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As global demands for rare-earth elements (REEs) continue to grow, the biological recovery of REEs has been explored as a promising strategy, driven by potential economic and environmental benefits. It is known that calcium-binding domains, including helix-loop-helix EF hands and repeats-in-toxin (RTX) domains, can bind lanthanide ions due to their similar ionic radii and coordination preference to calcium. Recently, the lanmodulin protein from Methylorubrum extorquens was reported, which has evolved a high affinity for lanthanide ions over calcium. Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile, which has been explored for use in bioleaching for metal recovery. In this report, A. ferrooxidans was engineered for the recombinant intracellular expression of lanmodulin. In addition, an RTX domain from the adenylate cyclase protein of Bordetella pertussis , which has previously been shown to bind Tb 3+ , was expressed periplasmically via fusion with the endogenous rusticyanin protein. The binding of lanthanides (Tb 3+ , Pr 3+ , Nd 3+ , and La 3+ ) was improved by up to 4-fold for cells expressing lanmodulin and 13-fold for cells expressing the RTX domains in both pure and mixed metal solutions. Interestingly, the presence of lanthanides in the growth media enhanced protein expression, likely by influencing protein stability. Both engineered cell lines exhibited higher recoveries and selectivities for four tested lanthanides (Tb 3+ , Pr 3+ , Nd 3+ , and La 3+ ) over non-REEs (Fe 2+ and Co 2+ ) in a synthetic magnet leachate, demonstrating the potential of these new strains for future REE reclamation and recycling applications.

High-potential iron-sulfur proteins (HiPIPs) from extremely acidophilic chemolithotrophic non-photosynthetic Acidithiobacillus commonly play a crucial role in ferrous or sulfurous biooxidation. Acidithiobacillus exhibit important industrial applications for bioleaching valuable metals from sulfide ores. In this study, two HiPIP genes from thermophilic Acidithiobacillus caldus SM-1 were cloned and successfully expressed, and their proteins were purified. The proteins displayed a brownish color with an optical absorbance peak at approximately 385 nm and an electronic paramagnetic resonance (EPR) g value of approximately 2.01, which confirmed that the iron-sulfur cluster was correctly inserted into the active site when the proteins were generated in E. coli. The proteins were more thermostable than HiPIPs from mesophilic Acidithiobacillus. The direct electron transfer (DET) between HiPIPs and electrode was achieved by the 2-mercaptopyrimidine (MP) surface-modified gold electrodes; the redox potentials of the HiPIP1 and HiPIP2 measured by cyclic voltammetry were approximately 304.5 mV and 400.5 mV, respectively. The electron transfer rate constant was estimated to be 0.75 s -1 and 0.66 s -1 , respectively. The MP/Au electrode and Au electrode showed consistent differences in heterogeneous electron transfer rates and electron transfer resistances. Bioinformatics and molecular simulations further explained the direct electron transfer between the proteins and surface-modified electrode., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies. Acidithiobacillus ferrooxidans is an iron- and sulfur-oxidizing chemolithoautotroph that is known to participate in metal bioleaching, and its metabolic capabilities have been exploited for industrial-scale copper and gold biomining. In addition to bioleaching, microorganisms could also be engineered for selective metal binding, enabling new opportunities for metal bioseparation and recovery. Here, we explored the ability of polyhistidine (polyHis) tags appended to two recombinantly expressed endogenous proteins to enhance the metal binding capacity of A. ferrooxidans . The genetically engineered cells achieved enhanced cobalt and copper binding capacities, and the Langmuir isotherm captures their interaction behavior with these divalent metals. Additionally, the cellular localization of the recombinant proteins correlated with kinetic modeling of the binding interactions, where the outer membrane-associated polyHis-tagged licanantase peptide bound the metals faster than the periplasmically expressed polyHis-tagged rusticyanin protein. The selectivity of the polyHis sequences for cobalt over copper from mixed metal solutions suggests potential utility in practical applications, and further engineering could be used to create metal-selective bioleaching microorganisms.

Cyc2 is the key protein in the outer membrane of Acidithiobacillus ferrooxidans that mediates electron transfer between extracellular inorganic iron and the intracellular central metabolism. This cytochrome c is specific for iron and interacts with periplasmic proteins to complete a reversible electron transport chain. A structure of Cyc2 has not yet been characterized experimentally. Here we describe a structural model of Cyc2, and associated proteins, to highlight a plausible mechanism for the ferrous iron electron transfer chain. A comparative modeling protocol specific for trans membrane beta barrel (TMBB) proteins in acidophilic conditions (pH ~ 2) was applied to the primary sequence of Cyc2. The proposed structure has three main regimes: Extracellular loops exposed to low-pH conditions, a TMBB, and an N-terminal cytochrome-like region within the periplasmic space. The Cyc2 model was further refined by identifying likely iron and heme docking sites. This represents the first computational model of Cyc2 that accounts for the membrane microenvironment and the acidity in the extracellular matrix. This approach can be used to model other TMBBs which can be critical for chemolithotrophic microbial growth., (© 2021 The Protein Society.)

A novel isolated strain Acidithiobacillus ferrooxidans BMSNITK17 has been investigated for its bioleaching potential from lateritic soil and the results are presented. System conditions like pH, feed mineral particle size, pulp density, temperature, rotor speed influences bioleaching potential of Acidithiobcillus ferrooxidans BMSNITK17 in leaching out iron from laterite soil. Effect of sulfate addition on bioleaching efficiency is studied. The bioleached laterite iron (BLFe's) on evaluation for its catalytic role in Fenton's oxidation for the degradation of ametryn and dicamba exhibits 94.24% of ametryn degradation and 92.45% of dicamba degradation efficiency. Fenton's oxidation performed well with the acidic pH 3. The study confirms the role of Acidithiobacillus ferrooxidans in leaching iron from lateritic ore and the usage of bioleached lateritic iron as catalyst in the Fenton's Oxidation., Competing Interests: The authors have declared that no competing interests exist.

Although silver ion in the solution is an important factor affecting the biodissolution of chalcopyrite, the effect of silver ion on the release of copper ion from chalcopyrite to the environment has not been explored until now. In order to fill this knowledge gap, the effect of silver ion on copper release from chalcopyrite in the presence of Acidithiobacillus ferrooxidans was investigated. The results indicate that silver ion significantly enhanced chalcopyrite biodissolution, thereby releasing more copper ion. In turn, this indicates that the release of copper ion from chalcopyrite to the environment was increased under these conditions. Biodissolution results, bacterial adsorption experiments, elemental composition analysis, and electrochemical analysis reveal that the enhancement of silver ion on copper ion release from chalcopyrite was mainly attributed to the improvement of electrochemical activity of chalcopyrite and the inhibition of the formation of passivation layer (S n 2- /S 0 ) on the chalcopyrite surface. This study provides a better understanding of the effect of silver ion on the release of copper ion from chalcopyrite to the environment. In the future, the influence of silver ion on chalcopyrite biodissolution should be considered in the evaluation of copper ion pollution to ensure reliability., Competing Interests: Declaration of Competing Interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Tooeleite is the only known ferric arsenite sulfate mineral and has environmental significance for arsenic remediation. This study investigated the formation and stability of biogenic tooeleite in Fe(II)-As(III)-SO 4 2- environment using Acidithiobacillus ferrooxidans under the ambient conditions. The results show that bacteria facilitated the formation and crystallization of tooeleite owing to the microbial oxidation of Fe(II) to Fe(III). Due to the better growth of bacteria, the higher removal of As(III) by tooeleite formation was achieved under 8.978-10.806 g/L initial Fe(II) concentration and 2.00-3.00 initial pH, and the highest efficiency was ~95%. Fe(III) and As(III) precipitated simultaneously into two types of tooeleite. The relatively stable tooeleite is featured by the developed (020) crystal face and the bulk-like structure with thick flakes. This study yields a better understanding of biogenic tooeleite, and the importance of tooeleite formation in As(III)-rich environment for arsenic remediation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: This study aims to investigate the attachment of Acidithiobacillus ferrooxidans to pyrite in two different environments: fresh and saline water (water with 35 g/L of NaCl or 0.6 M). Adsorption isotherms were analyzed using the Langmuir and Freundlich models. Saline water is water with 35 g/L of NaCl (0.6 M), which is the concentration of NaCl in seawater. The use of raw seawater in mining is becoming relevant in leaching and flotation process. At the same time the use of microorganisms in both processes is gaining attention. For this reason, it is important to study the behavior of adherence of microorganisms to minerals in saline aqueous environments, similar to seawater., Results: The bacteria showed a higher level of attachment to pyrite in fresh water than in saline water. The Langmuir model fitted better the experimental data obtained in fresh water than in saline water with a coefficient of determination (R 2 ) of 0.85 and 0.61 for fresh and saline water, respectively., Conclusions: This suggests that the bacteria tend to adhere more as a monolayer in fresh than in saline water in the early stage of adhesion.

Increasing awareness toward environmental remediation and renewable energy has led to a vigorous demand for exploring a win-win strategy to realize the eco-efficient conversion of pollutants ("trash") into energy-storage nanomaterials ("treasure"). Inspired by the biological metabolism of bacteria, Acidithiobacillus ferrooxidans (A. ferrooxidans) is successfully exploited as a promising eco-friendly sustainable biofactory for the controllable fabrication of α-Fe 2 O 3 nanorods via the oxidation of soluble ferrous irons to insoluble ferric substances (Jarosite, KFe 3 (SO 4 ) 2 (OH) 6 ) and a facile subsequent heat treatment. It is demonstrated that the stable solid electrolyte interphase layers and marvelous cracks in situ formed in biometabolic α-Fe 2 O 3 nanorods play important roles that not only significantly enhance the structure stability but also facilitate electron and ion transfer. Consequently, these biometabolic α-Fe 2 O 3 nanorods deliver a superior stable capacity of 673.9 mAh g -1 at 100 mA g -1 over 200 cycles and a remarkable multi-rate capability that observably prevails over the commercial counterpart. It is highly expected that such biological synthesis strategies can shed new light on an emerging field of research interconnecting biotechnology, energy technology, environmental technology, and nanotechnology., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

ArsR As(III)-responsive transcriptional repressors, members of the ArsR/SmtB family of metalloregulatory proteins, have been characterized biochemically but, to date, no As(III)-bound structure has been solved. Here we report two crystal structures of ArsR repressors from Acidithiobacillus ferrooxidans (AfArsR) and Corynebacterium glutamicum (CgArsR) in the As(III)-bound form. AfArsR crystallized in P2 1 space group and diffracted up to 1.86 Å. CgArsR crystallized in P2 1 2 1 2 1 and diffracted up to 1.6 Å. AfArsR showed one As(III) bound in one subunit of the homodimer, while the CgArsR structure showed two As(III) bound with S 3 coordination, one in each monomer. Previous studies indicated that in AfArsR As(III) binds to Cys95, Cys96 and Cys102 from the same monomer, while, in CgArsR, to Cys15, Cys16 from one monomer and Cys55 from the other monomer. The dimer interfaces of these structures showed distinct differences from other members of the ArsR/SmtB family of proteins, which potentially renders multiple options for evolving metal(loid) binding sites in this family of proteins. Also, CgArsR presents a new α2-N binding site, not the previously predicted α3-N site. Despite differences in the location of the binding cysteines in the primary sequences of these proteins, the two metal binding sites are almost congruent on their structures, an example of convergent evolution. Analyses of the electrostatic surface of the proteins at the DNA binding domain indicate that there two different modes of derepression in the ArsR/SmtB family of metalloregulatory proteins., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The importance of microorganisms and biotechnology in space exploration and future planets colonization has been discussed in the literature. Meteorites are interesting samples to study microbe-mineral interaction focused on space exploration. The chemolithotropic bacterium Acidithiobacillus ferrooxidans has been used as model to understand the iron and sulfur oxidation. In this work, capillary electrophoresis with capacitively coupled contactless conductivity detection and UV detection was used to monitor bacterial growth in a meteorite simulant by measuring the conversion of Fe 2+ into Fe +3 . The effect of Co 2+ and Ni 2+ (metals also found in meteorites) on the bacterial growth was also evaluated. The presented method allowed the analyses of all metals in a single run (less than 8 min). The background electrolyte was composted of 10 mmol/L α-hydroxyisobutyric acid/Histidine. For comparison purpose, the samples were also analyzed by UV-Vis spectrophotometry. The Fe 2+ conversion into Fe 3+ by A. ferrooxidans was observed up to 36 h with the growth rate constant of 0.19/h and 0.21/h in Tuovinen and Kelly (T&K) and in meteorite simulant media, respectively. The developed method presents favorable prospect to monitor the growth of other chemolithotropic microorganisms for biotechnology applications., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Microbiologically influenced corrosion (MIC) of S32654 (654SMO) super austenitic stainless steel (SASS) by acid producing bacterium (APB), Acidithiobacillus caldus SM-1, a strain of sulfur-oxidizing bacteria (SOB) used in biohydrometallurgy field, was investigated using electrochemical measurements and surface characterizations during a 14-day immersion test. The results indicated that S32654 SASS was susceptible to MIC by APB, and A. caldus SM-1 was capable of producing an aggressive acidic environment underneath the biofilm, resulting in the dissolution of the passive film and severe pitting attacks against S32654 SASS, which is commonly regarded as a corrosion resistant material., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The mineral sulfide-oxidising Acidithiobacillus ferrooxidans has been extensively studied over many years but some fundamental aspects of its metabolism remain uncertain, particularly with regard to its anaerobic oxidation of sulfur. This label-free, liquid chromatography-electron spray ionisation-mass spectrometry-based proteomic analysis estimated relative protein abundance during aerobic and anaerobic growth of At. ferrooxidans. One of its two bc1 complexes, that encoded by the petII operon, was strongly implicated in anaerobic ferric iron-coupled sulfur oxidation, probably in conjunction with two cytochromes. These two cytochromes are homologs of the Cyc2 and Cyc1 proteins that are involved in ferrous iron oxidation. The previously undetected cytochromes apparently associated with anaerobic growth in At. ferrooxidans appear to be absent in many other ferrous iron-oxidising acidophiles that can also reduce ferric iron, which suggests a diversity in the ferric-iron-coupled sulfur oxidation pathways. For aerobic growth of At. ferrooxidans, this analysis was consistent with the generally accepted mechanism for its oxidation of ferrous iron. Unexpectedly, proteins encoded by the petI operon were not abundant and generally not detected in the proteomic analyses of cells grown aerobically on sulfur, although there was some expression of genes of the petI and petII operons in these cells.

Pure phospholipids and membrane fragments from bacterial cells living under various conditions were studied against the influence of the surrounding acidity on the internal dynamics. For that we compared mean square displacements extracted from elastic incoherent neutron scattering data, measured both at low and at neutral pH, of the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine and of samples from neutralophilic and acidophilic micro-organisms (some being hyperthermophilic and others mesophilic). The lipids showed a slight shift in the phase transition temperature of about 4 degrees under pH variation and became slightly more mobile at lower pH. The membrane fragments not used to extreme acidic conditions were significantly more sensitive to variations in the pH values, whereas the acidophilic and -tolerant samples were much less influenced by this parameter. They presented the higher softness at low pH, which was closer to their native condition. Such findings might be a hint for adaptation mechanisms to different acidity conditions.

Biogenic flocculant produced by Acidithiobacillus ferrooxidans was used for sludge conditioning to improve the dewaterability of anaerobically-digested sludge, and its efficiency was compared with commercial cationic polyacrylamide (PAM). Biogenic flocculant rapidly reduced the pH and increased the oxidation-reduction potential of sludge. Capillary suction time (CST) and specific resistant to filtration (SRF) of sludge was decreased by 74% and 89%, respectively, compared with control; and the reductions were 58% CST and 67% SRF higher when compared with commercial polymer. Biogenic treatment improved the sludge calorific value by 13%, and also reduced the unpleasant odor. The small-scale mechanical filter press study showed that the biogenic flocculant can reduce the moisture content of sludge to 70%, and improve the clarity of the filtrate in terms of removal of total suspended solids and total dissolved solids when compared with synthetic polymer treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study presents the corrosion behavior of the copper-nickel (Cu-Ni) alloy in the presence of Acidithiobacillus ferrooxidans (A. ferrooxidans) and glucose oxidase (GOx) enzyme. In both the cases ferric ions played an important role in weight loss and thereby to carry out the corrosion of the Cu-Ni alloy. A corrosion rate of 0.6 (±0.008), 2.11 (±0.05), 3.69 (±0.26), 0.7 (±0.006) and 0.08 (±0.002) mm/year was obtained in 72 h using 9K medium with ferrous sulfate, A. ferrooxidans culture supernatant, A. ferrooxidans cells, GOx enzyme and hydrogen peroxide (H2O2) solution respectively. The scanning electron microscopy (SEM) micrographs showed that a variable extent of corrosion was caused by 9K medium with ferrous sulfate, GOx and A. ferrooxidans cells. An arithmetic average surface roughness (Ra) of 174.78 nm was observed for the control work-piece using optical profilometer. The change in Ra was observed with the treatment of the Cu-Ni alloy using various systems. The Ra for 9K medium with ferrous sulfate, GOx and A. ferrooxidans cells was 374.54, 607.32 and 799.48 nm, respectively, after 24 h. These results suggest that A. ferrooxidans cells were responsible for more corrosion of the Cu-Ni alloy than other systems used.

Argentojarosite (AgFe3(SO4)2(OH)6) is formed as a secondary phase in Ag-catalyzed bioleaching of chalcopyrite (CuFeS2), but to date very little is known about the paragenesis or characteristics of this silver-containing compound. The purpose of this study was to synthesize argentojarosite via biological oxidation of 120mM ferrous sulfate by Acidithiobacillus ferrooxidans. Because of its toxicity to A. ferrooxidans, Ag(+) (as AgNO3) was added to spent culture media (pH2) after complete oxidation of ferrous sulfate. Schwertmannite (ideally Fe8O8(OH)6(SO4)) was precipitated during the iron oxidation phase, and subsequent Ag(+) addition resulted in the formation of argentojarosite. Contact time (8h, 5d, and 14d) and Ag(+) concentration (0, 5, 20, and 40mM) were used as variables in these experiments. Synthesis of argentojarosite, schwertmannite and other mineral phases was confirmed through X-ray diffraction analysis. Additional analyses of solid-phase oxidation products included elemental composition, color and specific surface area. The sample synthesized in the presence of 40mM Ag(+) and with 14d contact time yielded an X-ray diffraction pattern of well crystallized argentojarosite, and its elemental composition closely matched the calculated Ag, Fe, and S contents of ideal argentojarosite. The color and surface area of the remaining samples were influenced by the presence of residual schwertmannite. This phase remained stable over the time course of 14d when no Ag(+) was present in the system. When equilibrations were extended to 42d, partial conversion of reference schwertmannite to goethite was noted in the absence of Ag. In the presence of 20mM or 40mM Ag over the same time course, some formation of argentojarosite was also noted. In this case, schwertmannite was the only source of Fe and SO4 for argentojarosite formation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In extremely acidic environments, ferric iron can be a thermodynamically favorable electron acceptor during elemental sulfur oxidation by some Acidithiobacillus spp. under anoxic conditions. Quantitative 2D-PAGE proteomic analysis of a resting cell suspension of a sulfur-grown Acidithiobacillus ferrooxidans CCM 4253 subculture that had lost its iron-reducing activity revealed 147 protein spots that were downregulated relative to an iron-reducing resting cell suspension of the antecedent sulfur-oxidizing culture and 111 that were upregulated. Tandem mass spectrometric analysis of strongly downregulated spots identified several physiologically important proteins that apparently play roles in ferrous iron oxidation, including the outer membrane cytochrome Cyc2 and rusticyanin. Other strongly repressed proteins were associated with sulfur metabolism, including heterodisulfide reductase, thiosulfate:quinone oxidoreductase and sulfide:quinone reductase. Transcript-level analyses revealed additional downregulation of other respiratory genes. Components of the iron-oxidizing system thus apparently play central roles in anaerobic sulfur oxidation coupled with ferric iron reduction in the studied microbial strain., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Unlabelled: Acidithiobacillus ferrooxidans is used in industrial bioleaching of minerals to extract valuable metals. A. ferrooxidans strain ATCC 53993 is much more resistant to copper than other strains of this microorganism and it has been proposed that genes present in an exclusive genomic island (GI) of this strain would contribute to its extreme copper tolerance. ICPL (isotope-coded protein labeling) quantitative proteomics was used to study in detail the response of this bacterium to copper. A high overexpression of RND efflux systems and CusF copper chaperones, both present in the genome and the GI of strain ATCC 53993 was found. Also, changes in the levels of the respiratory system proteins such as AcoP and Rus copper binding proteins and several proteins with other predicted functions suggest that numerous metabolic changes are apparently involved in controlling the effects of the toxic metal on this acidophile., Significance: Using quantitative proteomics we overview the adaptation mechanisms that biomining acidophiles use to stand their harsh environment. The overexpression of several genes present in an exclusive genomic island strongly suggests the importance of the proteins coded in this DNA region in the high tolerance of A. ferrooxidans ATCC 53993 to metals., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Acidithiobacillus ferrooxidans is an acidophile that thrives in metal-contaminated environments and tolerates high levels of uranium. To gain a better understanding of the processes involved in U(VI) resistance, comparative proteomics was used. The proteome of A. ferrooxidans was grown in the presence and absence of 0.5 mM U(VI); expression of 17 proteins was upregulated and one was downregulated. Most proteins with increased expression are part of the general stress response or are involved in reactive oxygen species detoxification. Four novel proteins showed increased expression in the presence of U(VI) and may contribute to U(VI) resistance via thiol homoeostasis and U(VI) binding., (Copyright © 2016 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Sulfide:quinone oxidoreductase (SQR) is a peripheral membrane enzyme that catalyzes the oxidation of sulfide and the reduction of ubiquinone. Ubiquinone binds to a conserved hydrophobic domain and shuttles electrons from a noncovalent flavin adenine dinucleotide cofactor to the membrane-bound quinone pool. Utilizing the structure of decylubiquinone bound to Acidithiobacillus ferrooxidans SQR, we combined site-directed mutagenesis and kinetic approaches to analyze quinone binding. SQR can reduce both benzoquinones and naphthoquinones. The alkyl side-chain of ubiquinone derivatives enhances binding to SQR but limits the enzyme turnover. Pentachlorophenol and 2-n-heptyl-4-hydroxyquinoline-N-oxide are potent inhibitors of SQR with apparent inhibition constants (Ki) of 0.46 μmol·L(-1) and 0.58 μmol·L(-1), respectively. The highly conserved amino acids surrounding the quinone binding site play an important role in quinone reduction. The phenyl side-chains of Phe357 and Phe391 sandwich the benzoquinone head group and are critical for quinone binding. Importantly, conserved amino acids that define the ubiquinone-binding site also play an important role in sulfide oxidation/flavin reduction.

Present study aimed to resolve the conflict between cell growth and magnetosome formation of Acidithiobacillus ferrooxidans (A. ferrooxidans) in batch experiments by applying response surface methodology (RSM) integrated a desirability function approach. The effects of several operating parameters on cell growth (OD600) and magnetosome production (Cmag) were evaluated. The maximum overall desirability (D) of 0.923 was achieved at iron concentration of 125.07mM, shake speed of 122.37rpm and nitrogen concentration of 2.40g/L. Correspondingly, the OD600 and Cmag were 0.522 and 1.196, respectively. The confirmation experiment confirmed that the optimum OD600 and Cmag obtained were in good agreement with the predicted values. The inductively coupled plasma atomic emission spectrometer (ICP-AES) and transmission electron microscopy (TEM) analyses revealed that the production of magnetosomes could be improved via optimization. X-ray diffraction (XRD) showed the magnetosomes are magnetite. Results indicated that RSM with a desirability function was a useful technique to get the maximum OD600 and Cmag simultaneously., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Calcium oxide was added into ferrous ion oxidation system in the presence of Acidithiobacillus ferrooxidans at concentrations of 0-4.00 g/L. The pH, ferrous ion oxidation efficiency, total iron precipitation efficiency, and phase of the solid minerals harvested from different treatments were investigated during the ferrous ion oxidation process. In control check (CK) system, pH of the solution decreased from 2.81 to 2.25 when ferrous ions achieved complete oxidation after 72 h of Acidithiobacillus ferrooxidans incubation without the addition of calcium oxide, and total iron precipitation efficiency reached 20.2%. Efficiency of ferrous ion oxidation and total iron precipitation was significantly improved when the amount of calcium oxide added was ≤1.33 g/L, and the minerals harvested from systems were mainly a mixture of jarosite and schwertmannite. For example, the ferrous ion oxidation efficiency reached 100% at 60 h and total iron precipitation efficiency was increased to 32.1% at 72 h when 1.33 g/L of calcium oxide was added. However, ferrous ion oxidation and total iron precipitation for jarosite and schwertmannite formation were inhibited if the amount of calcium oxide added was above 2.67 g/L, and large amounts of calcium sulfate dihydrate were generated in systems.

Bioleaching using an iron-oxidizing bacterium, Acidithiobacillus ferrooxidans, and its biogenic flocculants was evaluated to improve the dewaterability of chemically enhanced primary treatment (CEPT) sewage sludge. CEPT sludge in flasks was inoculated with A. ferrooxidans culture, medium-free cells and the cell-free culture filtrate with and without the energy substance Fe(2+), and periodically the sludge samples were analysed for the dewaterability. This investigation proves that bioleaching effectively improved the sludge dewaterability as evidenced from drastic reduction in capillary suction time (≤20 seconds) and specific resistance to filtration (≥90%); however, it requires an adaptability period of 1-2 days. On the other hand, the biogenic flocculant produced by A. ferrooxidans greatly decreased the time-to-filtration and facilitated the dewaterability within 4 h. Results indicate that rapid dewatering of CEPT sludge by biogenic flocculants provides an opportunity to replace the synthetic organic polymer for dewatering.

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 nm(2) 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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Acidithiobacillus ferroxidans strains were isolated from acidophilic microbial communities of Kazakhstan sulfide ore deposits. Their biotechnologically important properties (optimal and maximal growth temperatures and resistance to NaCl) were determined. While temperature optima of the strains were the same (30-32 degrees C), temperature ranges were different. Thus, strain TFBK oxidized iron very poorly at 37 degrees C, while for strain TFV, the iron oxidation rate at this temperature was insignificantly lower than at lesser temperatures. NaCl inhibited the oxidative activity of both strains. Iron oxidation by strain TFV was inhibited at 5 g/L NaCl and was suppressed almost completely at 20 g/L. Iron oxidation by strain TFBK was inhibited by NaCl to a lesser degree, so that iron oxidation rate was relatively high at 10 g/L, while at 20 g/L NaCl the process was not suppressed completely, although the oxidation rate was low. Sulfur oxidation by these strains was less affected by NaCl than oxidation of ferrous iron. Sulfur oxidation by strain TFV was considerably inhibited only at 20 g/L NaCl, but was not suppressed completely. Sulfur oxidation by strain TFBK was more affected by NaCl. At 10 g/L NaCl the oxidation rate was much lower than at lower NaCl concentrations (sulfate concentrations after 6 days of oxidation at 5 and 10 g/L NaCl were -130 and -100 mM, respectively). While sulfur oxidation by strain TFBK was considerably inhibited at 10 and 20 g/L NaCl, similar to strain TFV it was not suppressed completely. Our results indicate the adaptation of the species A. ferrooxidans to a broad range of growth conditions.

The response of Acidithiobacillus ferrooxidans ATCC 23270 to copper was analyzed in sulfur-grown cells by using quantitative proteomics. Forty-seven proteins showed altered levels in cells grown in the presence of 50 mM copper sulfate. Of these proteins, 24 were up-regulated and 23 down-regulated. As seen before in ferrous iron-grown cells, there was a notorious up-regulation of RND-type Cus systems and different RND-type efflux pumps, indicating that these proteins are very important in copper resistance. Copper also triggered the down-regulation of the major outer membrane porin of A. ferrooxidans in sulfur-grown bacteria, suggesting they respond to the metal by decreasing the influx of cations into the cell. On the contrary, copper in sulfur-grown cells caused an overexpression of putative TadA and TadB proteins known to be essential for biofilm formation in bacteria. Surprisingly, sulfur-grown microorganisms showed increased levels of proteins related with energy generation (rus and petII operons) in the presence of copper. Although rus operon is overexpressed mainly in cells grown in ferrous iron, the up-regulation of rusticyanin in sulfur indicates a possible role for this protein in copper resistance as well. Finally, copper response in A. ferrooxidans appears to be influenced by the substrate being oxidized by the microorganism., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Attachments of Acidithiobacillus ferrooxidans ATCC 23270 onto elemental sulfur, quartz and complex chalcopyrite were investigated by analysis of its extracellular polymeric substances as well as applying Langmuir and Freundlich equations. The two equations fitted the adsorption equilibrium data with significant correlation coefficient over 0.9. This indicated that bacterial attachment is complicated and involves Langmuir and Freundlich characterizations. Sulfur-grown cells showed the highest affinity for the three solid substrates. The investigated complex chalcopyrite possessed a higher maximum adsorption capacity for A. ferrooxidans than elemental sulfur or quartz. The Freundlich fitting parameters suggested that quartz had a weaker adsorption capacity and smaller adsorption areas than elemental sulfur or the complex chalcopyrite. It is not the content of total carbohydrates or proteins in EPS but their ratios that determine the affinity differences between cells and substrates.

Understanding the magnetic properties of magnetotactic bacteria (MTBs) is of great interest in fields of life sciences, geosciences, biomineralization, biomagnetism, and planetary sciences. Acidithiobacillus ferrooxidans (At. ferrooxidans), obtaining energy through the oxidation of ferrous iron and various reduced inorganic sulfur compounds, can synthesize intracellular magnetite magnetosomes. However, the magnetic properties of such microorganism remain unknown. Here we used transmission electronmicroscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) assay, vibrating sample magnetometer (VSM), magneto-thermogravimetric analysis (MTGA), and low temperature magnetometry to comprehensively investigate the magnetic characteristics of At. ferrooxidans. Results revealed that each cell contained only 1 to 3 magnetite magnetosomes, which were arranged irregularly. The magnetosomes were generally in a stable single-domain (SD) state, but superparamagnetic (SP) magnetite particles were also found. The calcined bacteria exhibited a ferromagnetic behavior with a Curie Temperature of 454 °C and a coercivity of 16.36 mT. Additionally, the low delta ratio (δFC/δZFC=1.27) indicated that there were no intact magnetosome chains in At. ferrooxidans. Our results provided the new insights on the biomineralization of bacterial magnetosomes and magnetic properties of At. ferrooxidans., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

The differential expression of extracellular thiol groups by Acidithiobacillus ferrooxidans grown on substrates Fe(2+) and S(0) was investigated by using synchrotron radiation based scanning transmission X-ray microscopy (STXM) imaging and microbeam X-ray fluorescence (μ-XRF) mapping. The extracellular thiol groups (SH) were first alkylated by iodoacetic acid forming Protein-SCH2COOH and then the P-SCH2COOH was marked by calcium ions forming P-SCH2COOCa. The STXM imaging and μ-XRF mapping of SH were based on analysis of SCH2COO-bonded Ca(2+). The results indicated that the thiol group content of A. ferrooxidans grown on S(0) is 3.88 times to that on Fe(2+). Combined with selective labeling of SH by Ca(2+), the STXM imaging and μ-XRF mapping provided an in situ and rapid analysis of differential expression of extracellular thiol groups., (© 2013.)

Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile that oxidizes ferrous iron or sulfur compounds to obtain energy in the presence of various ions. To investigate the potassium ion response of A. ferrooxidans, we conducted a proteomics analysis. We identified eight proteins that were differentially expressed in the presence of high potassium concentration, including four up-regulated and four down-regulated proteins. Transcription levels of the genes encoding differential expressed proteins were subsequently analyzed by Northern blot in the presence of high potassium concentration. Among the up-regulated proteins, GDP-mannose 4,6-dehydratase, ribose 5-phosphate isomerase A and ribose-phosphate pyrophosphokinase were known to be implicated in the synthesis of glycocalyx, suggesting that the formation of glycocalyx might be involved in the A. ferrooxidans response to high potassium concentration. Thickening of the glycocalyx layer was also observed in cells cultivated under high potassium concentration via transmission electronic microscopy (TEM) analysis. Among the down-regulated proteins, ATP synthase F1 delta subunit and ATP synthase F1 beta subunit were two important components of ATP synthase. ATP synthase (P-ATPase) is directly linked to the transport of potassium into the cell, thus Acidithiobacillus ferrooxidans might just reduce the quantity of ATP synthase to offset the high potassium level in the culture medium. Therefore, the results obtained here provide some new clues to improve our understanding of the response of A. ferrooxidans to high potassium concentration., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes, but the mechanism of its function in the biogenesis of iron-sulfur cluster remains elusive. In this paper, we demonstrate that Acidithiobacillus ferrooxidans IscA is a [4Fe-4S] cluster binding protein, and it can bind iron in the presence of DTT with an apparent iron association constant of 4·10(20) M(-1). The iron binding in IscA can be promoted by oxygen through oxidizing ferrous iron to ferric iron. Furthermore, we show that the iron bound form of IscA can be converted to iron-sulfur cluster bound form in the presence of IscS and L-cysteine in vitro. Substitution of the invariant cysteine residues Cys35, Cys99, or Cys101 in IscA abolishes the iron binding activity of the protein; the IscA mutants that fail to bind iron are unable to assemble the iron-sulfur clusters. Further studies indicate that the iron-loaded IscA could act as an iron donor for the assembly of iron-sulfur clusters in the scaffold protein IscU in vitro. Taken together, these findings suggest that A. ferrooxidans IscA is not only an iron-sulfur protein, but also an iron binding protein that can act as an iron donor for biogenesis of iron-sulfur clusters.

The sorption of Fe(II) and Fe(III) by extracellular polymeric substances (EPS) of acidophilic bacteria Acidiphilium 3.2Sup(5) and Acidithiobacillus ferrooxidans, harvested from the ecosystem of the Tinto River (Huelva, Spain), was investigated. EPS from mixed cultures of both bacteria (EPS(mixed)) and pure cultures of A. 3.2Sup(5) (EPS(pure)) were extracted with ethylenediamine tetraacetic acid (EDTA) and were characterized by Fourier-transform infrared (FTIR), electron photoemission (XPS), x-ray diffraction (DRX), and energy dispersive x-ray (EDX) spectroscopy and scanning electron microscopy (SEM). EPS pure were loaded, in sorption tests, with Fe(II) and Fe(III). The results obtained indicate that the biochemical composition and structure of EPS(mixed) was very similar to that of EPS(pure). Besides, results indicate that EPS(mixed) adsorbed Fe(II) and Fe(III) by preferential interaction with the carboxyl group, which favored the formation of Fe(II)/Fe(III) oxalates. These species were also formed in EPS(pure) loaded with Fe(II)/Fe(III). All this behavior suggested that the sorption of iron by EPS(mixed) was similar to sorption of EPS(pure), which fitted the Freundlich model. Thus, the iron uptake of EPS(mixed) reached 516.7 ± 23.4 mg Fe/g-EPS at an initial concentration of 2.0 g/L of Fe(total) and Fe(II)/Fe(III) ratio of 1.0.

The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was predicted to have novel features that work in reverse to catalyse the sulfane sulfur of GSnH species (n > 1) into sulfite in sulfur oxidation. There are two different highly upregulated genes potentially encoding the HdrC subunit in A. ferrooxidans grown in sulfur-containing medium. An HdrC containing iron-sulfur cluster from A. ferrooxidans corresponding to one of the genes had been expressed and biophysically characterized. Comparatively, here we report the cloning, expression, and characterization of another HdrC from A. ferrooxidans. This HdrC was expressed in inclusion bodies in all conditions tested. This purified HdrC displayed brown color and contained the [4Fe-4S] cluster confirmed by the UV-scanning and EPR spectra. This HdrC owned two identical motifs (Cx(2)Cx(2)Cx(3)C) including total of eight cysteine residues for [4Fe-4S] cluster binding. To our surprise, the site-directed mutagenesis results of these eight residues revealed that respective removal of the sulfhydryl group of Cys73, Cys76, Cys79, and Cys37 resulted in the cluster loss, but those of Cys27, Cys30, Cys33, and Cys83 had no influence, which demonstrated that this HdrC bound only one cluster, and it might be responsible for causing the HdrABC in A. ferrooxidans working in reverse. Molecular modeling results also supported the above results and showed that this cluster was ligated by Cys73, Cys76, and Cys79 in one motif and Cys37, however, in another motif.

The efficiency of copper leaching is improved by bacteria attached to chalcopyrite. Therefore, the study of the attachment mechanism to control leaching is important. The adhesion of three species of leaching microorganisms including Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans to chalcopyrite was investigated by using atomic force microscopy (AFM). The forces were measured with tip-immobilized cells approached to and retracted from the mineral. The results show that both the surface charge and the hydrophobicity of bacteria cells influence the adhesion force. Furthermore, the adhesion force decreased in case the extracellular polymeric substances (EPS) had been removed. In addition, the data indicate that the amount of attached cells increased with increasing adhesion force., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Protein acidostability is a common problem in biopharmaceutical and other industries. However, it remains a great challenge to engineer proteins for enhanced acidostability because our knowledge of protein acidostabilization is still very limited. In this paper, we present a comparative study of proteins from bacteria with acidic (AP) and neutral cytoplasms (NP) using an integrated statistical and machine learning approach. We construct a set of 393 non-redundant AP-NP ortholog pairs and calculate a total of 889 sequence based features for these proteins. The pairwise alignments of these ortholog pairs are used to build a residue substitution propensity matrix between APs and NPs. We use Gini importance provided by the Random Forest algorithm to rank the relative importance of these features. A scoring function using the 10 most significant features is developed and optimized using a hill climbing algorithm. The accuracy of the score function is 86.01% in predicting AP-NP ortholog pairs and is 76.65% in predicting non-ortholog AP-NP pairs, suggesting that there are significant differences between APs and NPs which can be used to predict relative acidostability of proteins. The overall trends uncovered in the study can be used as general guidelines for designing acidostable proteins. To best of our knowledge, this work represents the first systematic comparative study of the acidostable proteins and their non-acidostable orthologs.

The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the reaction between phosphoenolpyruvate and arabinose 5-phosphate (A5P) in the first committed step in the biosynthetic pathway for the formation of 3-deoxy-D-manno-octulosonate, an important component in the cell wall of Gram-negative bacteria. KDO8P synthase is evolutionarily related to the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase, which uses erythrose 4-phosphate in place of A5P. The A5P binding site in KDO8P synthase is formed by three long loops that extend from the core catalytic (β/α)(8) barrel, β2α2, β7α7, and β8α8. The extended β7α7 loop is always present in KDO8P synthase yet is not observed for DAH7P synthase. Modeling of this loop indicated interactions between this loop and the extended β2α2 loop; both loops provide key hydrogen-bonding contacts with A5P. The two absolutely conserved residues on the β7α7 loop (Gln and Ser) were mutated to Ala in both the metal-dependent KDO8P synthase from Acidithiobacillus ferrooxidans and the metal-independent KDO8P synthase from Neisseria meningitidis. In addition, mutants were constructed for both enzymes with the extended β7α7 loop excised to match the DAH7P synthase architecture. Removal of the loop extension severely hindered efficient catalysis, dramatically increasing the K(m)(A5P) and reducing the k(cat) for both enzymes. Excision of the complete loop was far more detrimental to catalysis than the double mutations of the two conserved Gln and Ser residues. Therefore, the presence of the entire extended β7α7 loop is important for efficient catalysis by KDO8P synthase, with the loop acting to promote efficient and productive binding of A5P.

The arsH gene is one of the arsenic resistance system in bacteria and eukaryotes. The ArsH protein was annotated as a NADPH-dependent flavin mononucleotide (FMN) reductase with unknown biological function. Here we report for the first time that the ArsH protein showed high ferric reductase activity. Glu104 was an essential residue for maintaining the stability of the FMN cofactor. The ArsH protein may perform an important role for cytosolic ferric iron assimilation in vivo.

Metal tolerance has been found to vary among Acidithiobacillus ferrooxidans strains and this can impact the efficiency of biomining practices. To explain observed strain variability for differences in metal tolerance we examined the effects of Cu(2+) and Ni(2+) concentrations (1-200 mM) on cytoplasmic membrane properties of two A. ferrooxidans type strains (ATCC 23270 and 19859) and four strains isolated from AMD water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), and fatty acid profiles indicated that three different modes of adaptation were present and could separate between strains showing moderate, or high metal tolerance from more sensitive strains. To compensate for the membrane ordering effects of the metals, significant remodelling of the membrane was used to either maintain homeoviscous adaptation in the moderately tolerant strains or to increase membrane fluidity in the sensitive strains. Shifts in the gel-to-liquid crystalline transition temperature in the moderately tolerant strains led to multiple phase transitions, increasing the potential for phase separation and compromised membrane integrity. The metal-tolerant strain however, was able to tolerate increases in membrane order without significant compensation via fatty acid composition. Our multivariate analyses show a common adaptive response which involves changes in the abundant 16:0 and 18:1 fatty acids. However, fatty acid composition and membrane properties showed no difference in response to either copper or nickel suggesting that adaptive response was non-specific and tolerance dependent. We demonstrate that strain variation can be evaluated using differences in membrane properties as intrinsic determinants of metal susceptibility., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Background: Acidithiobacillus ferrooxidans is chemolithoautotrophic γ-proteobacterium that thrives at extremely low pH (pH 1-2). Although a substantial amount of information is available regarding CO2 uptake and fixation in a variety of facultative autotrophs, less is known about the processes in obligate autotrophs, especially those living in extremely acidic conditions, prompting the present study., Results: Four gene clusters (termed cbb1-4) in the A. ferrooxidans genome are predicted to encode enzymes and structural proteins involved in carbon assimilation via the Calvin-Benson-Bassham (CBB) cycle including form I of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO, EC 4.1.1.39) and the CO2-concentrating carboxysomes. RT-PCR experiments demonstrated that each gene cluster is a single transcriptional unit and thus is an operon. Operon cbb1 is divergently transcribed from a gene, cbbR, encoding the LysR-type transcriptional regulator CbbR that has been shown in many organisms to regulate the expression of RubisCO genes. Sigma70-like -10 and -35 promoter boxes and potential CbbR-binding sites (T-N11-A/TNA-N7TNA) were predicted in the upstream regions of the four operons. Electrophoretic mobility shift assays (EMSAs) confirmed that purified CbbR is able to bind to the upstream regions of the cbb1, cbb2 and cbb3 operons, demonstrating that the predicted CbbR-binding sites are functional in vitro. However, CbbR failed to bind the upstream region of the cbb4 operon that contains cbbP, encoding phosphoribulokinase (EC 2.7.1.19). Thus, other factors not present in the assay may be required for binding or the region lacks a functional CbbR-binding site. The cbb3 operon contains genes predicted to encode anthranilate synthase components I and II, catalyzing the formation of anthranilate and pyruvate from chorismate. This suggests a novel regulatory connection between CO2 fixation and tryptophan biosynthesis. The presence of a form II RubisCO could promote the ability of A. ferrooxidans to fix CO2 at different concentrations of CO2., Conclusions: A. ferrooxidans has features of cbb gene organization for CO2-assimilating functions that are characteristic of obligate chemolithoautotrophs and distinguish this group from facultative autotrophs. The most conspicuous difference is a separate operon for the cbbP gene. It is hypothesized that this organization may provide greater flexibility in the regulation of expression of genes involved in inorganic carbon assimilation.

Tetrathionate hydrolase (4THase) plays an important role in dissimilatory sulfur metabolism in the acidophilic chemolithoautotrophic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans. We have already identified the gene encoding 4THase (Af-tth) in this bacterium. The heterologous expression of Af-tth in Escherichia coli resulted in the formation of inclusion bodies of the protein in an inactive form. The recombinant protein (Af-Tth) was successfully activated after an in vitro refolding treatment. The specific activity of the refolded Af-Tth obtained was 21.0+/-9.4 U mg(-1) when the protein solubilized from inclusion bodies by 6 M guanidine hydrochloride solution was refolded in a buffer containing 10 mM beta-alanine, 2 mM dithiothreitol, 0.4 M ammonium sulfate, and 30% v/v glycerol with the pH adjusted to 4.0 by sulfuric acid for 14 h at 4 degrees C. The in vitro refolding experiments revealed that Af-Tth required exposure to an acidic environment during protein folding for activation. This property reflects a physiological characteristic of the Af-Tth localized in the outer membrane of the acidophilic A. ferrooxidans. No cofactor such as pyrroloquinoline quinone (PQQ) was required during the refolding process in spite of the similarity in the primary structure of Af-Tth to the PQQ family of proteins.

A bioleaching study aimed at recovering metals from hazardous spent hydroprocessing catalysts was carried out. The exhaust catalyst was rich in nickel (4.5 mg/g), vanadium (9.4 mg/g) and molybdenum (4.4 mg/g). Involved microorganisms were iron/sulphur oxidizing bacteria. Investigated factors were elemental sulphur addition, ferrous iron addition and actions contrasting a possible metal toxicity (either adding powdered activated charcoal or simulating a cross current process by means of periodical filtration). Ferrous iron resulted to be essential for metal extraction: nickel and vanadium extraction yields were 83% and 90%, respectively, while about 50% with no iron. The observed values for molybdenum extraction yields were not as high as Ni and V ones (the highest values were around 30-40%). The investigated actions aimed at contrasting a possible metal toxicity resulted not to be effective; in contrast, sequential filtration of the liquor leach had a significant negative effect on metals extraction. Nickel and vanadium dissolution kinetics resulted to be significantly faster than molybdenum dissolution ones. Furthermore, a simple first order kinetic model was successfully fitted to experimental data. All the observed results supported the important role of the indirect mechanism in bioleaching of LC-Finer catalysts., (Copyright 2010 Elsevier B.V. All rights reserved.)

The Iro protein is a member of HiPIP family with the [Fe(4)S(4)] cluster for electron transfer. Many reports proposed that the conserved aromatic residues might be responsible for the stability of the iron-sulfur cluster in HiPIP. In this study, Tyr10 was found to be a critical residue for the stability of the [Fe(4)S(4)] cluster according to site-directed mutagenesis results. Tyr10, Phe26 and Phe48 were essential for the stability of the [Fe(4)S(4)] cluster under acidic condition. Trp44 were not involved in the stability of the [Fe(4)S(4)] cluster. Molecular structure modeling for the mutant Tyr10 proteins revealed that the aromatic group of Tyr10 may form a hydrophobic barrier to protect the [Fe(4)S(4)] cluster from solvent.

By proteomic analysis, we found a rhodanese-like protein(RhdA) from Acidithiobacillus ferrooxidans ATCC 23270 whose C-terminal contained a cysteine motif (Cys-XX-Trp-XX-Cys), known to bind iron-sulfur clusters. But so far, there were no articles to confirm the existence of iron-sulfur cluster in RhdA. In this study, RhdA gene from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The UV-Vis scanning and EPR spectra results indicated that the wild-type proteins contained an iron-sulfur cluster. Site-directed mutagenesis results revealed that the four cysteines Cys92, Cys101, Cys197, and Cys203 were crucial residues for iron-sulfur cluster binding.

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.

Adenosine 5'-phosphosulfate (APS) reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation in the biological sulfur cycle. In this study, the gene of APS reductase from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. The molecular mass of the recombinant APS reductase was determined to be 28 kDa using SDS-PAGE. According to optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster inserted into the active site of the protein. Site-directed mutation for the enzyme revealed that Cys110, Cys111, Cys193, and Cys196 were in ligation with the iron-sulfur cluster. The [Fe4S4] cluster could be assembled in vitro, and exhibited electron transport and redox catalysis properties. As we know so far, this is the first report of expression in E. coli of APS reductase from A. ferrooxidans.

Two sphalerite samples with different iron/sulphur (Fe/S) ratios, Shuikousan ore (Fe/S 0.2) and Dachang ore (Fe/S 0.52), were processed using three microbial species, Leptospirillum ferriphilum, Acidithiobacillus ferrooxidans and Acidithiobacillus caldus. Following 20 days of bioleaching in shake flask cultures, a higher zinc (Zn) extraction (96%) was achieved with Shuikousan ore than with Dachange ore (72%). The extraction efficiency increased when elemental S was added to Dachang ore to attain the same Fe/S ratio as that for Shuikousan ore. Following the addition of S, the redox potential, pH and total dissolved Fe for Dachang ore demonstrated similar behaviors to those of Shuikousan ore. Acidithiobacillus caldus and L. ferriphilum became the dominant species during the bioleaching of sphalerite with a high Fe/S ratio. In contrast, the dominant species were A. ferrooxidans and A. caldus during the bioleaching of sphalerite with a low Fe/S ratio. These results show that the Fe/S ratio has a significant influence on the bioleaching behavior of sphalerite and the composition of the microbial community.

In this work, the microbe-mediated oxidative dissolution of enargite surfaces (Cu(3)AsS(4)) was studied on powdered samples exposed to 9K nutrient solution (pH 2.3) inoculated by Acidithiobacillus ferrooxidans initially adapted to arsenopyrite. These conditions simulate the acid mine environment. The redox potential of the inoculated solutions increased up to +0.72 V vs normal hydrogen electrode (NHE), indicating the increase of the Fe(3+) to Fe(2+) ratio, and correspondingly the pH decreased to values as low as 1.9. In the sterile 9K control, the redox potential and pH remained constant at +0.52 V NHE and 2.34, respectively. Solution analyses showed that in inoculated medium Cu and As dissolved stoichiometrically with a dissolution rate of about three to five times higher compared to the sterile control. For the first time, X-ray photoelectron spectroscopy (XPS) was carried out on the bioleached enargite powder with the aim of clarifying the role of the microorganisms in the dissolution process. XPS results provide evidence of the formation of a thin oxidized layer on the mineral surface. Nitrogen was also detected on the bioleached surfaces and was attributed to the presence of an extracellular polymer substance layer supporting a mechanism of bacteria attachment via the formation of a biofilm a few nanometers thick, commonly known as nanobiofilm.

IscR (iron-sulfur cluster regulator) has been reported to be a repressor of the iscRSUA operon, and in vitro transcription reactions have revealed that IscR has a repressive effect on the iscR promoter in the case of [Fe(2)S(2)] cluster loading. In the present study, the iscR gene from A. ferrooxidans ATCC 23270 was cloned and successfully expressed in Escherichia coli, and then purified by one-step affinity chromatography to homogeneity. The molecular mass of the IscR was 18 kDa by SDS-PAGE. The optical and EPR spectra results for the recombinant IscR confirmed that an iron-sulfur cluster was correctly inserted into the active site of the protein. However, no [Fe(2)S(2)] cluster was assembled in apoIscR with ferrous iron and sulfide in vitro. Therefore, the [Fe(2)S(2)] cluster assembly in IscR in vivo would appear to require scaffold proteins and follow the Isc "AUS" pathway.