Role of a Ferric Ion-Reducing System in Sulfur Oxidation of Thiobacillus ferrooxidans

The properties of a ferric ion-reducing system which catalyzes the reduction of ferric ion with elemental sulfur was investigated with a pure strain of Thiobacillus ferrooxidans. In anaerobic conditions, washed intact cells of the strain reduced 6 mol of Fe³⁺ with 1 mol of elemental sulfur to give 6 mol of Fe²⁺, 1 mol of sulfate, and a small amount of sulfite. In aerobic conditions, the 6 mol of Fe²⁺ produced was immediately reoxidized by the iron oxidase of the cell, with a consumption of 1.5 mol of oxygen. As a result, Fe²⁺ production was never observed under aerobic conditions. However, in the presence of 5 mM cyanide, which completely inhibits the iron oxidase of the cell, an amount of Fe²⁺ production comparable to that formed under anaerobic conditions was observed under aerobic conditions. The ferric ion-reducing system had a pH optimum between 2.0 and 3.8, and the activity was completely destroyed by 10 min of incubation at 60°C. A short treatment of the strain with 0.5% phenol completely destroyed the ferric ion-reducing system of the cell. However, this treatment did not affect the iron oxidase of the cell. Since a concomitant complete loss of the activity of sulfur oxidation by molecular oxygen was observed in 0.5% phenol-treated cells, it was concluded that the ferric ion-reducing system plays an important role in the sulfur oxidation activity of this strain, and a new sulfur-oxidizing route is proposed for T. ferrooxidans.     

Synthesis of an Iron-Oxidizing System during Growth of Thiobacillus ferrooxidans on Sulfur-Basal Salts Medium

It was found that the de novo synthesis of not only sulfur:ferric ion oxidoreductase (ferric ion-reducing system) but also iron oxidase was absolutely required when Thiobacillus ferrooxidans AP19-3 was grown on sulfur-salts medium. The results strongly suggest that iron oxidase is involved in sulfur oxidation. This bacterium could not grow on sulfur-salts medium under anaerobic conditions with Fe³⁺ as a terminal electron acceptor, suggesting that energy conservation by electron transfer between elemental sulfur and Fe³⁺ is not available for this bacterium.     

Anaerobic Growth of Thiobacillus ferrooxidans

The obligately autotrophic acidophile Thiobacillus ferrooxidans was grown on elemental sulfur in anaerobic batch cultures, using ferric iron as an electron acceptor. During anaerobic growth, ferric iron present in the growth media was quantitatively reduced to ferrous iron. The doubling time in anaerobic cultures was approximately 24 h. Anaerobic growth did not occur in the absence of elemental sulfur or ferric iron. During growth, a linear relationship existed between the concentration of ferrous iron accumulated in the cultures and the cell density. The results suggest that ferric iron may be an important electron acceptor for the oxidation of sulfur compounds in acidic environments.     

Role of Thiobacillus ferrooxidans and sulphur (sulphide)-dependent ferric-ion-reducing activity in the oxidation of sulphide minerals

 Thiobacillus ferrooxidans oxidized the sulphide minerals e.g., pyrite, pyrrhotite and copper concentrate under anaerobic conditions in the presence of ferric ion as sole electron acceptor. Copper and iron were solubilized from sulphide ores by the sulphur (sulphide)-dependent ferric-ion oxidoreductase activity. Treatment of resting cells of T. ferrooxidans with 0.5% phenol for 30 min completely destroyed the iron- and copper-solubilizing activity. The above treatment destroyed the sulphur(sulphide)-dependent ferric-ion-reducing activity completely but did not affect the iron-oxidizing activity. The results suggest that sulphur(sulphide)-dependent ferric-ion-reducing activity actively participates in the oxidation of sulphide minerals under anaerobic conditions. The activity of sulphur(sulphide)-dependent ferric ion reduction in the solubilization of iron and copper from the sulphide ores were also observed under aerobic conditions in presence of sodium azide (0.1 μmol), which completely inhibits the iron-oxidizing activity. Received: 23 May 1995/Received revision: 10 October 1995/Accepted: 16 October 1995     

Ferric Iron Reduction by Acidophilic Heterotrophic Bacteria

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

Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge,a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions

Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. ¹³C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using ³H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions.     

Stoichiometry of bacterial anaerobic oxidation of elemental sulfur by ferric iron

The conventional stoichiometry of the oxidation of elemental sulfur by ferric iron in Acidithiobacillus ferrooxidans was not in agreement with our experimental data in terms of ferrous iron and proton formation. Reaction modelling under the actual conditions of bacterial activity resulted in a different stoichiometry, where additional iron species participate in the process to affect the number of released protons. The suggested reaction equation may more accurately predict the intensity of environmental acidification during the anaerobic bioprocess.     

Anaerobic leaching of covellite by Thiobacillus ferrooxidans

Thiobacillus ferrooxidans was able to grow under anaerobic conditions on copper sulphide with ferric ion as the electron acceptor. The dissolution of covellite under these conditions (68% after 35 days) was higher than values observed aerobically in cultures with similar media composition and almost as high as under aerobic conditions without iron. From these results we propose a mechanism for anaerobic bioleaching of covellite in the presence of ferric iron and speculate that it may occur in leach dumps where the oxygen concentration is, as reported elsewhere, very low. Received: 3 September 1996 / Received revision: 13 January 1997 / Accepted: 24 January 1997     

Amino Acid uptake by pea leaf fragments: specificity, energy sources, and mechanism

Amino acid uptake into leaf fragments of Pisum sativum depended on metabolism. Glycine uptake was optimal at 30 C and could be supported by respiration and by photosynthesis. Based on studies with an electron flow cofactor, inhibitors, and uncouplers, the energy source for glycine uptake was apparently ATP.     

Extraction of copper from an oxidized (lateritic) ore using bacterially catalysed reductive dissolution

An oxidized lateritic ore which contained 0.8 % (by weight) copper was bioleached in pH- and temperature-controlled stirred reactors under acidic reducing conditions using pure and mixed cultures of the acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans. Sulfur was provided as the electron donor for the bacteria, and ferric iron present in goethite (the major ferric iron mineral present in the ore) acted as electron acceptor. Significantly more copper was leached by bacterially catalysed reductive dissolution of the laterite than in aerobic cultures or in sterile anoxic reactors, with up to 78 % of the copper present in the ore being extracted. This included copper that was leached from acid-labile minerals (chiefly copper silicates) and that which was associated with ferric iron minerals in the lateritic ore. In the anaerobic bioreactors, soluble iron in the leach liquors was present as iron (II) and copper as copper (I), but both metals were rapidly oxidized (to iron (III) and copper (II)) when the reactors were aerated. The number of bacteria added to the reactors had a critical role in dictating the rate and yield of copper solubilised from the ore. This work has provided further evidence that reductive bioprocessing, a recently described approach for extracting base metals from oxidized deposits, has the potential to greatly extend the range of metal ores that can be biomined.     

Genetics and environmental regulation of Shigella iron transport systems

Shigella spp. have transport systems for both ferric and ferrous iron. The iron can be taken up as free iron or complexed to a variety of carriers. All Shigella species have both the Feo and Sit systems for acquisition of ferrous iron, and all have at least one siderophore-mediated system for transport of ferric iron. Several of the transport systems, including Sit, Iuc/IutA (aerobactin synthesis and transport), Fec (ferric di-citrate uptake), and Shu (heme transport) are encoded within pathogenicity islands. The presence and the genomic locations of these islands vary considerably among the Shigella species, and even between isolates of the same species. The expression of the iron transport systems is influenced by the concentration of iron and by environmental conditions including the level of oxygen. ArcA and FNR regulate iron transport gene expression as a function of oxygen tension, with the sit and iuc promoters being highly expressed in aerobic conditions, while the feo ferrous iron transporter promoter is most active under anaerobic conditions. The effects of oxygen are also seen in infection of cultured cells by Shigella flexneri; the Sit and Iuc systems support plaque formation under aerobic conditions, whereas Feo allows plaque formation anaerobically.     

Purification and some properties of sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans.

A sulfur:ferric ion oxidoreductase that utilizes ferric ion (Fe3+) as an electron acceptor of elemental sulfur was purified from iron-grown Thiobacillus ferrooxidans to an electrophoretically homogeneous state. Under anaerobic conditions in the presence of Fe3+, the enzyme reduced 4 mol of Fe3+ with 1 mol of elemental sulfur to give 4 mol of Fe2+ and 1 mol of sulfite, indicating that it corresponds to a ferric ion-reducing system (T. Sugio, C. Domatsu, O. Munakata, T. Tano, and K. Imai, Appl. Environ. Microbiol. 49:1401-1406, 1985). Under aerobic conditions, sulfite, but not Fe2+, was produced during the oxidation of elemental sulfur by this enzyme because the Fe2+ produced was rapidly reoxidized chemically by molecular oxygen. The possibility that Fe3+ serves as an electron acceptor under aerobic conditions was ascertained by adding o-phenanthroline, which chelates Fe2+, to the reaction mixture. Sulfur:ferric ion oxidoreductase had an apparent molecular weight of 46,000, and it is composed of two identical subunits (Mr = 23,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sulfur oxidation by this enzyme was absolutely dependent on the presence of reduced glutathione. The enzyme had an isoelectric point and a pH optimum at pH 4.6 and 6.5, respectively. Almost all the activity of sulfur:ferric ion oxidoreductase was observed in the osmotic shock fluid of the cells, suggesting that it was localized in the periplasmic space of the cells.     

The ability of aquatic macrophytes to increase root porosity and radial oxygen loss determines their resistance to sediment anoxia

Radial oxygen loss (ROL) has been suggested to be a major process to protect plants exposed to root anaerobic stress. In the present study, we aimed to test the importance of root porosity and radial oxygen loss on the aquatic macrophyte resistance to sediment anoxia. We expected that species living in eutrophic environments characterized by anaerobic conditions in sediments exhibited higher root porosity and radial oxygen loss than species restrained to oligotrophic environments. In this way, we compared the responses to sediment anoxia of two hydrophyte species growing under meso-eutrophic conditions in the field (Myriophyllum spicatum L. and Vallisneria spiralis L.) and three species growing under oligotrophic conditions (Potamogeton coloratus Horne, Elodea canadensis Michx and Sparganium emersum Michx.). Under laboratory conditions, ROL, root porosity, plant metabolism (aerobic respiration, photosynthesis, root fermentative activity) and plant growth were analysed after 3?months of acclimation in anaerobic sediments and compared with control values obtained from aerobic sediments. The results showed that two meso-eutrophic species (M. spicatum and V. spiralis) survived in anaerobic sediments and maintained similar photosynthesis rates than those measured under aerobic conditions. In contrast, the three oligotrophic species (P. coloratus, E. canadensis and S. emersum) suffered net biomass loss and depressed their photosynthesis rates under anaerobic conditions. All variables associated with plant tolerance to anaerobic conditions (maintenance of photosynthesis, aerobic respiration and growth rate, and limitation of root fermentative activity) were positively linked to root porosity and ROL. According to our hypothesis, species that could survive to anaerobic conditions were the species able to increase their root porosity and ROL under these conditions. Thus, in ecological studies, it would be useful to use the root porosity and ROL plasticity as biological traits in order to model the distribution of macrophytes in river floodplains.     

Inhibition of Sulfur Use by Sulfite Ion in Thiobacillus ferrooxidans

In Thiobacillus ferrooxidans AP19-3, elemental sulfur is oxidized by the cooperation of three enzymes, namely, hydrogen sulfide: ferric ion oxidoreductase (SFORase), sulfite: ferric ion oxidoreductase, and iron oxidase. Sulfite ions are one of the products when elemental sulfur is oxidized by SFORase. Under the conditions in which sulfite ions are accumulated in the cells, use of sulfur as an energy source by this strain was strongly inhibited. So the mechanism of inhibition by sulfite ions in T. ferrooxidans AP19-3 was studied. The activities of SFORase and iron oxidase were completely inhibited by 0.8 mm and 1.5 mm NaHSO₃, respectively. ¹⁴CO₂ uptake into washed intact cells was also completely inhibited by 1mm NaHSO₃ when ferrous ion or elemental sulfur was used as an energy source. However, the activities of ribulose-1,5-bisphosphate carboxylase, phosphoribulokinase, and ribosephosphate isomerase measured with a cell-free extract were not inhibited by NaHSO₃ at 1 mm, indicating that sulfite ions didn’t inhibit key enzymes of the Calvin cycle. Since the activity of CO₂ uptake into washed intact cells was absolutely dependent on Fe^{2 +} - or S0-oxidation, mechanism of inhibition of sulfur use by sulfite ions is proposed as follows: sulfite ions inhibit SFORase and iron oxidase, as a result T. ferrooxidans AP19-3 can not obtain a carbon source for CO₂ fixation and stops cell growth on sulfur-salts medium.     

Understanding aerobic/anaerobic metabolism in Caldibacillus debilis through a comparison with model organisms

Caldibacillus debilis GB1 is a facultative anaerobe isolated from a thermophilic aero-tolerant cellulolytic enrichment culture. There is a lack of representative proteomes of facultative anaerobic thermophilic Bacillaceae, exploring aerobic/anaerobic expression. The C. debilis GB1 genome was sequenced and annotated, and the proteome characterized under aerobic and anaerobic conditions while grown on cellobiose. The draft sequence of C. debilis GB1 contains a 3,340,752 bp chromosome and a 5,386 bp plasmid distributed over 49 contigs. Two-dimensional liquid chromatography mass spectrometry/mass spectrometry was used with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) to compare protein expression profiles, focusing on energy production and conversion pathways. Under aerobic conditions, proteins in glycolysis and pyruvate fermentation pathways were down-regulated. Simultaneously, proteins within the tricarboxylic acid cycle, pyruvate dehydrogenase, the electron transport chain, and oxygen scavenging pathways showed increased amounts. Under anaerobic conditions, protein levels in fermentation pathways were consistent with the generated end-products: formate, acetate, ethanol, lactate, and CO₂. Under aerobic conditions CO₂ and acetate production was consistent with incomplete respiration. Through a direct comparison with gene expression profiles from Escherichia coli, we show that global regulation of core metabolism pathways is similar in thermophilic and mesophilic facultative anaerobes of the Phylum Proteobacteria and Firmicutes.     

Existence of a Hydrogen Sulfide:Ferric Ion Oxidoreductase in Iron-Oxidizing Bacteria

The existence of a hydrogen sulfide:ferric ion oxidoreductase, which catalyzes the oxidation of elemental sulfur with ferric ions as an electron acceptor to produce ferrous and sulfite ions, was assayed with washed intact cells and cell extracts of various kinds of iron-oxidizing bacteria, such as Thiobacillus ferrooxidans 13598, 13661, 14119, 19859, 21834, 23270, and 33020 from the American Type Culture Collection, Leptospirillum ferrooxidans 2705 and 2391 from the Deutsche Sammlung von Mikroorganismen, L. ferrooxidans BKM-6-1339 and P3A, and moderately thermophilic iron-oxidizing bacterial strains BC1, TH3, and Alv. It was found that hydrogen sulfide:ferric ion oxidoreductase activity comparable to that of T. ferrooxidans AP19-3 was present in all iron-oxidizing bacteria tested, suggesting a wide distribution of this enzyme in iron-oxidizing bacteria.     

Anaerobic growth on elemental sulfur using dissimilar iron reduction by autotrophic Thiobacillus ferrooxidans

Abstract Anaerobic growth on elemental sulfur using dissimilar iron reduction by Thiobacillus ferrooxidans has been demonstrated. The ferric ion reducing activity (FIR) of the anaerobic cells was double that of the aerobic cells. Significant differences in inhibition of FIR by respiratory inhibitors were observed between aerobic and anaerobic cells. A higher amount of cytochrome was detected in anaerobic cells compared to aerobic cells. Absorption minima developed with the addition of ferric sulfate in the dithionite reduced cell suspension demonstrated that the ferric ion could accept electrons from the cytochrome system of this bacterium. The possibility of two different electron transport chains in ferric ion reduction is discussed.     

Energy-dependent accumulation of iron by isolated rat liver mitochondria V. Effect on factors controlling respiration and oxidative phosphorylation

1. Depending on the metabolic state, the addition of iron(III)-sucrose induces an inhibition or a stimulation of the respiration rate when added to isolated rat liver mitochondria.2. Under conditions identical to those used in the accumulation studies (Romslo, I. and Flatmark, T. (1973) Biochim. Biophys. Acta 305, 29?40), the ferric complex induces a decrease in the oxygen uptake concomitant to an oxidation of cytochromes c (+c₁) and a (+a₃). These results suggest that ferric iron is reduced to ferrous iron by the respiratory chain prior to or simultaneously with its energy-dependent accumulation.3. On the other hand, the addition of iron(III)-sucrose induces a stimulation of respiration in State 4 and State 3 provided Mg²⁺ is present in the suspending medium. In contrast to Ca²⁺, iron stimulates State 4 respiration in a cyclic process only within narrow concentration limits; at concentrations of iron above 100 μM the respiration remains in the activated state until anaerobiosis. The stimulation of State 4 respiration is more pronounced with succinate than with NAD-linked substrates, a difference which partly may be attributed to a stimulation of the succinate dehydrogenase complex.4. The stimulation of respiration by iron is approx. 3 times higher in State 3 than in State 4 and this difference can be attributed to a stimulation of the adenine nucleotide exchange reaction in State 3 with a concomitant increase in the rate of oxidative phosphorylation, although the $\text{P}\text{O}$ ratio is slightly diminished.