Oxidation and Reduction of Iron by Acidophilic Bacteria

Abstract

Redox reactions of iron in acidic environments are of economic and environmental significance, for example, for the leaching of metal ores and for the formation of acid mine drainage and acid sulfate soils. Until recently, research on microbial iron metabolism in acidic environments has mainly been focused on the role of aerobic, autotrophic ferrous iron‐oxidizing bacteria. In the present paper, recent new developments in the field of acidophilic iron metabolism are reviewed. In addition to the well‐known autotrophic ferrous iron‐oxidizing organisms, new heterotrophic isolates have been described that are capable of oxidizing ferrous iron. Microorganisms can also play an important role in the reductive part of the iron cycle. Both heterotrophic and autotrophic organisms may also be involved in this process. The contribution of heterotrophic organisms to acidophilic iron cycling can be twofold: In addition to their direct role as a catalyst, these organisms may scavenge organic compounds that inhibit their autotrophic counterparts. Detailed studies of acidophilic ecosystems are needed to assess the significance of the various types of microorganisms for the overall rate of iron cycling in these extreme environments.     

Role of sulfate‐reducing bacteria in corrosion of mild steel: A review

The influence of sulfate‐reducing bacteria on corrosion of mild steel is reviewed, with special emphasis on the effects of biofilm structure and function, medium composition (dissolved oxygen and ferrous ion concentrations) and the physical and chemical properties of iron sulfides. A summary of different corrosion mechanisms is critically discussed, based on electrochemical and rate process analyses. A mechanism is proposed which explains the high corrosion rates observed in the field.     

Effect of divers anions on the electron-transfer reaction between iron and rusticyanin from Thiobacillus ferrooxidans

Rusticyanin is a soluble blue copper protein found in abundance in the periplasmic space of Thiobacillus ferrooxidans, an acidophilic bacterium capable of growing chemolithotrophically on soluble ferrous sulfate. The one-electron-transfer reactions between soluble iron and purified rusticyanin were studied by stopped-flow spectrophotometry in acidic solutions containing each of 14 different anions. The second-order rate constants for both the Fe(II)-dependent reduction and the Fe(III)-dependent oxidation of the rusticyanin varied as a function of the identity of the principal anion in solution. Analogous electron-transfer reactions between soluble iron and bis(dipicolinato)cobaltate(III) or bis(dipicolinato)ferrate(II) were studied by stopped-flow spectrophotometry under solution conditions identical with those of the rusticyanin experiments. Similar anion-dependent reactivity patterns were obtained with soluble iron whether the other reaction partner was rusticyanin or either of the two organometallic complexes. The Marcus theory of outer-sphere electron transfer reactions was applied to this set of kinetic data to demonstrate that the rusticyanin may possess at least two electron-transfer pathways for liganded iron, one where the pattern of electron-transfer reactivity is controlled largely by protein-independent activation parameters and one where the protein exhibits an anion-dependent kinetic specificity. The exact role of rusticyanin in the iron-dependent respiratory electron transport chain of T. ferrooxidans remains unclear.     

Respiratory enzymes of Thiobacillus ferrooxidans. A kinetic study of electron transfer between iron and rusticyanin in sulfate media

Thiobacillus ferrooxidans is a chemolithotrophic bacterium capable of fulfilling all of its energy requirements from the oxidation of soluble ferrous sulfate. Rusticyanin is a soluble blue copper protein found in abundance in the periplasmic space of this bacterium. The one-electron transfer reaction between soluble iron and purified rusticyanin has been studied by stopped flow spectrophotometry in acidic solutions containing sulfate. Second order rate constants for the reduction of rusticyanin by Fe2+, FeHSO4+, and FeSO4(0) were 0.022, 0.73, and 2.30 M-1 s-1, respectively. The pseudo-first order rate constant for the reduction of rusticyanin exhibited substrate saturation when the concentration of the total ferrous ion was varied in solutions of limiting sulfate. This saturation behavior was quantitatively described using the values of the second order rate constants listed above and the distribution of the total ferrous ion into its water-, bisulfate-, and sulfate-coordinated forms. Second order rate constants for the oxidation of rusticyanin by Fe3+ and FeSO4+ were 0.73 and 0.26 M-1 s-1, respectively. The electron transfer reactions between iron and rusticyanin monitored in vitro were far too slow to support the hypothesis that rusticyanin is the primary oxidant of ferrous ions in the iron-dependent respiratory electron transport chain of T. ferrooxidans.     

Ferric iron reduction by Thiobacillus ferrooxidans at extremely low pH-values

When ferrous iron and sulfur were supplied, cells of T. ferrooxidans in a well-aerated medium started growth by oxidizing ferrous iron. After ferrous iron depletion a lagphase followed before sulfur oxidation started. During sulfur oxidation at pH-values below 1.3 (±0,2) the ferrous iron concentration increased again, although the oxygen saturation of the medium amounted to more than 95%. The number of viable cells did not increase. Thus resting cells of T. ferrooxidans, which are oxidizing sulfur to maintain their proton balance, reduce ferric to ferrous iron. The ferrous iron-oxidizing system seemed to be inhibited at pH-values below 1.3. At a pH-value of 1.8 the ferrous iron was reoxidized at once. A scheme for the linkage of iron- and sulfur metabolism is discussed.     

Enzymes of aerobic respiration on iron

Abstract: Bacteria capable of aerobic respiration on ferrous ions are spread throughout eubacterial and archaebacterial phyla. Comparative spectroscopic analyses revealed that phylogenetically distinct organisms expressed copious quantities of spectrally distinct redox-active biomolecules during autotrophic growth on soluble iron. Thiobaeillus ferrooxidans, Leptospirillum ferrooxidans, Sulfobacillus thermosulfidooxidans , and Metallosphaera sedula possessed iron respiratory chains dominated by a blue copper protein, a novel red cytochrome, a novel yellow protein, and a novel yellow cytochrome, respectively. Further investigation of each type of respiratory chain will be necessary to deduce the advantages and disadvantages of each.     

Acid-stable cytochromes in ferrous ion oxidizing cell-free preparations from Thiobacillus ferrooxidans

Abstract Cell-free preparations from ferrous ion- and sulfur-grown Thiobacillus ferrooxidans prepared under neutral (pH 7.5) or acidic conditions (pH 2.0) were compared. Under neutral conditions the ferrous ion-oxidizing system of T. ferrooxidans was membrane-bound. At acidic conditions, the enzyme system became partially solubilized. In ferrous ion-oxidizing membranes of ferrous ion-grown cells (neutral conditions) a₁-, c- and b-type cytochromes were present. The acidic preparations contained only cytochrome a₁ and c. At least three acid-stable c-type cytochromes (M_r 60 000, 30 000 and 25 000), an acid-stable protein with non-convalently bound heme group (M_r probably rusticyanin, were detected in ferrous ion oxidizing preparations. Membranes of sulfur-grown cells prepared under neutral conditions still oxidized ferrous ions, and contained a₁-, b-, c- and in addition an aa₃-type cytochrome. Cytochrome b and aa₃ were acid-labile.     

革兰氏阴性菌亚铁离子转运系统的组成及作用机制

几乎所有细菌的生长都离不开铁元素。在有氧的环境中,三价铁离子几乎无法被细菌直接利用。但是在宿主胃肠道中,铁元素主要以可溶性的亚铁离子形式存在,它们可通过革兰氏阴性菌外膜直接进入胞周质,在周质通过亚铁离子转运系统,将铁离子转运至胞浆供细菌利用。绝大多数阴性菌主要是通过Feo转运系统利用亚铁离子,大肠杆菌的Feo转运系统由feoA、feoB和feoC3个基因组成。除Feo转运系统外,还发现Yfe转运系统、Efe转运系统、Sit转运系统等。本文重点介绍革兰氏阴性菌Feo转运系统的组成及作用机制,以期为进一步研究细菌亚铁离子的转运机制提供参考。     

Efficient Reduction of Iron Oxides by Paenibacillus spp. Strains Isolated from Tropical Soils

The genus Paenibacillus was hardly described as a Fe(III)-reducing agent, only limited to reduce soluble forms or Fe inserted in poorly crystallized structures. In this study, three Paenibacillus strains capable of reducing manganese oxides in addition to iron oxides were isolated from Cameroonian and Brazilian soils. These strains reduced iron minerals from poorly crystallized 2-line ferrihydrite to well-crystallized Al-substituted and pure goethite with a significant production of soluble ferrous iron. These Paenibacillus strains, inhabitants from ferralitic temporarily waterlogged soils, could play an important role in the bioweathering of minerals and metal mobility in soils.     

Solubilization and speciation of iron during pyrite oxidation by Thiobacillus ferrooxidans

Various species of soluble iron in pyrite‐grown cultures of Thiobacillus ferrooxidans were determined by colorimetry, atomic absorption spectrometry, and ultraviolet spectroscopy. All the cultures were incubated for six weeks before iron analysis. The effects of the following factors were investigated: particle size, initial pH, shaking (aeration), concentration of pyrite, and concentration of yeast extract. Shaking, but not initial pH nor particle size, influenced the relative proportion of different iron species. Polynomial regressions could be used to describe the functional relationship between the different iron species and concentration of pyrite; fewer relationships were evident with respect to concentration of yeast extract. The variance‐covariance matrices indicated a linear dependence among the different iron species. Canonical correlations indicated perfect correlations between group variables of iron, copper, and zinc, with the exception of an absence of significant correlation with the hydroxy complex of iron (FeOH²⁺).

The dissolved ferrous iron (dissociated and weakly chelated) always remained less than 7% of the total iron in solution. The total ferrous iron, which included complexed species, amounted to 7–34% of the total iron in solution. The concentrations of dissociated ferrous and ferric iron and their weak chelates (the dissolved iron) remained mostly constant, irrespective of the concentration of the total iron in solution. Most of the total iron was complexed as ferric species and the amount correlated with culture conditions. The hydroxy complex (FeOH²⁺), which was indicative of the relative amount of hydrolyzable ferric iron upon dilution in CO₂‐free water, usually ranged between 60 and 80% of the total iron. The amount of the total iron in uninoculated controls was less than 12% of that solu‐bilized in the presence of iron‐oxidizing thiobacilli.

T. ferrooxidans was enumerated by a most‐probable‐number technique after three and six weeks of growth on pyrite. The counts after three weeks indicated an increase in the number of free and loosely attached bacteria, followed by a decline of about one order of magnitude in bacterial numbers after six weeks. The technique for bacterial enumeration was deemed unsatisfactory because it could not account for cells attached on pyrite.     

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant

Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm² in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins'' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s⁻¹. The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment.     

Molecular aspects of the electron transfer system which participates in the oxidation of ferrous ion by Thiobacillus ferrooxidans

Abstract: The enzymes and redox proteins, which participate in the oxidation of ferrous ion by the acidophilic iron-oxidizing bacterium Thiobacillus ferrooxidans , have been isolated and characterized. They are Fe(II)-cytochrome c oxidoreductase, cytochromes c -552(s), c -552(m) and c -550(m), rusticyanin, and cytochrome c oxidase. On the basis of the interactions of these components, an electron transfer system has been proposed which seems to function in the oxidation of ferrous ion by the bacterium.     

Effects of ferrous iron on the performance and microbial community in aerobic granular sludge in relation to nutrient removal

Lab‐scale experiments were conducted to investigate the effects of ferrous iron on nutrient removal performance and variations in the microbial community inside aerobic granular sludge for 408 days. Two reactors were simultaneously operated, one without added ferrous iron (SBR1), and one with 10 mg Fe²⁺ L^?1 of added ferrous iron (SBR2). A total of 1 mg Fe²⁺ L^?1 of added ferrous iron was applied to SBR1 starting from the 191st day to observe the resulting variations in the nutrient removal performance and the microbial community. The results show that ammonia‐oxidizing bacteria (AOB) could not oxidize ammonia due to a lack of iron compounds, but they could survive in the aerobic granular sludge. Limited ferrous iron addition encouraged nitrification. Enhanced biological phosphorus removal (EBPR) from both reactors could not be maintained regardless of the amount of ferrous iron that was applied. EBPR was established in both reactors when the concentration of mixed liquor suspended solid (MLSS) and the percentage of Accumulibacteria increased. A total of 10 mg Fe²⁺ L^?1 of added ferrous iron had a relatively adverse effect on the growth of AOB species compared to 1 mg Fe²⁺ L^?1 of added ferrous iron, but it encouraged the growth of Nitrospira sp. and Accumulibacteria, which requires further study. It could be said that the compact and stable structure of aerobic granular sludge preserved AOB and NOB from Fe‐deficient conditions, and wash‐out during the disintegration period. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:716–725, 2017     

Stoichiometric model and metabolic flux analysis for Leptospirillum ferrooxidans

A metabolic model for Leptospirillum ferrooxidans was developed based on the genomic information of an analogous iron oxidizing bacteria and on the pathways of ferrous iron oxidation, nitrogen and CO₂ assimilation based on experimental evidence for L. ferrooxidans found in the literature. From this metabolic reconstruction, a stoichiometric model was built, which includes 86 reactions describing the main catabolic and anabolic aspects of its metabolism. The model obtained has 2 degrees of freedom, so two external fluxes were estimated to achieve a determined and observable system. By using the external oxygen consumption rate and the generation flux biomass as input data, a metabolic flux map with a distribution of internal fluxes was obtained. The results obtained were verified with experimental data from the literature, achieving a very good prediction of the metabolic behavior of this bacterium at steady state. Biotechnol. Bioeng. 2010;107:696–706. © 2010 Wiley Periodicals, Inc.     

Colony formation of free-living and particle-associated sulfate-reducing bacteria

Abstract Some of the components that are likely to be involved in the respiratory chains that transfer electrons from the ferrous iron substrate of various acidophilic bacteria have been revealed by spectroscopic analysis. An apparently unique, soluble and acid-stable cytochrome was found in Leptospirillum ferrooxidans which appeared to lack any of the major cytochromes or the rusticyanin of the better-studied iron-oxidizing mesophile, Thiobacillus ferrooxidans . A specific absorption peak, only found in iron-grown cells, was revealed in a whole cell spectra of thermoacidophilic archaebacteria of different genera.     

Acidithiobacillus ferrianus sp. nov.: an ancestral extremely acidophilic and facultatively anaerobic chemolithoautotroph

Strain MG, isolated from an acidic pond sediment on the island of Milos (Greece), is proposed as a novel species of ferrous iron- and sulfur-oxidizing Acidithiobacillus. Currently, four of the eight validated species of this genus oxidize ferrous iron, and strain MG shares many key characteristics with these four, including the capacities for catalyzing the oxidative dissolution of pyrite and for anaerobic growth via ferric iron respiration. Strain MG also grows aerobically on hydrogen and anaerobically on hydrogen coupled to ferric iron reduction. While the 16S rRNA genes of the iron-oxidizing Acidi-thiobacillus species (and strain MG) are located in a distinct phylogenetic clade and are closely related (98–99% 16S rRNA gene identity), genomic relatedness indexes (ANI/dDDH) revealed strong genomic divergence between strain MG and all sequenced type strains of the taxon, and placed MG as the first cultured representative of an ancestral phylotype of iron oxidizing acidithiobacilli. Strain MG is proposed as a novel species, Acidithiobacillus ferrianus sp. nov. The type strain is MG^T (= DSM 107098^T = JCM 33084^T). Similar strains have been found as isolates or indicated by cloned 16S rRNA genes from several mineral sulfide mine sites.

     

Differential Protein Expression during Growth of Acidithiobacillus ferrooxidans on Ferrous Iron, Sulfur Compounds, or Metal Sulfides

A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferrooxidans ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using two-dimensional polyacrylamide gel electrophoresis. N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A. ferrooxidans ATCC 23270. The genomic context around several of these genes suggests their involvement in the energetic metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function. The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome c₅₅₂, a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others. The results suggest in general a separation of the iron and sulfur utilization pathways. Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides. During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available.     

Inhibition of Iron-oxidizing Activity by Bisulfite Ion in Thiobacillus ferrooxidans

Growth of Thiobacillus ferrooxidans on iron- and sulfur-salts media and iron oxidizing activity of this bacterium were strongly inhibited by bisulfite ion. The mechanism of inhibition by bisulfite ion of iron-oxidizing activity was studied with the plasma membrane of T. ferrooxidans AP19-3. The c-type cytochrome in the plasma membrane was reduced by ferrous ion and the cytochrome reduced by Fe²⁺ was oxidized by cytochrome c oxidase in the plasma membrane. In contrast, c-type cytochrome was reduced by bisulfite ion, but it was not oxidized by cytochrome c oxidase in the membrane. Cytochrome c-oxidizing activity was also inhibited by the ion when mammalian cytochrome c was used as an electron donor, suggesting that cytochrome c oxidase, one of the component of iron oxidase, is the site of inhibition by bisulfite ion.     

Role of rusticyanin in the electron transport process in Thiobacillus ferrooxidans.

Effect of diethyl dithiocarbamate (DEDC), an antimicrobial agent, on growth of Thiobacillus ferrooxidans, possibly by inhibiting rusticyanin present in the periplasmic space of the microorganism, has been studied to gain more insight into the electron transport chain in the bioleaching process. DEDC is found to form a stable complex with rusticyanin in solution and also in polyacrylamide gel. The spectrum of the complex is identical to that of Cu-DEDC complex, suggesting binding of DEDC with copper moiety of rusticyanin and resulting in inhibition of growth. In vitro reduction of purified rusticyanin by Fe(II) in absence of acid-stable cytochrome c is very slow, indicating the importance of cytochrome c in electron transport. Thus, in the iron oxidation process, acid-stable cytochrome c is the primary acceptor of electron, transferring the electron to rusticyanin at pH 2.0, which, in turn, affects electron transfer to iron-cytochrome c reductase around pH 5.5.