Regulation of Fe3+-oxide Formation Among Fe2+-oxidizing Bacteria

Helical stalks (resembling Gallionella ferruginea, Mariprofundus ferrooxydans) and filamentous sheaths (resembling Leptothrix ochracea) of Fe²⁺-oxidizing bacteria (FeOB) are mineralized by hydrous ferric oxides (HFO). To perform both inter-species and inter-site size comparisons of HFO particles on stalks and sheaths we measured HFO particles in samples of natural bacteriogenic iron oxides (BIOS) from 3 contrasting field sites: the Loihi Seamount (southern Hawaii); Äspö Hard Rock Laboratory (eastern Sweden); and Chalk River Laboratories (northern Canada) representing seafloor saline, underground brackish, and surface freshwater aqueous conditions. Ambient temperatures were in the psychrophilic range and pHs measured for Loihi, CRL, and Äspö were 5.6, 6.9 and 7.4, respectively. Dissolved Fe was lowest for CRL (0.2 mg · L^?1) followed by Äspö (1.5 mg · L^?1), then Loihi (4.5–14.9 mg · L^?1). L. ochraceasheaths appear to have surface properties that restrict HFO particle growth in comparison to G.ferruginea-M.ferrooxydans stalks in the same environment, which we attribute to interfacial surface energy (γ). An inverse relationship between particle size and stalk/sheath length due to restrictions in reactive surface area was also observed, which may provide insight into FeOB survival strategies to alleviate oxidative stress arising from Fe³⁺ production.     

Dependence of In Situ Bacterial Fe(II)-Oxidation and Fe(III)-Precipitation on Sequential Reactive Transport

A study was conducted to determine in situ rates of Fe(II) oxidation and Fe(III) precipitation along a 5.0 m reach of a ferruginous groundwater discharge zone under two distinct conditions; (i) the natural state featuring abundant flocculent mats of bacteriogenic iron oxides (BIOS) produced by Fe(II)-oxidizing bacteria, and (ii) after a manual washout of the streambed to remove the microbial mat. Examination of mat samples by differential interference contrast light microscopy revealed tangled meshworks of filamentous Leptothrix sheaths and helical Gallionella stalks intermixed with fine-grained hydrous ferric oxide (HFO) precipitates. The greatest accumulation of BIOS mat was 1.0 m downstream of the groundwater spring. Redox potential (Eh) increased sharply from 200 mV to over 300 mV over the last 2.0 m of the reach. Similarly, dissolved oxygen increased from < 10% saturation to almost 100% saturation over the last 2.0 m of the reach, whereas pH increased from 6.4 to 7.3. Pseudo-first-order rate constants determined on the basis of analytical solutions to sequential partial differential advection-dispersion-reaction equations for the linear Fe(II)→Fe(III)→HFO reaction network yielded in situ Fe(II) oxidation rate constants (k_ox) of 1.70 ± 0.20 min^?1 in natural conditions and 0.48 ± 0.14 min^?1 after washout. Corresponding Fe(III)-precipitation rates (k_p) before and after washout were 3.45 ± 0.10 min^?1 and 0.90 ± 0.01 min^?1, respectively. These values for k_ox and k_p are higher than estimates obtained from closed batch microcosm and laboratory experiments, underscoring the crucial dependence of in situ Fe(II) oxidation and Fe(III) precipitation rates on advective and dispersive mass transport. The results also highlight the influence that BIOS microbial mats exert on the reaction kinetics of the multiple heterogeneous reactions contributing not only to Fe(II)/Fe(III) redox transformations in groundwater discharge zones, but also the precipitation of HFO.     

A Comparison of the Rates of Fe(III) Reduction in Synthetic and Bacteriogenic Iron Oxides by Shewanella putrefaciens CN32

The susceptibility of various bacteriogenic iron oxides (BIOS) to bacterial Fe(III) reduction was examined. Reduction resulted in complete dissolution of the iron mineral from the surfaces of the Fe-oxidizing consortium. Reduction rates were compared to that of synthetic ferrihydrite (HFO). The reduction rate of HFO (0.162 day^{? 1}) was significantly lower than that of Äspö (Gallionella dominated) BIOS (0.269 day^{? 1}). Two Canadian (Leptothrix dominated) BIOS samples showed statistically equivalent rates of reduction (0.541 day^?1 and 0.467 day^{? 1}), which were higher than both Äspö BIOS and HFO. BIOS produced by different iron-oxidizing genera have different susceptibilities to microbial reduction.     

Membrane biosensor for the determination of iron (II,III) based on immobilized cells ofThiobacillus ferrooxidans

The bacterial electrode for amperometric determination of iron ions is based on a Clark-type oxygen electrode, on the measuring part of which a paste containing a mixture of jarosite precipitate and iron-oxidizing bacteria is mounted with the aid of a cellophane membrane. In acidic media a biochemical oxidation of Fe²⁺ connected with oxygen consumption takes place in the biocatalytic layer. Fe³⁺ is determined after its reduction to Fe²⁺. The determination limit is 60 μmol/L, the stability of the electrode is two months at room temperature.     

The influence of pH on OH scavenger inhibition of damage to deoxyribose by Fenton reaction

Summary Hydroxyl radicals (OH') can be formed in aqueous solution by direct reaction of hydrogen peroxide (H₂O₂) with ferrous salt (Fenton reaction). OH' damage to deoxyribose, measured as formation of thiobarbituric acid-reactive material, was evaluated at different pHs to study the mechanism of action of classical OH' scavengers. OH' scavenger effect on Fe²⁺ oxidation was also evaluated in the same experimental conditions. In the absence of OH' scavengers, OH' damage to deoxyribose is higher at acidic compared to neutral and moderately basic pH. At acidic pH deoxiribose is per se able to inhibit Fe²⁺ oxidation by H₂0₂. Most of OH' scavengers tested inhibit deoxyribose damage and Fe²⁺ oxidation in a similar manner: both inhibitions are most relevant at acidic pH and decrease by increasing the pH. These results are not due to OH' scavenger inhibition of Fenton reaction. The influence of pH on the parameters studied appears to be due to the competition of deoxyribose and OH' scavengers for iron. These results suggest the prominent role of iron binding in the degradation of deoxyribose and in the OH' scavenging ability of different compounds. Results obtained with triethylenetetramine, a iron chelator with a low rate constant with OH', confirm that both deoxyribose and the OH' scavengers interact with iron bringing about a site specific Fenton reaction; that the OH' formed at these sites oxidize these molecules to their radical forms which in turn reduce the Fe^3– produced by Fenton reaction. The results presented indicate that most of classical OH' scavengers exert their effect predominantly by preventing the site specific reaction between Fe²⁺ and H₂0₂ on the deoxyribose molecule.     

Evaluation of Bacterial Community Structure and Its Influence on Sulfide Oxidation in a Bio-Leaching Environment

The mechanism of sulfide oxidation by adhering bacteria (direct oxidation mechanism) and by ferric ion in the aqueous phase was studied by quantitative assessment of bacterial activity on the sulfide surface. To probe for the principal bacterial species on the surface and in the supernatant, a library of DNA genes encoding portions of bacterial 16S rRNA was constructed. The PCR-amplified DNA from the bacterial populations was cloned employing PROMEGA's pGEM-T Easy Vector system. The clone frequency indicated that iron-oxidizing bacteria were dominant in the liquid phase, while Acidithiobacillus ferroixdans, which is both sulfur and iron oxidizer was the most prevalent on the sulfide surface. Samples of crystalline pyrite were exposed to the bacterial consortium to evaluate surface alterations caused by bacteria. Chemical (abiotic) oxidation experiments with ferric ion as the oxidant were carried out in parallel with the biological oxidation tests. Changes in the surface topography were monitored by atomic force microscopy (AFM) while changes in surface chemistry were examined by Raman spectroscopy. Bacterial attachment resulted in a 53% increase in the specific surface area in comparison to a 13% increase caused by chemical (ferric ion) oxidation. The oxidation rate was assessed by evaluating the iron release. After corrections for surface area changes, the specific abiotic (oxidation by Fe^{3 +}) and biotic oxidation rates with adhering bacteria were nearly the same (2.6 × 10^{? 9} mol O₂/s/m² versus 3.3 × 10^{? 9} mol O₂/s/m²) at pH = 2 and a temperature of 25°C. The equality of rates implies that the availability of ferric ion as the oxidant is rate limiting.     

Modelling of Fe2 + oxidation by Thiobacillus ferrooxidans

Summary The kinetics of oxidation of aqueous acidic ferrous sulphate by Thiobacillus ferrooxidans has been studied in a batch reactor. The contribution of cell wall envelopes to the oxidation rate has been shown to be negligible. A model which accounts for the oxidation of Fe^{2 +}, death of bacteria due to Fe^{3 +} poisoning, existence of an optimal pH and precipitation of Fe^{3 +} has been proposed. The model is able to predict the concentration of Fe^{2 +} and pH quite satisfactorily. The predictions of Fe^{3 +} are not so accurate because of simplifying assumptions made about its precipitation. Offprint requests to: R. Kumar     

Pyrite oxidation by Thiobacillus ferrooxidans with special reference to the sulphur moiety of the mineral

Available cultures of Thiobacillus ferrooxidans were found to be contaminated with bacteria very similar to Thiobacillus acidophilus. The experiments described were performed with a homogeneous culture of Thiobacillus ferrooxidans.Pyrite (FeS₂) was oxidized by Thiobacillus ferrooxidans grown on iron (Fe²⁺), elemental sulphur (S^o) or FeS₂.Evidence for the direct utilization of the sulphur moiety of pyrite by Thiobacillus ferrooxidans was derived from the following observations: a. Known inhibitors of Fe²⁺ and S^o oxidation, NaN₃ and NEM, respectively, partially abolished FeS₂ oxidation. b. A b-type cytochrome was detectable in FeS₂-and S^o-grown cells but not in Fe²⁺-grown cells. c. FeS₂ and S^o reduced b-type cytochromes in whole cells grown on S^o. d. CO₂ fixation at pH 4.0 per mole of oxygen consumed was the highest with S^o, lowest with Fe²⁺ and medium with FeS₂ as substrate. e. Bacterial Fe²⁺ oxidation was found to be negligible at pH 5.0 whereas both FeS₂ and S^o oxidation was still appreciable above this pH. f. Separation of pyrite and bacteria by means of a dialysis bag caused a pronounced drop of the oxidation rate which was similar to the reduction of pyrite oxidation by NEM; indirect oxidation of the sulphur moiety by Fe³⁺ was not affected by separation of pyrite and bacteria.Bacterial oxidation and utilization of the sulphur moiety of pyrite were relatively more important with increasing pH.     

Bacterial Oxidation of Dibromomethane and Methyl Bromide in Natural Waters and Enrichment Cultures

Bacterial oxidation of ¹⁴CH₂Br₂ and ¹⁴CH₃Br was measured in freshwater, estuarine, seawater, and hypersaline-alkaline samples. In general, bacteria from the various sites oxidized similar amounts of ¹⁴CH₂Br₂ and comparatively less ¹⁴CH₃Br. Bacterial oxidation of ¹⁴CH₃Br was rapid in freshwater samples compared to bacterial oxidation of ¹⁴CH₃Br in more saline waters. Freshwater was also the only site in which methyl fluoride-sensitive bacteria (e.g., methanotrophs or nitrifiers) governed brominated methane oxidation. Half-life calculations indicated that bacterial oxidation of CH₂Br₂ was potentially significant in all of the waters tested. In contrast, only in freshwater was bacterial oxidation of CH₃Br as fast as chemical removal. The values calculated for more saline sites suggested that bacterial oxidation of CH₃Br was relatively slow compared to chemical and physical loss mechanisms. However, enrichment cultures demonstrated that bacteria in seawater can rapidly oxidize brominated methanes. Two distinct cultures of nonmethanotrophic methylotrophs were recovered; one of these cultures was able to utilize CH₂Br₂ as a sole carbon source, and the other was able to utilize CH₃Br as a sole carbon source.     

Effects of protease inhibitors on liver regeneration

The oxidation of Fe²⁺ to Fe³⁺ by oxygen at pH 7.45 is a first order reaction with a 25 minute half life. In the presence of apotransferrin the oxidation rate is greatly enhanced and Fe³⁺-transferrin is formed. The apotransferrin mediated reaction reaches 50% completion in one minute; it does not follow simple first order kinetics. Iron-saturated transferrin does not exhibit the rate enhancement effect suggesting that the specific metal binding sites are the loci of the iron oxidation. Addition of H₂O₂, an agent which rapidly oxidizes Fe²⁺ to Fe³⁺, during the reaction of Fe²⁺ with apotransferrin greatly decreases the yield of Fe³⁺-transferrin. It is postulated that the basis of the rate enhancement effect is the binding of Fe²⁺ to the metal binding site of the transferrin molecule, followed by a rapid oxidation of the iron to the trivalent form.     

Proton NMR investigation of heme and surrounding proton in low-spin cyanide-ligated bacterial hemoglobin from Vitreoscilla

¹H NMR spectra of low-spin cyanide-ligated bacterial hemoglobin fromVitreoscilla (VtHb-CN) are reported. The assignments of the¹H NMR spectra of VtHb-CN have been made through MCOSY, NOESY, 1D TOE and SUPERWEFT experiments. Almost all resonance peaks of heme and ligated His85 are identified. The spin-lattice relaxation timeT ¹’s and the variation relationships of chemical shifts of these peaks with temperature have been acquired, from which the distances between the measured protons and Fe³⁺, and the diamagnetic chemical shifts have been acquired, respectively. The ionization constants of pK _a’s of ligated His85 are determined through pH titration of chemical shift, which is 4.95 for ligated His85 C₂H proton. The lower pK _a is attributed to the influence of the Fe³⁺ of carrying positive charge and the coordination of His85 and Fe³⁺ of heme.     

Anaerobic ammonia-oxidation coupled with Fe3+ reduction by an anaerobic culture from a piggery wastewater acclimated to NH4 +/Fe3+ medium

The oxidation of ammonia coupled with the reduction of iron is a unique pathway mostly reported in soils and sediments. An anaerobic sludge from a piggery wastewater treatment plant had been acclimated to an NH⁺/Fe³⁺-rich environment to secure an enrichment culture and investigate an anaerobic ammonia oxidation coupled with an iron reduction. The enrichment culture showed an average pH of 6.8 and the concentration of mixed liquor volatile suspended solid was measured as 1,120 mg/L. The mol ratio of oxidized NH₄ ⁺ and reduced Fe³⁺ was 0.33 mol NH₄ ⁺/mol Fe³⁺. It was suggested that the culture acclimated to NH₄ ⁺/Fe³⁺ contained the anaerobic ammonia oxidizing bacteria as well and thus NH₄ ⁺ was fully oxidized to NO₃ ⁻ by the bacterial consortia. In a batch experiment using the culture, the oxidation of NH₄ ⁺ was increased as the initial concentration increased. However, it was suspected from the experimental results that other iron reducing bacteria had grown under the environment applied for the enrichment culture. As a result, it was observed that heterotrophic and autotrophic iron reducers were competing for Fe³⁺.     

Ferroxidase activity of recombinant Desulfovibrio vulgaris rubrerythrin

 Rubrerythrin (Rr) is the trivial name given to a non-heme iron protein of unknown function which has been found in anaerobic sulfate-reducing bacteria. Rr is unique in containing both rubredoxin-type FeS₄ and diiron-oxo sites in the same protein. The results described here demonstrate for the recombinant protein that: (a) Rr catalyzes oxidation of Fe²⁺ to Fe³⁺ by O₂, i.e., Rr has ferroxidase activity, (b) both FeS₄ and diiron domains of the Rr protein are required for ferroxidase activity, (c) with excess Fe²⁺ and O₂ the initial rate of this oxidation appears to be first order in [Rr] and independent of starting [Fe²⁺] above 30 μM, (d) the Fe³⁺ is produced in a form which is capable of rapid incorporation into the iron-binding site of ovotransferrin, and (e) the ferroxidase activity of Rr is comparable to that of published ferroxidase activities of apoferritins on a subunit basis. Ferroxidase activity of Rr was monitored either by the rate of increase in absorbance at 315 nm (which lies near an isosbestic point for oxidized and reduced Rr) or by using apoovotransferrin as Fe³⁺ acceptor, and measuring the rate and extent of diferric transferrin formation at 460 nm. No polyironoxyhydroxide aggregates appeared to associate with Rr after the ferroxidase reaction. A truncated form of Rr containing only the diiron domain had little or no ferroxidase activity. Rr could function as one component of a set of enzymes which channels the reaction products of O₂ and Fe²⁺ onto a non-toxic pathway during transient exposure of the bacteria to air.     

Oxidation of high ferrous iron concentrations by chemolithotrophic Thiobacillus ferrooxidans in packed bed bioreactors

The bacterial oxidation of high ferrous iron concentrations in batch culture has been studied in a packed bed bioreactor. It has been found that aeration rates from 0.49 to 1.2 VVM did not influence the biofilm oxidation activity during the period of biofilm formation up to 30 g l⁻¹ initial ferrous iron concentration. The contribution of swimming, attached and fixed bacteria to the Fe²⁺ oxidation process has been evaluated. Kinetics data showed that the oxidation rate depends on the aeration rate, when the initial Fe²⁺ concentration exceeded 30 g l⁻¹. The maximum overall Fe²⁺ oxidation rate was 1.8 g l⁻¹ h⁻¹, when the initial ferrous iron concentration was in the range 30 to 45 g l⁻¹.     

Production of Metallogenium-like particles by heterotrophic manganese-oxidizing bacteria collected from a lake

As a model of chemically stratified structure of environment typical to the chemocline of lakes, a semisolid gradient medium was prepared to cultivate heterotrophic manganese-oxidizing bacteria originally collected from a lake. The bacteria growing under the conditions described produced extracellularly Metallogenium-like particles similar to those which are often detected in the chemocline of lakes. This suggested that the naturally occurring Metallogenium-like particles originated in activities of such heterotrophic manganese-oxidizing bacteria. The manganese oxidation activity usually appeared only at the stationary phase of bacterial growth. The oxidation of Mn²⁺ and the formation of Metallogenium-like particles by the bacteria were observed at neutral or slightly acidic pH. not under alkaline conditions, which is a conspicuous difference from the inorganic oxidation of Mn²⁺ by O₂. Bacterial manganese oxidation was stimulated by bicarbonate (50 or 500 M). An experiment of addition of H₂O₂ to the incubation tubes isolated from atmosphere failed to confirm the availability of externally added H₂O₂ as the electron acceptor, suggesting that the bacterial manganese oxidation required the presence of O₂.     

pK(a) values for the unfolded state under native conditions explain the pH-dependent stability of PGB1

Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pK_a values. However, pK_a values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pK_a values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pK_a values were determined for carboxyl groups by monitoring their pH-dependent ¹³C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pK_a values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pK_a values of the folded and unfolded states and compared to experimental stability data. The use of experimental pK_a values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.     

Rate Equations and Kinetic Parameters of the Reactions Involved in Pyrite Oxidation by Thiobacillus ferrooxidans

Rate equations and kinetic parameters were obtained for various reactions involved in the bacterial oxidation of pyrite. The rate constants were 3.5 μM Fe²⁺ per min per FeS₂ percent pulp density for the spontaneous pyrite dissolution, 10 μM Fe²⁺ per min per mM Fe³⁺ for the indirect leaching with Fe³⁺, 90 μM O₂ per min per mg of wet cells per ml for the Thiobacillus ferrooxidans oxidation of washed pyrite, and 250 μM O₂ per min per mg of wet cells per ml for the T. ferrooxidans oxidation of unwashed pyrite. The K_m values for pyrite concentration were similar and were 1.9, 2.5, and 2.75% pulp density for indirect leaching, washed pyrite oxidation by T. ferrooxidans, and unwashed pyrite oxidation by T. ferrooxidans, respectively. The last reaction was competitively inhibited by increasing concentrations of cells, with a K_i value of 0.13 mg of wet cells per ml. T. ferrooxidans cells also increased the rate of Fe²⁺ production from Fe³⁺ plus pyrite.     

Precipitation of iron,silica, and sulfate on bacterial cell surfaces

The present study documents the precipitation of Fe(III), silica, and sulfate in the presence of 3 different bacteria (Bacillus subtilus, Bacillus licheniformis, and Pseudomonas aeruginosa), under different total Fe(III) concentrations (10^?2 M, 10^?3 M, 10^?4 M) at constant pH (4.0). Morphology and chemical composition of the precipitates were compared with those formed in abiotic control systems, while chemical composition and precipitation of the precipitates were modeled according to solution chemistry data. Transmission electron microscopy (TEM) observations showed morphological differences between the biotic and abiotic systems. All systems contained small grains (diam. 2–50 nm), but amorphous material (i.e., material without any specific morphology) and nodules were present only in the cell systems. This is because bacterial surfaces and exopolymers provided numerous binding sites for metal and anion sorption and promoted heterogeneous nucleation of hydrous ferric oxides (HFO). The initial Fe/Si and Fe/SO₄ molar ratios of the solutions dictated the type of precipitates in most systems, since abiotic control systems were saturated to oversaturated with respect to amorphous silica, siliceous ferrihydrite, schwertmannite, ferrihydrite, goethite, or combinations of these. Of the three strains studied, B. licheniformis appeared to have the greatest influence on the chemical composition of the precipitates, especially in the presence of Si. B. licheniformis (a gram‐positive bacterium with a large capsule) favored the precipitation of HFO containing less Si than the predicted solids, because Si rather than Fe oxides was preferentially sorted to extracellular polymers (capsule). On the other hand, the formation of SO₄‐rich HFO (similar to schwertmannite) did not seem to be affected by the presence of bacteria.     

Studies of Hypervalent Iron

The iron (IV), (V) and (VI) oxidation states are of great interest because of their role in catalytic oxidation/ hydroxylation reactions. This report summarizes the information currently available on the kinetic and chemical properties of the water-soluble ions of FeO²₄-, FeO^3–₄ and FeO^4–₄, their prorogated forms. and/or simple complex derivatives. The discussion includes their radiation-induced formation, decay kinetics, reactivity with other compounds, determination of their respective pK_a, values as well as spectral properties.     

Lipid Peroxidation. Definition of Experimental Conditions for Selective Study of the Propagation and Termination Phases

To find experimental conditions to selectively study the propagation phase of lipoperoxidation we studied the lipoperoxidation, catalyzed by FeCl₂, of liposomes in a buffering condition where Fe²⁺ autoxidation and oxygen active species generation does not occur. Liposomes from egg yolk phosphatidylcholine. prepared by vortex mixing, do not oxidize Fe²⁺: on the contrary they oxidize Fe²⁺ when prepared by ultrasonic irradiation. Dimyristoyl phosphatidylcholine liposomes prepared by ultrasonic irradiation do not oxidize Fe²⁺. During sonication polyunsaturated fatty acid residues autoxidize and lipid hydroperoxides (LOOH) are generated. Only when LOOH are present in the liposimes Fe²⁺ oxidizes and its rate of oxidation depends on the amount of LOOH in the assay. The reaction results in the generation of both LOOH and thiobarbituric acid reactive material (TBAR): it is inhibited by butylated hydroxytoluene and has a acidic pH optimum; it is not inhibited by catalase and OH' scavengers. The reaction studied. thus, appears to be the chain branching and propagation phase of lipoperoxidation. When we studied the dependence of Fe²⁺ oxidation, LOOH and TBAR generation on FeCl₂ concentration, we observed that at high FeCl₂ concentrations the termination phase of lipoperoxidation was prevalent. Thus. by selecting the appropriate FeCl₂ concentration the proposed experimental system allows study of either the propagation or the termination phase of lipoperoxidation.