOH), and hematite (alpha-Fe(2)O(3)) adhered to the cell surface. Attempts to separate suspensions of cells and minerals with an 80% glycerin cushion proved that the sorbed minerals were tightly attached to the bacteria. The interaction between minerals and cells resulted in the formation of mineral-cell aggregates, which increased biomass density and provided better sedimentation of mineral Fe compared to suspensions of minerals alone. Transmission electron microscopy observations of cells prepared by whole-mount, conventional embedding, and freeze-substitution methods confirmed the close association between cells and minerals and suggested that in some instances, the mineral crystals had even penetrated the outer membrane and peptidoglycan layers. Given the abundance of these mineral types in natural environments, the data suggest that not all naturally occurring cell surface-associated minerals are necessarily formed de novo on the cell wall.     

Influence of pH on the balance between methanogenesis and iron reduction

Methanogenesis and iron reduction play major roles in determining global fluxes of greenhouse gases. Despite their importance, environmental factors that influence their interactions are poorly known. Here, we present evidence that pH significantly influences the balance between each reaction in anoxic environments that contain ferric (oxyhydr)oxide minerals. In sediment bioreactors that contained goethite as a source of ferric iron, both iron reduction and methanogenesis occurred but the balance between them varied significantly with pH. Compared to bioreactors receiving acidic media (pH 6), electron donor oxidation was 85% lower for iron reduction and 61% higher for methanogenesis in bioreactors receiving alkaline media (pH 7.5). Thus, methanogenesis displaced iron reduction considerably at alkaline pH. Geochemistry data collected from U.S. aquifers demonstrate that a similar pattern also exists on a broad spatial scale in natural settings. In contrast, in bioreactors that were not augmented with goethite, clay minerals served as the source of ferric iron and the balance between each reaction did not vary significantly with pH. We therefore conclude that pH can regulate the relative contributions of microbial iron reduction and methanogenesis to carbon fluxes from terrestrial environments. We further propose that the availability of ferric (oxyhydr)oxide minerals influences the extent to which the balance between each reaction is sensitive to pH. The results of this study advance our understanding of environmental controls on microbial methane generation and provide a basis for using pH and the occurrence of ferric minerals to refine predictions of greenhouse gas fluxes.     

Evidence for microbial iron reduction in a landfill leachate-polluted aquifer (Vejen, Denmark).

Aquifer sediment samples obtained from the anaerobic part of a landfill leachate plume in Vejen, Denmark, were suspended in groundwater or in an artificial medium and incubated. The strictly anaerobic suspensions were tested for reduction of ferric iron [Fe(III)] oxides, which was measured as an increase in the concentration of dissolved Fe(II). Iron reduction did not occur when the medium was inoculated with inactive sediment and when the organisms in the inoculated medium were killed by formaldehyde, by chloroform, or by pasteurization, whereas the level of iron reduction was significant when living bacteria were present. Mixed cultures were obtained from the sediment samples, and differences in apparent iron reduction rates among the different cultures were maintained during several transfers. In addition, iron reduction was observed in unamended incubation mixtures containing whole sediment and groundwater. Synthetic amorphous Fe(III) oxides, as well as naturally occurring sediment-bound Fe(III) oxides, could be reduced by the cultures. Together, our results provide evidence that iron-reducing bacteria are present and microbial iron reduction occurs in the polluted aquifer sediments which we studied.     

Fe(III)-enhanced anaerobic transformation of 2,4-dichlorophenoxyacetic acid by an iron-reducing bacterium Comamonas koreensis CY01

This work studied the ability of Comamonas koreensis CY01 to reduce Fe(III) (hydr)oxides by coupling the oxidation of electron donors and the enhanced biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the presence of Fe(III) (hydr)oxides. The experimental results suggested that strain CY01 can utilize ferrihydrite, goethite, lepidocrocite or hematite as the terminal electron acceptor and citrate, glycerol, glucose or sucrose as the electron donor. Strain CY01 could transform 2,4-D to 4-chlorophenol through reductive side-chain removal and dechlorination. Under the anaerobic conditions, Fe(III) reduction and 2,4-D biodegradation by strain CY01 occurred simultaneously. The presence of Fe(III) (hydr)oxides would significantly enhance 2,4-D biodegradation, probably due to the fact that the reactive mineral-bound Fe(II) species generated from Fe(III) reduction can abiotically reduce 2,4-D. This is the first report of a strain of C. koreensis capable of reducing Fe(III) (hydr)oxides and 2,4-D, which extends the diversity of iron-reducing bacteria associated with dechlorination.     

Melanin production and use as a soluble electron shuttle for Fe(III) oxide reduction and as a terminal electron acceptor by Shewanella algae BrY

Dissimilatory metal-reducing bacteria (DMRB) utilize numerous compounds as terminal electron acceptors, including insoluble iron oxides. The mechanism(s) of insoluble-mineral reduction by DMRB is not well understood. Here we report that extracellular melanin is produced by Shewanella algae BrY. The extracted melanin served as the sole terminal electron acceptor. Upon reduction the reduced, soluble melanin reduced insoluble hydrous ferric oxide in the absence of bacteria, thus demonstrating that melanin produced by S. algae BrY is a soluble Fe(III)-reducing compound. In the presence of bacteria, melanin acted as an electron conduit to Fe(III) minerals and increased Fe(III) mineral reduction rates. Growth of S. algae BrY occurred in anaerobic minimal medium supplemented with melanin extracted from previously grown aerobic cultures of S. algae BrY. Melanin produced by S. algae BrY imparts increased versatility to this organism as a soluble Fe(III) reductant, an electron conduit for iron mineral reduction, and a sole terminal electron acceptor that supports growth.     

Ferrihydrite-dependent growth of Sulfurospirillum deleyianum through electron transfer via sulfur cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

XAS analysis of a nanostructured iron polysaccharide produced anaerobically by a strain of Klebsiella oxytoca

A strain of Klebsiella oxytoca, isolated from acid pyrite-mine drainage, characteristically produces a ferric hydrogel, consisting of branched heptasaccharide repeating units exopolysaccharide (EPS), with metal content of 36 wt%. The high content of iron in the EPS matrix cannot be explained by a simple ferric ion bond to the sugar skeleton. The bio-generated Fe-EPS is investigated by X-ray absorption spectroscopy. Fe K-edge XANES analysis shows that iron is mostly in trivalent form, with a non-negligible amount of Fe(2+) in the structure. The Fe EXAFS results indicate that iron in the sample is in a mineralized form, prevalently in the form of nano-sized particles of iron oxides/hydroxides, most probably a mixture of different nano-crystalline forms. TEM shows that these nanoparticles are located in the interior of the EPS matrix, as in ferritin. The strain produces Fe-EPS to modulate Fe-ions uptake from the cytoplasm to avoid iron toxicity under anaerobic conditions. This microbial material is potentially applicable as iron regulator.