Effect of Oxidation Rate and Fe(II) State on Microbial Nitrate-Dependent Fe(III) Mineral Formation

A nitrate-dependent Fe(II)-oxidizing bacterium was isolated and used to evaluate whether Fe(II) chemical form or oxidation rate had an effect on the mineralogy of biogenic Fe(III) (hydr)oxides resulting from nitrate-dependent Fe(II) oxidation. The isolate (designated FW33AN) had 99% 16S rRNA sequence similarity to Klebsiella oxytoca. FW33AN produced Fe(III) (hydr)oxides by oxidation of soluble Fe(II) [Fe(II)_sol] or FeS under nitrate-reducing conditions. Based on X-ray diffraction (XRD) analysis, Fe(III) (hydr)oxide produced by oxidation of FeS was shown to be amorphous, while oxidation of Fe(II)_sol yielded goethite. The rate of Fe(II) oxidation was then manipulated by incubating various cell concentrations of FW33AN with Fe(II)_sol and nitrate. Characterization of products revealed that as Fe(II) oxidation rates slowed, a stronger goethite signal was observed by XRD and a larger proportion of Fe(III) was in the crystalline fraction. Since the mineralogy of Fe(III) (hydr)oxides may control the extent of subsequent Fe(III) reduction, the variables we identify here may have an effect on the biogeochemical cycling of Fe in anoxic ecosystems.     

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.     

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 microM uranium and 81 microM cobalt from solution. The adsorptive capacity of the biogenic Fe(III) oxides was lower than that of abiotically produced Fe(III) oxides (100 microM 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 microM) 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.     

Geochemical Controls on Microbial Nitrate-Dependent U(IV) Oxidation

After reductive immobilization of uranium, the element may be oxidized and remobilized in the presence of nitrate by the activity of dissimilatory nitrate-reducing bacteria. We examined controls on microbially mediated nitrate-dependent U(IV) oxidation in landfill leachate-impacted subsurface sediments. Nitrate-dependent U(IV)-oxidizing bacteria were at least two orders of magnitude less numerous in these sediments than glucose- or Fe(II)-oxidizing nitrate-reducing bacteria and grew more slowly than the latter organisms, suggesting that U(IV) is ultimately oxidized by Fe(III) produced by nitrate-dependent Fe(II)-oxidizing bacteria or by oxidation of Fe(II) by nitrite that accumulates during organotrophic dissimilatory nitrate reduction. We examined the effect of nitrate and reductant concentration on nitrate-dependent U(IV) oxidation in sediment incubations and used the initial reductive capacity (RDC = [reducing equivalents] - [oxidizing equivalents]) of the incubations as a unified measurement of the nitrate or reductant concentration. When we lowered the RDC with progressively higher nitrate concentrations, we observed a corresponding increase in the extent of U(IV) oxidation, but did not observe this relationship between RDC and U(IV) oxidation rate, especially when RDC > 0, suggesting that nitrate concentration strongly controls the extent, but not the rate of nitrate-dependent U(IV) oxidation. On the other hand, when we raised the RDC in sediment incubations with progressively higher reductant (acetate, sulfide, soluble Fe(II), or FeS) concentrations, we observed progressively lower extents and rates of nitrate-dependent U(IV) oxidation. Acetate was a relatively poor inhibitor of nitrate-dependent U(IV) oxidation, while Fe(II) was the most effective inhibitor. Based on these results, we propose that it may be possible to predict the stability of U(IV) in a bioremediated aquifer based on the geochemical characteristics of that aquifer.     

Microaerophilic, Fe(II)-dependent growth and Fe(II) oxidation by a Dechlorospirillum species

A species of Dechlorospirillum was isolated from an Fe(II)-oxidizing, opposing-gradient-culture enrichment using an inoculum from a circumneutral, freshwater creek that showed copious amounts of Fe(III) (hydr)oxide precipitation. In gradient cultures amended with a redox indicator to visualize the depth of oxygen penetration, Dechlorospirillum sp. strain M1 showed Fe(II)-dependent growth at the oxic-anoxic interface and was unable to utilize sulfide as an alternate electron donor. The bacterium also grew with acetate as an electron donor under both microaerophilic and nitrate-reducing conditions, but was incapable of organotrophic Fe(III) reduction or nitrate-dependent Fe(II) oxidation. Although members of the genus Dechlorospirillum are primarily known as perchlorate and nitrate reducers, our results suggest that some species are members of the microbial communities involved in iron redox cycling at the oxic-anoxic transition zones in freshwater sediments.     

Growth of Pseudomonas mendocina on Fe(III) (hydr)oxides

Although iron (Fe) is an essential element for almost all living organisms, little is known regarding its acquisition from the insoluble Fe(III) (hydr)oxides in aerobic environments. In this study a strict aerobe, Pseudomonas mendocina, was grown in batch culture with hematite, goethite, or ferrihydrite as a source of Fe. P. mendocina obtained Fe from these minerals in the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals appears to have occurred in excess, as evidenced by the growth of P. mendocina in the medium above that of the insoluble Fe(III) (hydr)oxide aggregates, and this release was independent of the mineral's surface area. These results demonstrate that an aerobic microorganism was able to obtain Fe for growth from several insoluble Fe minerals and did so with various growth rates.     

Anaerobic Biooxidation of Fe(II) by Dechlorosoma suillum

Anaerobic microbial oxidation of Fe(II) was only recently discovered and very little is known about this metabolism. We recently demonstrated that several dissimilatory perchlorate-reducing bacteria could utilize Fe(II) as an electron donor under anaerobic conditions. Here we report on a more in-depth analysis of Fe(II) oxidation by one of these organisms, Dechlorosoma suillum. Similarly to most known nitrate-dependent Fe(II) oxidizers, D. suillum did not grow heterotrophically or lithoautotrophically by anaerobic Fe(II) oxidation. In the absence of a suitable organic carbon source, cells rapidly lysed even though nitrate-dependent Fe(II) oxidation was still occurring. The coupling of Fe(II) oxidation to a particular electron acceptor was dependent on the growth conditions of cells of D. suillum. As such, anaerobically grown cultures of D. suillum did not mediate Fe(II) oxidation with oxygen as the electron acceptor, while conversely, aerobically grown cultures did not mediate Fe(II) oxidation with nitrate as the electron acceptor. Anaerobic washed cell suspensions of D. suillum rapidly produced an orange/brown precipitate which X-ray diffraction analysis identified as amorphous ferric oxyhydroxide or ferrihydrite. This is similar to all other identified nitrate-dependent Fe(II) oxidizers but is in contrast to what is observed for growth cultures of D. suillum, which produced a mixed-valence Fe(II)-Fe(III) precipitate known as green rust. D. suillum rapidly oxidized the Fe(II) content of natural sediments. Although the form of ferrous iron in these sediments is unknown, it is probably a component of an insoluble mineral, as previous studies indicated that soluble Fe(II) is a relatively minor form of the total Fe(II) content of anoxic environments. The results of this study further enhance our knowledge of a poorly understood form of microbial metabolism and indicate that anaerobic Fe(II) oxidation by D. suillum is significantly different from previously described forms of nitrate-dependent microbial Fe(II) oxidation.     

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.     

Growth of Pseudomonas mendocina on Fe(III) (Hydr)Oxides

Although iron (Fe) is an essential element for almost all living organisms, little is known regarding its acquisition from the insoluble Fe(III) (hydr)oxides in aerobic environments. In this study a strict aerobe, Pseudomonas mendocina, was grown in batch culture with hematite, goethite, or ferrihydrite as a source of Fe. P. mendocina obtained Fe from these minerals in the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals appears to have occurred in excess, as evidenced by the growth of P. mendocina in the medium above that of the insoluble Fe(III) (hydr)oxide aggregates, and this release was independent of the mineral's surface area. These results demonstrate that an aerobic microorganism was able to obtain Fe for growth from several insoluble Fe minerals and did so with various growth rates.     

Repeated anaerobic microbial redox cycling of iron

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.     

Anoxic and Oxic Oxidation of Rocks Containing Fe(II)Mg-Silicates and Fe(II)-Monosulfides as Source of Fe(III)-Minerals and Hydrogen. Geobiotropy.

In this article, anoxic and oxic hydrolyses of rocks containing Fe (II) Mg-silicates and Fe (II)-monosulfides are analyzed at 25 °C and 250–350 °C. A table of the products is drawn. It is shown that magnetite and hydrogen can be produced during low-temperature (25 °C) anoxic hydrolysis/oxidation of ferrous silicates and during high-temperature (250 °C) anoxic hydrolysis/oxidation of ferrous monosulfides. The high-T (350 °C) anoxic hydrolysis of ferrous silicates leads mainly to ferric oxides/hydroxides such as the hydroxide ferric trihydroxide, the oxide hydroxide goethite/lepidocrocite and the oxide hematite, and to Fe(III)-phyllosilicates. Magnetite is not a primary product. While the low-T (25 °C) anoxic hydrolysis of ferrous monosulfides leads to pyrite. Thermodynamic functions are calculated for elementary reactions of hydrolysis and carbonation of olivine and pyroxene and E-pH diagrams are analyzed. It is shown that the hydrolysis of the iron endmember is endothermic and can proceed within the exothermic hydrolysis of the magnesium endmember and also within the exothermic reactions of carbonations. The distinction between three products of the iron hydrolysis, magnetite, goethite and hematite is determined with E-pH diagrams. The hydrolysis/oxidation of the sulfides mackinawite/troilite/pyrrhotite is highly endothermic but can proceed within the heat produced by the exothermic hydrolyses and carbonations of ferromagnesian silicates and also by other sources such as magma, hydrothermal sources, impacts. These theoretical results are confirmed by the products observed in several related laboratory experiments. The case of radiolyzed water is studied. It is shown that magnetite and ferric oxides/hydroxides such as ferric trihydroxide, goethite/lepidocrocite and hematite are formed in oxic hydrolysis of ferromagnesian silicates at 25 °C and 350 °C. Oxic oxidation of ferrous monosulfides at 25 °C leads mainly to pyrite and ferric oxides/hydroxides such as ferric trihydroxide, goethite/lepidocrocite and hematite and also to sulfates, and at 250 °C mainly to magnetite instead of pyrite, associated to the same ferric oxides/hydroxides and sulfates. Some examples of geological terrains, such as Mawrth Vallis on Mars, the Tagish Lake meteorite and hydrothermal venting fields, where hydrolysis/oxidation of ferromagnesian silicates and iron(II)-monosulfides may occur, are discussed. Considering the evolution of rocks during their interaction with water, in the absence of oxygen and in radiolyzed water, with hydrothermal release of H₂ and the plausible associated formation of components of life, geobiotropic signatures are proposed. They are mainly Fe(III)-phyllosilicates, magnetite, ferric trihydroxide, goethite/lepidocrocite, hematite, but not pyrite.     

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.     

Chemical and biological interactions during nitrate and goethite reduction by Shewanella putrefaciens 200

Although previous research has demonstrated that NO(3)(-) inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO(3)(-) biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO(3)(-) and synthetic, high-surface-area goethite. Results showed that the presence of NO(3)(-) inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO(3)(-) reduction, a 10-fold decrease in the rate of NO(2)(-) reduction, and a 20-fold increase in the amounts of N(2)O produced. Nitrogen stable isotope experiments that utilized delta(15)N values of N(2)O to distinguish between chemical and biological reduction of NO(2)(-) revealed that the N(2)O produced during NO(2)(-) or NO(3)(-) reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO(3)(-) reduction produces NO(2)(-) and Fe(II), which then abiotically react to reduce NO(2)(-) to N(2)O with the subsequent oxidation of Fe(II) to Fe(III).     

Chemolithotrophic nitrate-dependent Fe(II)-oxidizing nature of actinobacterial subdivision lineage TM3

Anaerobic nitrate-dependent Fe(II) oxidation is widespread in various environments and is known to be performed by both heterotrophic and autotrophic microorganisms. Although Fe(II) oxidation is predominantly biological under acidic conditions, to date most of the studies on nitrate-dependent Fe(II) oxidation were from environments of circumneutral pH. The present study was conducted in Lake Grosse Fuchskuhle, a moderately acidic ecosystem receiving humic acids from an adjacent bog, with the objective of identifying, characterizing and enumerating the microorganisms responsible for this process. The incubations of sediment under chemolithotrophic nitrate-dependent Fe(II)-oxidizing conditions have shown the enrichment of TM3 group of uncultured Actinobacteria. A time-course experiment done on these Actinobacteria showed a consumption of Fe(II) and nitrate in accordance with the expected stoichiometry (1:0.2) required for nitrate-dependent Fe(II) oxidation. Quantifications done by most probable number showed the presence of 1 × 10⁴ autotrophic and 1 × 10⁷ heterotrophic nitrate-dependent Fe(II) oxidizers per gram fresh weight of sediment. The analysis of microbial community by 16S rRNA gene amplicon pyrosequencing showed that these actinobacterial sequences correspond to ∼0.6% of bacterial 16S rRNA gene sequences. Stable isotope probing using ¹³CO₂ was performed with the lake sediment and showed labeling of these Actinobacteria. This indicated that they might be important autotrophs in this environment. Although these Actinobacteria are not dominant members of the sediment microbial community, they could be of functional significance due to their contribution to the regeneration of Fe(III), which has a critical role as an electron acceptor for anaerobic microorganisms mineralizing sediment organic matter. To the best of our knowledge this is the first study to show the autotrophic nitrate-dependent Fe(II)-oxidizing nature of TM3 group of uncultured Actinobacteria.     

Oxidation of Fe(II)‐EDTA by nitrite and by two nitrate‐reducing Fe(II)‐oxidizing Acidovorax strains

The enzymatic oxidation of Fe(II) by nitrate‐reducing bacteria was first suggested about two decades ago. It has since been found that most strains are mixotrophic and need an additional organic co‐substrate for complete and prolonged Fe(II) oxidation. Research during the last few years has tried to determine to what extent the observed Fe(II) oxidation is driven enzymatically, or abiotically by nitrite produced during heterotrophic denitrification. A recent study reported that nitrite was not able to oxidize Fe(II)‐EDTA abiotically, but the addition of the mixotrophic nitrate‐reducing Fe(II)‐oxidizer, Acidovorax sp. strain 2AN, led to Fe(II) oxidation (Chakraborty & Picardal, 2013). This, along with other results of that study, was used to argue that Fe(II) oxidation in strain 2AN was enzymatically catalyzed. However, the absence of abiotic Fe(II)‐EDTA oxidation by nitrite reported in that study contrasts with previously published data. We have repeated the abiotic and biotic experiments and observed rapid abiotic oxidation of Fe(II)‐EDTA by nitrite, resulting in the formation of Fe(III)‐EDTA and the green Fe(II)‐EDTA‐NO complex. Additionally, we found that cultivating the Acidovorax strains BoFeN1 and 2AN with 10 mm nitrate, 5 mm acetate, and approximately 10 mm Fe(II)‐EDTA resulted only in incomplete Fe(II)‐EDTA oxidation of 47–71%. Cultures of strain BoFeN1 turned green (due to the presence of Fe(II)‐EDTA‐NO) and the green color persisted over the course of the experiments, whereas strain 2AN was able to further oxidize the Fe(II)‐EDTA‐NO complex. Our work shows that the two used Acidovorax strains behave very differently in their ability to deal with toxic effects of Fe‐EDTA species and the further reduction of the Fe(II)‐EDTA‐NO nitrosyl complex. Although the enzymatic oxidation of Fe(II) cannot be ruled out, this study underlines the importance of nitrite in nitrate‐reducing Fe(II)‐ and Fe(II)‐EDTA‐oxidizing cultures and demonstrates that Fe(II)‐EDTA cannot be used to demonstrate unequivocally the enzymatic oxidation of Fe(II) by mixotrophic Fe(II)‐oxidizers.     

Evidence for microbial Fe(III) reduction in anoxic, mining-impacted lake sediments (Lake Coeur d'Alene, Idaho)

Mining-impacted sediments of Lake Coeur d'Alene, Idaho, contain more than 10% metals on a dry weight basis, approximately 80% of which is iron. Since iron (hydr)oxides adsorb toxic, ore-associated elements, such as arsenic, iron (hydr)oxide reduction may in part control the mobility and bioavailability of these elements. Geochemical and microbiological data were collected to examine the ecological role of dissimilatory Fe(III)-reducing bacteria in this habitat. The concentration of mild-acid-extractable Fe(II) increased with sediment depth up to 50 g kg(-1), suggesting that iron reduction has occurred recently. The maximum concentrations of dissolved Fe(II) in interstitial water (41 mg liter(-1)) occurred 10 to 15 cm beneath the sediment-water interface, suggesting that sulfidogenesis may not be the predominant terminal electron-accepting process in this environment and that dissolved Fe(II) arises from biological reductive dissolution of iron (hydr)oxides. The concentration of sedimentary magnetite (Fe(3)O(4)), a common product of bacterial Fe(III) hydroxide reduction, was as much as 15.5 g kg(-1). Most-probable-number enrichment cultures revealed that the mean density of Fe(III)-reducing bacteria was 8.3 x 10(5) cells g (dry weight) of sediment(-1). Two new strains of dissimilatory Fe(III)-reducing bacteria were isolated from surface sediments. Collectively, the results of this study support the hypothesis that dissimilatory reduction of iron has been and continues to be an important biogeochemical process in the environment examined.     

Ecophysiology and the energetic benefit of mixotrophic Fe(II) oxidation by various strains of nitrate-reducing bacteria

In order to assess the importance of nitrate-dependent Fe(II) oxidation and its impact on the growth physiology of dominant Fe oxidizers, we counted these bacteria in freshwater lake sediments and studied their growth physiology. Most probable number counts of nitrate-reducing Fe(II)-oxidizing bacteria in the sediment of Lake Constance, a freshwater lake in Southern Germany, yielded about 10⁵ cells mL⁻¹ of the total heterotrophic nitrate-reducing bacteria, with about 1% (10³ cells mL⁻¹) of nitrate-reducing Fe(II) oxidizers. We investigated the growth physiology of Acidovorax sp. strain BoFeN1, a dominant nitrate-reducing mixotrophic Fe(II) oxidizer isolated from this sediment. Strain BoFeN1 uses several organic compounds (but no sugars) as substrates for nitrate reduction. It also reduces nitrite, dinitrogen monoxide, and O₂, but cannot reduce Fe(III). Growth experiments with cultures amended either with acetate plus Fe(II) or with acetate alone demonstrated that the simultaneous oxidation of Fe(II) and acetate enhanced growth yields with acetate alone (12.5 g dry mass mol⁻¹ acetate) by about 1.4 g dry mass mol⁻¹ Fe(II). Also, pure cultures of Pseudomonas stutzeri and Paracoccus denitrificans strains can oxidize Fe(II) with nitrate, whereas Pseudomonas fluorescens and Thiobacillus denitrificans strains did not. Our study demonstrates that nitrate-dependent Fe(II) oxidation contributes to the energy metabolism of these bacteria, and that nitrate-dependent Fe(II) oxidation can essentially contribute to anaerobic iron cycling.     

Inhibition of NO3- and NO2- reduction by microbial Fe(III) reduction: evidence of a reaction between NO2- and cell surface-bound Fe2+

A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO(3)(-) and NO(2)(-) (NO(x)(-)) reduction by Shewanella putrefaciens 200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe(2+) and NO(2)(-). This coating could then inhibit reduction of NO(x)(-) by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of approximately 3 mM NO(3)(-) reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe(2+) and NO(2)(-), demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe(2+) onto Paracoccus denitrificans inhibited NO(x)(-) reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO(2)(-) was chemically reduced to N(2)O by goethite or cell-sorbed Fe(2+), but not at appreciable rates by aqueous Fe(2+). Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe(2+) and NO(2)(-). The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.     

Enhanced growth of Acidovorax sp. strain 2AN during nitrate-dependent Fe(II) oxidation in batch and continuous-flow systems

Microbial nitrate-dependent, Fe(II) oxidation (NDFO) is a ubiquitous biogeochemical process in anoxic sediments. Since most microorganisms that can oxidize Fe(II) with nitrate require an additional organic substrate for growth or sustained Fe(II) oxidation, the energetic benefits of NDFO are unclear. The process may also be self-limiting in batch cultures due to formation of Fe-oxide cell encrustations. We hypothesized that NDFO provides energetic benefits via a mixotrophic physiology in environments where cells encounter very low substrate concentrations, thereby minimizing cell encrustations. Acidovorax sp. strain 2AN was incubated in anoxic batch reactors in a defined medium containing 5 to 6 mM NO₃⁻, 8 to 9 mM Fe²⁺, and 1.5 mM acetate. Almost 90% of the Fe(II) was oxidized within 7 days with concomitant reduction of nitrate and complete consumption of acetate. Batch-grown cells became heavily encrusted with Fe(III) oxyhydroxides, lost motility, and formed aggregates. Encrusted cells could neither oxidize more Fe(II) nor utilize further acetate additions. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced, and further additions of acetate and Fe(II)-EDTA could be oxidized. Experiments using a novel, continuous-flow culture system with low concentrations of substrate, e.g., 100 μM NO₃⁻, 20 μM acetate, and 50 to 250 μM Fe²⁺, showed that the growth yield of Acidovorax sp. strain 2AN was always greater in the presence of Fe(II) than in its absence, and electron microscopy showed that encrustation was minimized. Our results provide evidence that, under environmentally relevant concentrations of substrates, NDFO can enhance growth without the formation of growth-limiting cell encrustations.