Genetic Modification of Acidithiobacillus ferrooxidans for Rare-Earth Element Recovery under Acidic Conditions.

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 Tb3+, was expressed periplasmically via fusion with the endogenous rusticyanin protein. The binding of lanthanides (Tb3+, Pr3+, Nd3+, and La3+) 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 (Tb3+, Pr3+, Nd3+, and La3+) over non-REEs (Fe2+ and Co2+) in a synthetic magnet leachate, demonstrating the potential of these new strains for future REE reclamation and recycling applications.

Expression, purification, characterization and direct electrochemistry of two HiPIPs from Acidithiobacillus caldus SM-1.

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.

Engineering Polyhistidine Tags on Surface Proteins of Acidithiobacillus ferrooxidans: Impact of Localization on the Binding and Recovery of Divalent Metal Cations.

Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies.Acidithiobacillus ferrooxidansis 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.

Computational structure prediction provides a plausible mechanism for electron transfer by the outer membrane protein Cyc2 from Acidithiobacillus ferrooxidans.

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.

Bioleaching of iron from laterite soil using an isolated Acidithiobacillus ferrooxidans strain and application of leached laterite iron as Fenton's catalyst in selective herbicide degradation.

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.

Formation and stability of biogenic tooeleite during Fe(II) oxidation by Acidithiobacillus ferrooxidans.

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)-SO42- 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.

Catalytic effect of silver on copper release from chalcopyrite mediated by Acidithiobacillus ferrooxidans.

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 (Sn2-/S0) 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.

Attachment of Acidithiobacillus ferrooxidans to pyrite in fresh and saline water and fitting to Langmuir and Freundlich isotherms.

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 (R2) 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.

Biological Metabolism Synthesis of Metal Oxides Nanorods from Bacteria as a Biofactory toward High-Performance Lithium-Ion Battery Anodes.

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-Fe2 O3 nanorods via the oxidation of soluble ferrous irons to insoluble ferric substances (Jarosite, KFe3 (SO4 )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-Fe2 O3 nanorods play important roles that not only significantly enhance the structure stability but also facilitate electron and ion transfer. Consequently, these biometabolic α-Fe2 O3 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.

Structures of two ArsR As(III)-responsive transcriptional repressors: Implications for the mechanism of derepression.

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 P21 space group and diffracted up to 1.86 Å. CgArsR crystallized in P212121 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 S3 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.

Application of capillary electrophoresis combined with conductometric and UV detection to monitor meteorite simulant bioleaching by Acidithiobacillus ferrooxidans.

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 Fe2+ into Fe+3 . The effect of Co2+ and Ni2+ (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 Fe2+ conversion into Fe3+ 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.

Severe microbiologically influenced corrosion of S32654 super austenitic stainless steel by acid producing bacterium Acidithiobacillus caldus SM-1.

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.

Cytochromes in anaerobic growth of Acidithiobacillus ferrooxidans.

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.

The effect of pH on the dynamics of natural membranes.

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.

Comparative proteomic analysis of sulfur-oxidizing Acidithiobacillus ferrooxidans CCM 4253 cultures having lost the ability to couple anaerobic elemental sulfur oxidation with ferric iron reduction.

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.

Synthesis of argentojarosite with simulated bioleaching solutions produced by Acidithiobacillus ferrooxidans.

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.

Global response of Acidithiobacillus ferrooxidans ATCC 53993 to high concentrations of copper: A quantitative proteomics approach.

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.

Study of Acidithiobacillus ferrooxidans and enzymatic bio-Fenton process-mediated corrosion of copper-nickel alloy.

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.

Sludge conditioning using biogenic flocculant produced by Acidithiobacillus ferrooxidans for enhancement in dewaterability.

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.

Effect of calcium oxide on the efficiency of ferrous ion oxidation and total iron precipitation during ferrous ion oxidation in simulated acid mine drainage treatment with inoculation of Acidithiobacillus ferrooxidans.

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.