Dissimilatory iron reduction and odor indicator abatement by biofilm communities in swine manure microcosms

Animal waste odors arising from products of anaerobic microbial metabolism create community relations problems for livestock producers. We investigated a novel approach to swine waste odor reduction: the addition of FeCl(3), a commonly used coagulant in municipal wastewater treatment, to stimulate degradation of odorous compounds by dissimilatory iron-reducing bacteria (DIRB). Two hypotheses were tested: (i) FeCl(3) is an effective source of redox-active ferric iron (Fe(3+)) for dissimilatory reduction by bacteria indigenous to swine manure, and (ii) dissimilatory iron reduction results in significant degradation of odorous compounds within 7 days. Our results demonstrated that Fe(3+) from FeCl(3) was reduced biologically as well as chemically in laboratory microcosms prepared with prefiltered swine manure slurry and limestone gravel, which provided pH buffering and a substrate for microbial biofilm development. Addition of a 1-g liter(-1) equivalent concentration of Fe(3+) from FeCl(3), but not from presynthesized ferrihydrite, caused initial, rapid solids flocculation, chemical Fe(3+) reduction, and E(h) increase, followed by a 2-day lag period. Between 2 and 6 days of incubation, increases in Fe(2+) concentrations were accompanied by significant reductions in concentrations of volatile fatty acids used as odor indicators. Increases in Fe(2+) concentrations between 2 and 6 days did not occur in FeCl(3)-treated microcosms that were sterilized by gamma irradiation or amended with NaN(3), a respiratory inhibitor. DNA sequences obtained from rRNA gene amplicons of bacterial communities in FeCl(3)-treated microcosms were closely related to Desulfitobacterium spp., which are known representatives of DIRB. Use of iron respiration to abate wastewater odors warrants further investigation.     

Reduction of Soluble and Insoluble Iron Forms by Membrane Fractions of Shewanella oneidensis Grown under Aerobic and Anaerobic Conditions

The effect of iron substrates and growth conditions on in vitro dissimilatory iron reduction by membrane fractions of Shewanella oneidensis MR-1 was characterized. Membrane fractions were separated by sucrose density gradients from cultures grown with O₂, fumarate, and aqueous ferric citrate as the terminal electron acceptor. Marker enzyme assays and two-dimensional gel electrophoresis demonstrated the high degree of separation between the outer and cytosolic membrane. Protein expression pattern was similar between chelated iron- and fumarate-grown cultures, but dissimilar for oxygen-grown cultures. Formate-dependent ferric reductase activity was assayed with citrate-Fe³⁺, ferrozine-Fe³⁺, and insoluble goethite as electron acceptors. No activity was detected in aerobic cultures. For fumarate and chelated iron-grown cells, the specific activity for the reduction of soluble iron was highest in the cytosolic membrane. The reduction of ferrozine-Fe³⁺ was greater than the reduction of citrate-Fe³⁺. With goethite, the specific activity was highest in the total membrane fraction (containing both cytosolic and outer membrane), indicating participation of the outer membrane components in electron flow. Heme protein content and specific activity for iron reduction was highest with chelated iron-grown cultures with no heme proteins in aerobically grown membrane fractions. Western blots showed that CymA, a heme protein involved in iron reduction, expression was also higher in iron-grown cultures compared to fumarate- or aerobic-grown cultures. To study these processes, it is important to use cultures grown with chelated Fe³⁺ as the electron acceptor and to assay ferric reductase activity using goethite as the substrate.     

Effect of pH and Oxalate on Hydroquinone-Derived Hydroxyl Radical Formation during Brown Rot Wood Degradation

The redox cycle of 2,5-dimethoxybenzoquinone (2,5-DMBQ) is proposed as a source of reducing equivalent for the regeneration of Fe²⁺ and H₂O₂ in brown rot fungal decay of wood. Oxalate has also been proposed to be the physiological iron reductant. We characterized the effect of pH and oxalate on the 2,5-DMBQ-driven Fenton chemistry and on Fe³⁺ reduction and oxidation. Hydroxyl radical formation was assessed by lipid peroxidation. We found that hydroquinone (2,5-DMHQ) is very stable in the absence of iron at pH 2 to 4, the pH of degraded wood. 2,5-DMHQ readily reduces Fe³⁺ at a rate constant of 4.5 × 10³ M⁻¹s⁻¹ at pH 4.0. Fe²⁺ is also very stable at a low pH. H₂O₂ generation results from the autoxidation of the semiquinone radical and was observed only when 2,5-DMHQ was incubated with Fe³⁺. Consistent with this conclusion, lipid peroxidation occurred only in incubation mixtures containing both 2,5-DMHQ and Fe³⁺. Catalase and hydroxyl radical scavengers were effective inhibitors of lipid peroxidation, whereas superoxide dismutase caused no inhibition. At a low concentration of oxalate (50 μM), ferric ion reduction and lipid peroxidation are enhanced. Thus, the enhancement of both ferric ion reduction and lipid peroxidation may be due to oxalate increasing the solubility of the ferric ion. Increasing the oxalate concentration such that the oxalate/ferric ion ratio favored formation of the 2:1 and 3:1 complexes resulted in inhibition of iron reduction and lipid peroxidation. Our results confirm that hydroxyl radical formation occurs via the 2,5-DMBQ redox cycle.     

Apoplastic pH and Fe3+ Reduction in Intact Sunflower Leaves

It has been hypothesized that under NO₃⁻ nutrition a high apoplastic pH in leaves depresses Fe³⁺ reductase activity and thus the subsequent Fe²⁺ transport across the plasmalemma, inducing Fe chlorosis. The apoplastic pH in young green leaves of sunflower (Helianthus annuus L.) was measured by fluorescence ratio after xylem sap infiltration. It was shown that NO₃⁻ nutrition significantly increased apoplastic pH at distinct interveinal sites (pH ≥ 6.3) and was confined to about 10% of the whole interveinal leaf apoplast. These apoplastic pH increases presumably derive from NO₃⁻/proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH₄⁺ or NH₄NO₃ nutrition. Complementary to pH measurements, the formation of Fe²⁺-ferrozine from Fe³⁺-citrate was monitored in the xylem apoplast of intact leaves in the presence of buffers at different xylem apoplastic pH by means of image analysis. This analysis revealed that Fe³⁺ reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5.0. In analogy to the monitoring of Fe³⁺ reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe³⁺ reduction is depressed, inducing leaf chlorosis. The apoplastic pH in the xylem vessels remained low in the still-green veins of leaves with intercostal chlorosis.     

Fluorescent Pseudomonad Pyoverdines Bind and Oxidize Ferrous Ion

Major pyoverdines from Pseudomonas fluorescens 2-79 (Pf-B), P. aeruginosa ATCC 15692 (Pa-C), and P. putida ATCC 12633 (Pp-C) were examined by absorption and fluorescence spectroscopic techniques to investigate the interaction between ferrous ion and the pyoverdine ligand. At physiological pH, ferrous ion quenched the fluorescence of all three pyoverdines much faster than ferric ion did. Also, increased absorbance at 460 nm was observed to be much faster for Fe²⁺-pyoverdine than for Fe³⁺-pyoverdine. At pH 7.4, about 90% of Fe³⁺ was bound by pyoverdine Pa-C after 24 h whereas Fe²⁺ was bound by the pyoverdine completely in only 5 min. The possibility that Fe²⁺ underwent rapid autoxidation before being bound by pyoverdine was considered unlikely, since the Fe²⁺ concentration in pyoverdine-free samples remained constant over a 3-min period at pH 7.4. Incubating excess Fe²⁺ with pyoverdine in the presence of 8-hydroxyquinoline, an Fe³⁺-specific chelating agent, resulted in the formation of a Fe³⁺-hydroxyquinoline complex, suggesting that the iron in the Fe²⁺-pyoverdine complex existed in the oxidized form. These results strongly suggested that pyoverdines bind and oxidize the ferrous ion.     

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 NO₃⁻ and NO₂⁻ (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 Fe²⁺ and NO₂⁻. 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 ~3 mM NO₃⁻ 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 Fe²⁺ and NO₂⁻, demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe²⁺ onto Paracoccus denitrificans inhibited NO_x⁻ reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO₂⁻ was chemically reduced to N₂O by goethite or cell-sorbed Fe²⁺, but not at appreciable rates by aqueous Fe²⁺. Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe²⁺ and NO₂⁻. The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.     

Anaerobic Killing of Oral Streptococci by Reduced, Transition Metal Cations

Reduced, transition metal cations commonly enhance oxidative damage to cells caused by hydroperoxides formed as a result of oxygen metabolism or added externally. As expected, the cations Fe²⁺ and Cu⁺ enhanced killing of Streptococcus mutans GS-5 by hydroperoxides. However, unexpectedly, they also induced lethal damage under fully anaerobic conditions in a glove box with no exposure to O₂ or hydroperoxides from initial treatment with the cations. Sensitivities to anaerobic killing by Fe²⁺ varied among the organisms tested. The oral streptococci Streptococcus gordonii ATCC 10558, Streptococcus rattus FA-1, and Streptococcus sanguis NCTC 10904 were approximately as sensitive as S. mutans GS-5. Enterococcus hirae ATCC 9790, Actinomyces viscosus OMZ105E, and Actinomyces naeslundii WVU45 had intermediate sensitivity, while Lactobacillus casei ATCC 4646 and Escherichia coli B were insensitive. Killing of S. mutans GS-5 in response to millimolar levels of added Fe²⁺ occurred over a wide range of temperatures and pH. The organism was able to take up ferrous iron, but ferric reductase activity could not be detected. Chelators, uric acid, and thiocyanate were not effective inhibitors of the lethal damage. Sulfhydryl compounds, ferricyanide, and ferrocyanide were protective if added prior to Fe²⁺ exposure. Fe²⁺, but not Fe³⁺, acted to reduce the acid tolerance of glycolysis by intact cells of S. mutans. The reduction in acid tolerance appeared to be related directly to Fe²⁺ inhibition of F-ATPase, which could be assayed with permeabilized cells, isolated membranes, or F₁ enzyme separated from membranes. Cu⁺ and Cu²⁺ also inhibited F-ATPase and sensitized glycolysis by intact cells to acid. All of these damaging actions occurred anaerobically and thus did not appear to involve reactive oxygen species.     

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 (Fe₃O₄), 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 × 10⁵ 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.     

Oxidative Inactivation of Mitochondrial Aconitase Results in Iron and H2O2-Mediated Neurotoxicity in Rat Primary Mesencephalic Cultures

Background

Mitochondrial oxidative stress is a contributing factor in the etiology of numerous neuronal disorders. However, the precise mechanism(s) by which mitochondrial reactive oxygen species (ROS) modify cellular targets to induce the death of neurons remains unknown. The goal of this study was to determine if oxidative inactivation of mitochondrial aconitase (m-aconitase) resulted in the release of redox-active iron (Fe²⁺) and hydrogen peroxide (H₂O₂) and whether this contributes to cell death.

Methodology/Principal Findings

Incubation of rat primary mesencephalic cultures with the redox cycling herbicide paraquat (PQ²⁺) resulted in increased production of H₂O₂ and Fe²⁺ at times preceding cell death. To confirm the role of m-aconitase as a source of Fenton reagents and death, we overexpressed m-aconitase using an adenoviral construct thereby increasing the target available for inactivation by ROS. Co-labeling studies identified astrocytes as the predominant cell type expressing transduced m-aconitase although neurons were identified as the primary cell type dying. Oxidative inactivation of m-aconitase overexpressing cultures resulted in exacerbation of H₂O₂ production, Fe²⁺ accumulation and increased neuronal death. Increased cell death in m-aconitase overexpressing cultures was attenuated by addition of catalase and/or a cell permeable iron chelator suggesting that neuronal death occurred in part via astrocyte-derived H₂O₂.

Conclusions

These results suggest a role of ROS-sensitive m-aconitase as a source of Fe²⁺ and H₂O₂ and as a contributing factor to neurotoxicity.     

Bioenergetic Aspects of Halophilism

Examinination of microbial diversity in environments of increasing salt concentrations indicates that certain types of dissimilatory metabolism do not occur at the highest salinities. Examples are methanogenesis for H₂ + CO₂ or from acetate, dissimilatory sulfate reduction with oxidation of acetate, and autotrophic nitrification. Occurrence of the different metabolic types is correlated with the free-energy change associated with the dissimilatory reactions. Life at high salt concentrations is energetically expensive. Most bacteria and also the methanogenic archaea produce high intracellular concentrations of organic osmotic solutes at a high energetic cost. All halophilic microorganisms expend large amounts of energy to maintain steep gradients of NA⁺ and K⁺ concentrations across their cytoplasmic membrane. The energetic cost of salt adaptation probably dictates what types of metabolism can support life at the highest salt concentrations. Use of KCl as an intracellular solute, while requiring far-reaching adaptations of the intracellular machinery, is energetically more favorable than production of organic-compatible solutes. This may explain why the anaerobic halophilic fermentative bacteria (order Haloanaerobiales) use this strategy and also why halophilic homoacetogenic bacteria that produce acetate from H₂ + CO₂ exist whereas methanogens that use the same substrates in a reaction with a similar free-energy yield do not.     

Induction of Extracellular Hydroxyl Radical Production by White-Rot Fungi through Quinone Redox Cycling

A simple strategy for the induction of extracellular hydroxyl radical (OH) production by white-rot fungi is presented. It involves the incubation of mycelium with quinones and Fe³⁺-EDTA. Succinctly, it is based on the establishment of a quinone redox cycle catalyzed by cell-bound dehydrogenase activities and the ligninolytic enzymes (laccase and peroxidases). The semiquinone intermediate produced by the ligninolytic enzymes drives OH production by a Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺). H₂O₂ production, Fe³⁺ reduction, and OH generation were initially demonstrated with two Pleurotus eryngii mycelia (one producing laccase and versatile peroxidase and the other producing just laccase) and four quinones, 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and 2-methyl-1,4-naphthoquinone (menadione [MD]). In all cases, OH radicals were linearly produced, with the highest rate obtained with MD, followed by DBQ, MBQ, and BQ. These rates correlated with both H₂O₂ levels and Fe³⁺ reduction rates observed with the four quinones. Between the two P. eryngii mycelia used, the best results were obtained with the one producing only laccase, showing higher OH production rates with added purified enzyme. The strategy was then validated in Bjerkandera adusta, Phanerochaete chrysosporium, Phlebia radiata, Pycnoporus cinnabarinus, and Trametes versicolor, also showing good correlation between OH production rates and the kinds and levels of the ligninolytic enzymes expressed by these fungi. We propose this strategy as a useful tool to study the effects of OH radicals on lignin and organopollutant degradation, as well as to improve the bioremediation potential of white-rot fungi.White-rot fungi are unique in their ability to degrade a wide variety of organopollutants (36, 47), mainly due to the secretion of a low-specificity enzyme system whose natural function is the degradation of lignin (11). Components of this system include laccase and/or one or two types of peroxidase, such as lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP) (31). Besides acting directly, the ligninolytic enzymes can bring about lignin and pollutant degradation through the generation of low-molecular-weight extracellular oxidants, including (i) Mn³⁺, (ii) free radicals from some fungal metabolites and lignin depolymerization products (7, 22), and (iii) oxygen free radicals, mainly hydroxyl radicals (OH) and lipid peroxidation radicals (21). Although OH radicals are the strongest oxidants found in cultures of white-rot fungi (1), studies of their involvement in pollutant degradation are scarce. One of the reasons is that the mechanisms proposed for OH production still await in vivo validation.Several potential sources of extracellular OH based on the Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺) have been postulated for white-rot fungi. In one case, an extracellular fungal glycopeptide has been shown to reduce O₂ and Fe³⁺ to H₂O₂ and Fe²⁺ (45). Enzymatic sources include cellobiose dehydrogenase, LiP, and laccase. Among these, only cellobiose dehydrogenase is able to directly catalyze the formation of Fenton''s reagent (33). The ligninolytic enzymes, however, act as an indirect source of OH through the generation of Fe³⁺ and O₂ reductants, such as formate (CO₂⁻) and semiquinone (Q⁻) radicals. The first time evidence was provided that a ligninolytic enzyme was involved in OH production, oxalate was used to generate CO₂⁻ in a LiP reaction mediated by veratryl alcohol (4). The proposed mechanism consisted of the following cascade of reactions: production of veratryl alcohol cation radical (Valc⁺) by LiP, oxidation of oxalate to CO₂⁻ by Valc⁺, reduction of O₂ to O₂⁻ by CO₂⁻, and a superoxide-driven Fenton reaction (Haber-Weiss reaction) in which Fe³⁺ was reduced by O₂⁻. The OH production mechanism assisted by Q⁻ was inferred from the oxidation of 2-methoxy-1,4-benzohydroquinone (MBQH₂) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH₂) by Pleurotus eryngii laccase in the presence of Fe³⁺-EDTA. The ability of Q⁻ radicals to reduce both Fe³⁺ to Fe²⁺ and O₂ to O₂⁻, which dismutated to H₂O₂, was demonstrated (14). In this case, OH radicals were generated by a semiquinone-driven Fenton reaction, as Q⁻ radicals were the main agents accomplishing Fe³⁺ reduction. The first evidence of the likelihood of this OH production mechanism being operative in vivo had been obtained from incubations of P. eryngii with 2-methyl-1,4-naphthoquinone (menadione [MD]) and Fe³⁺-EDTA (15). Extracellular OH radicals were produced on a constant basis through quinone redox cycling, consisting of the reduction of MD by a cell-bound quinone reductase (QR) system, followed by the extracellular oxidation of the resulting hydroquinone (MDH₂) to its semiquinone radical (MD⁻). The production of extracellular O₂⁻ and H₂O₂ by P. eryngii via redox cycling involving laccase was subsequently confirmed using 1,4-benzoquinone (BQ), 2-methyl-1,4-benzoquinone, and 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone), in addition to MD (16). However, the demonstration of OH production based on the redox cycling of quinones other than MD was still required.In the present paper, we describe the induction of extracellular OH production by P. eryngii upon its incubation with BQ, 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ), and MD in the presence of Fe³⁺-EDTA. The three benzoquinones were selected because they are oxidation products of p-hydroxyphenyl, guaiacyl, and syringyl units of lignin (MD was included as a positive control). Along with laccase, the involvement of P. eryngii VP in the production of O₂⁻ and H₂O₂ from hydroquinone oxidation has also been reported (13). Since hydroquinones are substrates of all known ligninolytic enzymes, quinone redox cycling catalysis could involve any of them. Here, we demonstrate OH production by P. eryngii under two different culture conditions, leading to the production of laccase or laccase and VP. We also show that quinone redox cycling is widespread among white-rot fungi by using a series of well-studied species that produce different combinations of ligninolytic enzymes.     

Enhancing the Production of Hydroxyl Radicals by Pleurotus eryngii via Quinone Redox Cycling for Pollutant Removal

The induction of hydroxyl radical (OH) production via quinone redox cycling in white-rot fungi was investigated to improve pollutant degradation. In particular, we examined the influence of 4-methoxybenzaldehyde (anisaldehyde), Mn²⁺, and oxalate on Pleurotus eryngii OH generation. Our standard quinone redox cycling conditions combined mycelium from laccase-producing cultures with 2,6-dimethoxy-1,4-benzoquinone (DBQ) and Fe³⁺-EDTA. The main reactions involved in OH production under these conditions have been shown to be (i) DBQ reduction to hydroquinone (DBQH₂) by cell-bound dehydrogenase activities; (ii) DBQH₂ oxidation to semiquinone (DBQ⁻) by laccase; (iii) DBQ⁻ autoxidation, catalyzed by Fe³⁺-EDTA, producing superoxide (O₂⁻) and Fe²⁺-EDTA; (iv) O₂⁻ dismutation, generating H₂O₂; and (v) the Fenton reaction. Compared to standard quinone redox cycling conditions, OH production was increased 1.2- and 3.0-fold by the presence of anisaldehyde and Mn²⁺, respectively, and 3.1-fold by substituting Fe³⁺-EDTA with Fe³⁺-oxalate. A 6.3-fold increase was obtained by combining Mn²⁺ and Fe³⁺-oxalate. These increases were due to enhanced production of H₂O₂ via anisaldehyde redox cycling and O₂⁻ reduction by Mn²⁺. They were also caused by the acceleration of the DBQ redox cycle as a consequence of DBQH₂ oxidation by both Fe³⁺-oxalate and the Mn³⁺ generated during O₂⁻ reduction. Finally, induction of OH production through quinone redox cycling enabled P. eryngii to oxidize phenol and the dye reactive black 5, obtaining a high correlation between the rates of OH production and pollutant oxidation.The degradation of lignin and pollutants by white-rot fungi is an oxidative and rather nonspecific process based on the production of substrate free radicals (36). These radicals are produced by ligninolytic enzymes, including laccase and three kinds of peroxidases: lignin peroxidase, manganese peroxidase, and versatile peroxidase (VP) (23). The H₂O₂ required for peroxidase activities is provided by several oxidases, such as glyoxal oxidase and aryl-alcohol oxidase (AAO) (9, 18). This free-radical-based degradative mechanism leads to the production of a broad variety of oxidized compounds. Common lignin depolymerization products are aromatic aldehydes and acids, and quinones (34). In addition to their high extracellular oxidation potential, white-rot fungi show strong ability to reduce these lignin depolymerization products, using different intracellular and membrane-bound systems (4, 25, 39). Since reduced electron acceptors of oxidized compounds are donor substrates for the above-mentioned oxidative enzymes, the simultaneous actions of both systems lead to the establishment of redox cycles (35). Although the function of these redox cycles is not fully understood, they have been hypothesized to be related to further metabolism of lignin depolymerization products that require reduction to be converted in substrates of the ligninolytic enzymes (34). A second function attributed to these redox cycles is the production of reactive oxygen species, i.e., superoxide anion radicals (O₂⁻), H₂O₂, and hydroxyl radicals (OH), where lignin depolymerization products and fungal metabolites act as electron carriers between intracellular reducing equivalents and extracellular oxygen. This function has been studied in Pleurotus eryngii, whose ligninolytic system is composed of laccase (26), VP (24), and AAO (9). Incubation of this fungus with different aromatic aldehydes has been shown to provide extracellular H₂O₂ on a constant basis, due to the establishment of a redox cycle catalyzed by an intracellular aryl-alcohol dehydrogenase (AAD) and the extracellular AAO (7, 10). The process was termed aromatic aldehyde redox cycling, and 4-methoxybenzaldehyde (anisaldehyde) serves as the main Pleurotus metabolite acting as a cycle electron carrier (13). A second cyclic system, involving a cell-bound quinone reductase activity (QR) and laccase, was found to produce O₂⁻ and H₂O₂ during incubation of P. eryngii with different quinones (11). The process was described as the cell-bound divalent reduction of quinones (Q) by QR, followed by extracellular laccase oxidation of hydroquinones (QH₂) into semiquinones (Q⁻), which autoxidized to some extent, producing O₂⁻ (Q⁻ + O₂ ⇆ Q + O₂⁻). H₂O₂ was formed by O₂⁻ dismutation (O₂⁻ + HO₂ + H⁺ → O₂ + H₂O₂). In an accompanying paper, we describe the extension of this O₂⁻ and H₂O₂ generation mechanism to OH radical production by the addition of Fe³⁺-EDTA to incubation mixtures of several white-rot fungi with different quinones (6). Among them, those derived from 4-hydroxyphenyl, guaiacyl, and syringyl lignin units were used: 1,4-benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), and 2,6-dimethoxy-1,4-benzoquinone (DBQ), respectively. Semiquinone autoxidation under these conditions was catalyzed by Fe³⁺-EDTA instead of being a direct electron transfer to O₂. The intermediate Fe²⁺-EDTA reduced not only O₂, but also H₂O₂, leading to OH radical production by the Fenton reaction (H₂O₂ + Fe²⁺ → OH + OH⁻ + Fe³⁺).Although OH radicals are the strongest oxidants produced by white-rot fungi (2, 14), studies of their involvement in pollutant degradation are quite scarce. In this context, the objectives of this study were to (i) determine possible factors enhancing the production of OH radicals by P. eryngii via quinone redox cycling and (ii) test the validity of this inducible OH production mechanism as a strategy for pollutant degradation. Our selection of possible OH production promoters was guided by two observations (6). First, the redox cycle of benzoquinones working with washed P. eryngii mycelium is rate limited by hydroquinone oxidation, since the amounts of the ligninolytic enzymes that remained bound to the fungus under these conditions were not large. Second, H₂O₂ is the limiting reagent for OH production by the Fenton reaction.With these considerations in mind, anisaldehyde and Mn²⁺ were selected to increase H₂O₂ production. As mentioned above, anisaldehyde induces H₂O₂ production in P. eryngii via aromatic aldehyde redox cycling (7). Mn²⁺ has been shown to enhance H₂O₂ production during the oxidation of QH₂ by P. eryngii laccase by reducing the O₂⁻ produced in the semiquinone autoxidation reaction (Mn²⁺ + O₂⁻ → Mn³⁺ + H₂O₂ + 2 H⁺) (26). Mn²⁺ was also selected to increase the hydroquinone oxidation rate, since this reaction has been shown to be propagated by the Mn³⁺ generated in the latter reaction (QH₂ + Mn³⁺ → Q⁻ + Mn²⁺ + 2 H⁺). The replacement of Fe³⁺-EDTA by Fe³⁺-oxalate was also planned in order to increase the QH₂ oxidation rate above that resulting from the action of laccase. Oxalate is a common extracellular metabolite of wood-rotting fungi to which the function of chelating iron and manganese has been attributed (16, 45). The use of Fe³⁺-oxalate and nonchelated Fe³⁺, both QH₂ oxidants, has been proven to enable quinone redox cycling in fungi that do not produce ligninolytic enzymes, such as the brown-rot fungus Gloeophyllum trabeum (17, 40, 41). Finally, phenol and the azo dye reactive black 5 (RB5) were selected as model pollutants.     

Ferrous Iron-Dependent Volatilization of Mercury by the Plasma Membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe²⁺ medium (pH 2.5) supplemented with 6 μM Hg²⁺. In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 μM Hg²⁺. When incubated for 3 h in a salt solution (pH 2.5) with 0.7 μM Hg²⁺, resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe²⁺ was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30°C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe²⁺-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 μM Hg²⁺ and 1 mM Fe²⁺, plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe²⁺-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe²⁺-dependent mercury volatilization activity of the plasma membrane.     

A paradox: Fe2+-containing agents decreased ROS and apoptosis induced by CoNPs in vascular endothelial cells by inhibiting HIF-1α

Cobalt nanoparticles (CoNPs) released from hip joint implants are known to have a toxic effect on several organs probably through increasing reactive oxygen species (ROS). Ferrous ion (Fe²⁺) is well-known to enhance oxidative stress by catalysing the production of ROS. However, in our pilot study, we found that Fe²⁺ conversely inhibited the ROS production induced by CoNPs. To elucidate the underlying mechanism, the present study treated vascular endothelial HUVEC and HMEC-1 cells with CoNPs alone or in combination with ferrous lactate [Fe(CH₃CHOHCOO)₂], ferrous succinate [Fe(CH₂COO)₂], and ferrous chloride (FeCl₂). CoNP toxicity was evaluated by measuring cell viability, rate of apoptosis and lactose dehydrogenase (LDH) release, and intracellular ROS levels. Treatment with CoNPs decreased cell viability, LDH release, and ROS production and increased apoptosis. CoNPs increased hypoxia-inducible factor-1α (HIF-1α) protein level and mRNA levels of vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1) downstream of HIF-1α signalling. Silencing HIF-1α attenuated CoNP toxicity, as seen by recovery of cell viability, LDH release, and ROS levels and reduced apoptosis. CoNPs caused a pronounced reduction of Fe²⁺ in cells, but supplementation with Fe(CH₃CHOHCOO)₂, Fe(CH₂COO)₂, and FeCl₂ restored Fe²⁺ levels and inhibited HIF-1α activation. Moreover, all three Fe²⁺-containing agents conferred protection from CoNPs; Fe(CH₃CHOHCOO)₂ and Fe(CH₂COO)₂ more effectively than FeCl₂. In summary, the present study revealed that CoNPs exert their toxicity on human vascular endothelial cells by depleting intracellular Fe²⁺ level, which causes activation of HIF-1α signalling. Supplements of Fe²⁺, especially in the form of Fe(CH3CHOHCOO)₂ and Fe(CH₂COO)₂, mitigated CoNP toxicity.     

Riboflavin Biosynthesis Is Associated with Assimilatory Ferric Reduction and Iron Acquisition by Campylobacter jejuni

One of the pathways involved in the acquisition of the essential metal iron by bacteria involves the reduction of insoluble Fe³⁺ to soluble Fe²⁺, followed by transport of Fe²⁺ to the cytoplasm. Flavins have been implicated as electron donors in this poorly understood process. Ferrous iron uptake is essential for intestinal colonization by the important pathogen Campylobacter jejuni and may be of particular importance under low-oxygen conditions. In this study, the links among riboflavin biosynthesis, ferric reduction, and iron acquisition in C. jejuni NCTC11168 have been investigated. A riboflavin auxotroph was generated by inactivation of the ribB riboflavin biosynthesis gene (Cj0572), and the resulting isogenic ribB mutant only grew in the presence of exogenous riboflavin or the riboflavin precursor diacetyl but not in the presence of the downstream products flavin adenine dinucleotide and flavin mononucleotide. Riboflavin uptake was unaffected in the ribB mutant under iron-limited conditions but was lower in both the wild-type strain and the ribB mutant under iron-replete conditions. Mutation of the fur gene, which encodes an iron uptake regulator of C. jejuni, resulted in an increase in riboflavin uptake which was independent of the iron content of the medium, suggesting a role for Fur in the regulation of the as-yet-unknown riboflavin transport system. Finally, ferric reduction activity was independent of iron availability in the growth medium but was lowered in the ribB mutant compared to the wild-type strain and, conversely, increased in the fur mutant. Taken together, the findings confirm close relationships among iron acquisition, riboflavin production, and riboflavin uptake in C. jejuni.     

Iron-Corroding Methanogen Isolated from a Crude-Oil Storage Tank

Microbiologically influenced corrosion of steel in anaerobic environments has been attributed to hydrogenotrophic microorganisms. A sludge sample collected from the bottom plate of a crude-oil storage tank was used to inoculate a medium containing iron (Fe⁰) granules, which was then incubated anaerobically at 37°C under an N₂-CO₂ atmosphere to enrich for microorganisms capable of using iron as the sole source of electrons. A methanogen, designated strain KA1, was isolated from the enrichment culture. An analysis of its 16S rRNA gene sequence revealed that strain KA1 is a Methanococcus maripaludis strain. Strain KA1 produced methane and oxidized iron much faster than did the type strain of M. maripaludis, strain JJ^T, which produced methane at a rate expected from the abiotic H₂ production rate from iron. Scanning electron micrographs of iron coupons that had been immersed in either a KA1 culture, a JJ^T culture, or an aseptic medium showed that only coupons from the KA1 culture had corroded substantially, and these were covered with crystalline deposits that consisted mainly of FeCO₃.Iron (Fe⁰) is an inexpensive metal and is widely used in many industrial processes and industrial/commercial products. When iron contacts an aqueous electrolyte, it readily corrodes. This happens because, as a result of metallurgical and environmental heterogeneities, the electrolytes are not evenly distributed across the surface of the metal and consequently the electric potential is also unevenly distributed. Therefore, electrons flow within the metal from an area of higher electrical potential (the anode) to an area of lower electrical potential (the cathode). At the anode, iron atoms lose electrons and dissolve into ferrous ions (Fe²⁺), whereas cations or elements dissolved in solution (e.g., H⁺ under anaerobic conditions or O₂ under aerobic conditions) are reduced by electrons at the cathode.The corrosion of structures that contain iron is economically devastating. It has been estimated that in the United States alone, the cost of corrosion is 276 billion dollars annually (17). Iron is corroded not only by physiochemical processes but also by the metabolic activity of microorganisms; this metabolic process is termed microbiologically influenced corrosion (MIC). Some 10% of all corrosion damage may be the result of microbial activity (15), and sulfate-reducing bacteria (SRB) are widely regarded as the causative agents of MIC in anaerobic environments (11, 12, 18, 21). The mechanism by which SRB stimulate iron corrosion may occur via the uptake of electrons at the cathodic surface of iron (cathodic depolarization) in conjunction with sulfate reduction (8e⁻ + SO₄²⁻ + 10H⁺ → H₂S + 4H₂O) (27), while at the anionic surface, iron atoms are oxidized to ferrous ions (Fe → Fe²⁺ + 2e⁻). In fact, certain SRB use not only hydrogen but also iron as a source of electrons for sulfate reduction (1, 9, 22). Because not all SRB grow as fast in the presence of iron as they do in the presence of hydrogen (9), fast-growing SRB on iron may have a specific enzyme(s) that removes electrons from iron.Because some methanogens are viable in a hydrogen atmosphere, as are most SRBs, these methanogens may also cause iron corrosion under anaerobic conditions. Several methanogens have been shown to grow and produce methane in medium containing iron as the sole source of electrons (5). The extent of the corrosion by these methanogens, however, was not substantial (2). Others have reported that methanogens do not increase the rate of iron corrosion in comparison with aseptic solutions (6, 7). Recently Dinh and colleagues (9) isolated a methanogen (strain IM1) that produces methane more rapidly than does Methanococcus maripaludis (DSMZ 2771) when cultured with iron granules. Although the rate of iron oxidation was not measured in their experiments, their results suggests that strain IMI oxidizes iron more rapidly than does strain DSMZ 2771.We report herein that a methanogen that was isolated from the sludge of an oil storage tank can unequivocally oxidize iron.     

Reduction of the 2,2′-Azinobis(3-Ethylbenzthiazoline-6-Sulfonate) Cation Radical by Physiological Organic Acids in the Absence and Presence of Manganese

Laccase is a copper-containing phenoloxidase, involved in lignin degradation by white rot fungi. The laccase substrate range can be extended to include nonphenolic lignin subunits in the presence of a noncatalytic cooxidant such as 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), with ABTS being oxidized to the stable cation radical, ABTS^·+, which accumulates. In this report, we demonstrate that the ABTS^·+ can be efficiently reduced back to ABTS by physiologically occurring organic acids such as oxalate, glyoxylate, and malonate. The reduction of the radical by oxalate results in the formation of H₂O₂, indicating the formation of O₂^·− as an intermediate. O₂^·− itself was shown to act as an ABTS^·+ reductant. ABTS^·+ reduction and H₂O₂ formation are strongly stimulated by the presence of Mn²⁺, with accumulation of Mn³⁺ being observed. Additionally, 4-methyl-O-isoeugenol, an unsaturated lignin monomer model, is capable of directly reducing ABTS^·+. These data suggest several mechanisms for the reduction of ABTS^·+ which would permit the effective use of ABTS as a laccase cooxidant at catalytic concentrations.     

Uptake of iron byGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum (isolate 1–9) pathogenic on citrus fruits, appears to lack siderophore production. Iron uptake byG. candidum is mediated by two distinct iron-regulated, energy-and temperature-dependent transport systems that require sulfhydryl groups. One system exhibits specificity for either ferric or ferrous iron, whereas the other exhibits specificity for ferrioxamine-B-mediated iron uptake and presumably other hydroxamate siderophores. Radioactive iron uptake from⁵⁹FeCl₃ showed an optimum at pH 6 and 35° C, and Michaelis-Menten kinetics (apparentK _m = 3 m,V _max = 0.054 nmol · mg^–1 · min^–1). The maximal rate of Fe²⁺ uptake was higher than Fe³⁺ (V _max = 0.25 nmol · mg^–1 · min^–1) but theK _m was identical. Reduction of ferric to ferrous iron prior to transport could not be detected. The ferrioxamine B system exhibits an optimum at pH 6 and 40° C and saturation kinetics (K _m = 2 M,V _max = 0.22 nmol · mg^–1 · min^–1). The two systems were distinguished as two separate entities by negative reciprocal competition, and on the basis of differential response to temperature and phenazine methosulfate. Mössbauer studies revealed that cells fed with either⁵⁷FeCl₃ or⁵⁷FeCl₂ accumulated unknown ferric and ferrous binding metabolites.     

Laccase-Catalyzed Oxidation of Mn2+ in the Presence of Natural Mn3+ Chelators as a Novel Source of Extracellular H2O2 Production and Its Impact on Manganese Peroxidase

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn²⁺ to Mn³⁺, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn³⁺ complexes were produced at 0.15, 0.05, and 0.10 μmol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H₂O₂ formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H₂O₂ production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn²⁺ oxidation and abiotic decomposition of these organic chelators by the resulting Mn³⁺, which leads to formation of superoxide and its subsequent reduction to H₂O₂. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn³⁺ complexes in assays containing 1 mM Mn²⁺ and 100 mM oxalate or malonate, but omitting an additional H₂O₂ source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn²⁺ oxidation in providing H₂O₂ for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.     

Investigation of an Iron-Oxidizing Microbial Mat Community Located near Aarhus, Denmark: Laboratory Studies

We constructed a small flow chamber in which suboxic medium containing 60 to 120 μM FeCl₂ flowed up through a sample well into an aerated reservoir, thereby creating an suboxic-oxic interface similar to the physicochemical conditions that exist in natural iron seeps. When microbial mat material from the Marselisborg iron seep that contained up to 10⁹ bacterial cells per cm³ (D. Emerson and N. P. Revsbech, Appl. Environ. Microbiol. 60:4022-4031, 1994) was placed in the sample well of the chamber, essentially all of the Fe²⁺ flowing through the sample well was oxidized at rates of up to 1,200 nmol of Fe²⁺ oxidized per h per cm³ of mat material. The oxidation rates of samples of the mat that were pasteurized prior to inoculation were only about 20 to 50% of the oxidation rates of unpasteurized samples. Sodium azide also significantly inhibited oxidation. These results suggest that at least 50% and up to 80% of the Fe oxidation in the chamber were actively mediated by the microbes in the mat. It also appeared that Fe stimulated the growth of the community since chambers fed with FeCl₂ accumulated masses of either filamentous or particulate growth, both in the sample well and attached to the walls of the chamber. Control chambers that did not receive FeCl₂ showed no sign of such growth. Furthermore, after 4 to 5 days the chambers fed with FeCl₂ contained 35 to 75% more protein than chambers not supplemented with FeCl₂. Leptothrix ochracea and, to a lesser extent, Gallionella spp. were responsible for the filamentous growth, and the sheaths and stalks, respectively, of these two organisms harbored large numbers of Fe-encrusted, nonappendaged unicellular bacteria. In chambers where particulate growth predominated, the unicellular bacteria alone appeared to be the primary agents of iron oxidation. These results provide the first clear evidence that the “iron bacteria” commonly found associated with neutral-pH iron seeps are responsible for most of the iron oxidation and that the presence of ferrous iron appears to stimulate the growth of these organisms.