Acidithiobacillus acidisediminis sp. nov., an acidophilic sulphur-oxidizing chemolithotroph isolated from acid mine drainage sediment.

Strain S30A2T, isolated from the acid mine drainage sediment of Mengzi Copper Mine, Yunnan, is proposed to represent a novel species of the sulphur-oxidizing genus Acidithiobacillus. Cells were Gram-stain-negative, non-endospore forming, highly motile with one or two monopolar flagella and rod-shaped. The strain was mesophilic, growing at 30-50 °C (optimum, 38 °C), acidophilic, growing at pH 2.0-4.5 (optimum, pH 2.5), and tolerant of 0-4 % (w/v; 684 mol l-1) NaCl. The 16S rRNA gene-based sequence analysis showed that strain S30A2T belongs to the genus Acidithiobacillus and shows the largest similarity of 96.6 % to the type strain Acidithiobacillus caldus KUT. The genomic DNA G+C content of strain S30A2T was 59.25 mol%. The average nucleotide identity ANIb and ANIm values between strain S30A2T and A. caldus KUT were 70.95 and 89.78 %, respectively and the digital DNA-DNA hybridization value was 24.9 %. Strain S30A2T was strictly aerobic and could utilize elementary sulphur and tetrathionate to support chemolithotrophic growth. The major cellular fatty acid of S30A2T was C19 : 1ω7c. The respiratory quinones were ubiquinone-8 and ubiquinone-7. Based upon its phylogenetic, genetic, phenotypic, physiologic and chemotaxonomic characteristics, strain S30A2T is considered to represent a novel species of the genus Acidithiobacillus, for which the name Acidithiobacillus acidisediminis sp. nov. is proposed. The type strain is S30A2T (=CGMCC 1.17059T=KCTC 72580T).

Techno-economic assessment of bioleaching for metallurgical by-products.

This study focused on the economic feasibility of two potential industrial-scale bioleaching technologies for metal recovery from specific metallurgical by-products, mainly basic oxygen steelmaking dust (BOS-D) and goethite. The investigation compared two bioleaching scaling technology configurations, including an aerated bioreactor and an aerated and stirred bioreactor across different scenarios. Results indicated that bioleaching using Acidithiobacillus ferrooxidans proved financially viable for copper extraction from goethite, particularly when 5% and 10% pulp densities were used in the aerated bioreactor, and when 10% pulp density was used in the aerated and stirred bioreactor. Notably, a net present value (NPV) of $1,275,499k and an internal rate of return (IRR) of 65% for Cu recovery from goethite were achieved over 20-years after project started using the aerated and stirred bioreactor plant with a capital expenditure (CAPEX) of $119,816,550 and an operational expenditure (OPEX) of $5,896,580/year. It is expected that plant will start to make profit after one year of operation. Aerated and stirred bioreactor plant appeared more reliable alternative compared to the aerated bioreactor plant as the plant consists of 12 reactors which can allow better management and operation in small volume with multiple reactors. Despite the limitations, this techno-economic assessment emphasized the significance of selective metal recovery and plant design, and underscored the major expenses associated with the process.

Copper recovery from low-grade copper sulfides using bioleaching and its community structure succession in the presence of Sargassum.

Bioleaching characteristics and bacterial community structure were studied during low-grade copper sulfide ores bioleaching in the presence of pretreated Sargassum (PSM). Results indicated that proportion of attached bacteria and copper recovery were improved by using appropriate-dosage PSM. High copper recovery (82.99%) and low Fe3+ concentration were obtained when 150 mg L-1 PSM was used. Precipitation, such as KFe3(SO4)2(OH)6 and (H3O)Fe3(SO4)2(OH)6, was not found in samples used PSM according to XRD, FTIR and TG analyses, which may result from less passivation layer formed by Fe3+ hydrolysis. I- contained in PSM can act as the reductant to convert Fe3+ into Fe2+, which can reduce Fe3+ hydrolysis and adjust Eh value. Bacterial community structure was influenced significantly by PSM according to the 16 S rDNA analysis. Acidithiobacillus ferrooxidans dominated proportion of bacterial community throughout bioleaching process, whose proportion reached 89.1091% after 14 days in sample added 150 mg L-1 PSM.

Insertion sequence contributes to the evolution and environmental adaptation of Acidithiobacillus.

BACKGROUND: The genus Acidithiobacillus has been widely concerned due to its superior survival and oxidation ability in acid mine drainage (AMD). However, the contribution of insertion sequence (IS) to their biological evolution and environmental adaptation is very limited. ISs are the simplest kinds of mobile genetic elements (MGEs), capable of interrupting genes, operons, or regulating the expression of genes through transposition activity. ISs could be classified into different families with their own members, possessing different copies. RESULTS: In this study, the distribution and evolution of ISs, as well as the functions of the genes around ISs in 36 Acidithiobacillus genomes, were analyzed. The results showed that 248 members belonging to 23 IS families with a total of 10,652 copies were identified within the target genomes. The IS families and copy numbers among each species were significantly different, indicating that the IS distribution of Acidithiobacillus were not even. A. ferrooxidans had 166 IS members, which may develop more gene transposition strategies compared with other Acidithiobacillus spp. What's more, A. thiooxidans harbored the most IS copies, suggesting that their ISs were the most active and more likely to transpose. The ISs clustered in the phylogenetic tree approximately according to the family, which were mostly different from the evolutionary trends of their host genomes. Thus, it was suggested that the recent activity of ISs of Acidithiobacillus was not only determined by their genetic characteristics, but related with the environmental pressure. In addition, many ISs especially Tn3 and IS110 families were inserted around the regions whose functions were As/Hg/Cu/Co/Zn/Cd translocation and sulfur oxidation, implying that ISs could improve the adaptive capacities of Acidithiobacillus to the extremely acidic environment by enhancing their resistance to heavy metals and utilization of sulfur. CONCLUSIONS: This study provided the genomic evidence for the contribution of IS to evolution and adaptation of Acidithiobacillus, opening novel sights into the genome plasticity of those acidophiles.

Convergent Community Assembly among Globally Separated Acidic Cave Biofilms.

Acidophilic bacteria and archaea inhabit extreme geochemical "islands" that can tell us when and how geographic barriers affect the biogeography of microorganisms. Here, we describe microbial communities from extremely acidic (pH 0 to 1) biofilms, known as snottites, from hydrogen sulfide-rich caves. Given the extreme acidity and subsurface location of these biofilms, and in light of earlier work showing strong geographic patterns among snottite Acidithiobacillus populations, we investigated their structure and diversity in order to understand how geography might impact community assembly. We used 16S rRNA gene cloning and fluorescence in situ hybridization (FISH) to investigate 26 snottite samples from four sulfidic caves in Italy and Mexico. All samples had very low biodiversity and were dominated by sulfur-oxidizing bacteria in the genus Acidithiobacillus. Ferroplasma and other archaea in the Thermoplasmatales ranged from 0 to 50% of total cells, and relatives of the bacterial genera Acidimicrobium and Ferrimicrobium were up to 15% of total cells. Rare phylotypes included Sulfobacillus spp. and members of the phyla "Candidatus Dependentiae" and "Candidatus Saccharibacteria" (formerly TM6 and TM7). Although the same genera of acidophiles occurred in snottites on separate continents, most members of those genera represent substantially divergent populations, with 16S rRNA genes that are only 95 to 98% similar. Our findings are consistent with a model of community assembly where sulfidic caves are stochastically colonized by microorganisms from local sources, which are strongly filtered through environmental selection for extreme acid tolerance, and these different colonization histories are maintained by dispersal restrictions within and among caves. IMPORTANCE Microorganisms that are adapted to extremely acidic conditions, known as extreme acidophiles, are catalysts for rock weathering, metal cycling, and mineral formation in naturally acidic environments. They are also important drivers of large-scale industrial processes such as biomining and contaminant remediation. Understanding the factors that govern their ecology and distribution can help us better predict and utilize their activities in natural and engineered systems. However, extremely acidic habitats are unusual in that they are almost always isolated within circumneutral landscapes. So where did their acid-adapted inhabitants come from, and how do new colonists arrive and become established? In this study, we took advantage of a unique natural experiment in Earth's subsurface to show how isolation may have played a role in the colonization history, community assembly, and diversity of highly acidic microbial biofilms.

Molecular Insights into a Novel Cu(I)-Sensitive ArsR/SmtB Family Repressor in Extremophile Acidithiobacillus caldus.

Acidithiobacillus caldus is a common bioleaching bacterium that is inevitably exposed to extreme copper stress in leachates. The ArsR/SmtB family of metalloregulatory repressors regulates homeostasis and resistance in bacteria by specifically responding to metals. Here, we characterized A. caldus Cu(I)-sensitive repressor (AcsR) and gained molecular insights into this new member of the ArsR/SmtB family. Transcriptional analysis indicated that the promoter (PIII) of acsR was highly active in Escherichia coli but inhibited upon AcsR binding to the PIII-acsR region. Size exclusion chromatography and circular dichroism spectra revealed that CuI-AcsR shared an identical assembly state with apo-AcsR, as a dimer with fewer α helices, more extended strands, and more ß turns. Mutation of the cysteine site in AcsR did not affect its assembly state. Copper(I) titrations revealed that apo-AcsR bound two Cu(I) molecules per monomer in vitro with an average dissociation constant (KD) for bicinchoninic acid competition of 2.55 × 10-9 M. Site-directed mutation of putative Cu(I)-binding ligands in AcsR showed that replacing Cys64 with Ala reduces copper binding ability from two Cu(I) molecules per monomer to one, with an average KD of 6.05 × 10-9 M. Electrophoretic mobility shift assays revealed that apo-AcsR has high affinity for the 12-2-12 imperfect inverted repeats P2245 and P2270 in the acsR gene cluster and that Cu-loaded AcsR had lower affinity for DNA fragments than apo-AcsR. We developed a hypothetical working model of AcsR to better understand Cu resistance mechanisms in A. caldus. IMPORTANCE Copper (Cu) resistance among various microorganisms is attracting interest. The chemolithoautotrophic bacterium A. caldus, which can tolerate extreme copper stress (≥10 g/L Cu ions), is typically used to bioleach chalcopyrite (CuFeS2). Understanding of Cu resistance in A. caldus is limited due to scant investigation and the absence of efficient gene manipulation tools. Here, we characterized a new member of the ArsR/SmtB family of prokaryotic metalloregulatory transcriptional proteins that repress operons linked to stress-inducing concentrations of heavy metal ions. This protein can bind two Cu(I) molecules per monomer and negatively regulate its gene cluster. Members of the ArsR/SmtB family have not been investigated in A. caldus until now. The discovery of this novel protein enriches understanding of Cu homeostasis in A. caldus.

Disorder and amino acid composition in proteins: their potential role in the adaptation of extracellular pilins to the acidic media, where Acidithiobacillus thiooxidans grows.

There are few biophysical studies or structural characterizations of the type IV pilin system of extremophile bacteria, such as the acidophilic Acidithiobacillus thiooxidans. We set out to analyze their pili-comprising proteins, pilins, because these extracellular proteins are in constant interaction with protons of the acidic medium in which At. thiooxidans grows. We used the web server Operon Mapper to analyze and identify the cluster codified by the minor pilin of At. thiooxidans. In addition, we carried an in-silico characterization of such pilins using the VL-XT algorithm of PONDR® server. Our results showed that structural disorder prevails more in pilins of At. thiooxidans than in non-acidophilic bacteria. Further computational characterization showed that the pilins of At. thiooxidans are significantly enriched in hydroxy (serine and threonine) and amide (glutamine and asparagine) residues, and significantly reduced in charged residues (aspartic acid, glutamic acid, arginine and lysine). Similar results were obtained when comparing pilins from other Acidithiobacillus and other acidophilic bacteria from another genus versus neutrophilic bacteria, suggesting that these properties are intrinsic to pilins from acidic environments, most likely by maintaining solubility and stability in harsh conditions. These results give guidelines for the application of extracellular proteins of acidophiles in protein engineering.

Genetic Engineering of Acidithiobacillus ferridurans Using CRISPR Systems To Mitigate Toxic Release in Biomining.

Biomining processes utilize microorganisms, such as Acidithiobacillus, to extract valuable metals by producing sulfuric acid and ferric ions that dissolve sulfidic minerals. However, excessive production of these compounds can result in metal structure corrosion and groundwater contamination. Synthetic biology offers a promising solution to improve Acidithiobacillus strains for sustainable, eco-friendly, and cost-effective biomining, but genetic engineering of these slow-growing microorganisms is challenging with current inefficient and time-consuming methods. To address this, we established a CRISPR-dCas9 system for gene knockdown in A. ferridurans JAGS, successfully downregulating the transcriptional levels of two genes involved in sulfur oxidation. More importantly, we constructed an all-in-one CRISPR-Cas9 system for fast and efficient genome editing in A. ferridurans JAGS, achieving seamless gene deletion (HdrB3), promoter substitution (Prus to Ptac), and exogenous gene insertion (GFP). Additionally, we created a HdrB-Rus double-edited strain and performed biomining experiments to extract Ni from pyrrhotite tailings. The engineered strain demonstrated a similar Ni recovery rate to wild-type A. ferridurans JAGS but with significantly lower production of iron ions and sulfuric acid in leachate. These high-efficient CRISPR systems provide a powerful tool for studying gene functions and creating useful recombinants for synthetic biology-assisted biomining applications in the future.

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.

Biolixiviation of Metals from Computer Printed Circuit Boards by Acidithiobacillus ferrooxidans and Bioremoval of Metals by Mixed Culture Subjected to a Magnetic Field.

Crushed and ground printed circuit board (PCB) samples were characterized to evaluate copper, lead, and aluminum using X-ray fluorescence spectroscopy (XRF) and the morphology was done by scanning electron microscopy (SEM). The XRF characterizations showed 0.12% lead, 3.72% copper, and 12.73% aluminum in the PCBs. The metal solubilization experiments using Acidithiobacillus ferrooxidans indicated higher values of total metal solubilization when the initial pH of the inoculum was adjusted. However, these experiments did not show higher metal solubilization by bioleaching. The sequential bioremoval experiments using mixed culture after bioleaching assays with A. ferrooxidans with initial adjustment of inoculum pH and without applying a magnetic field removed 100% of Al, 27.34% of Cu, and 96.43% of Pb from the lixiviate medium; with magnetic field application, 100% of Al, 83.82% of Cu, and 98.27% of Pb were removed. A similar bioleaching experiment without inoculum pH adjustment and without field application achieved 99.74% removal for Cu and 91.92% for Pb. When the magnetic field was applied, 100% of Cu and 95.76% of Pb were removed. Bioreactors with a magnetic field do not show significantly better removal of any of the metals analyzed.

Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase.

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-ß4 loop, pore loop 1, and the presensor 1-ß hairpin (PS1-ßH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO2 fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures.

Bioleaching performance of vanadium-bearing smelting ash by Acidithiobacillus ferrooxidans for vanadium recovery.

The bioleaching process is widely used in the treatment of ores or solid wastes, but little is known about its application in the treatment of vanadium-bearing smelting ash. This study investigated bioleaching of smelting ash with Acidithiobacillus ferrooxidans. The vanadium-bearing smelting ash was first treated with 0.1 M acetate buffer and then leached in the culture of Acidithiobacillus ferrooxidans. Comparison between one-step and two-step leaching process indicated that microbial metabolites could contribute to the bioleaching. The Acidithiobacillus ferrooxidans demonstrated a high vanadium leaching potential, solubilizing 41.9% of vanadium from the smelting ash. The optimal leaching condition was determined, which was 1% pulp density, 10% inoculum volume, an initial pH of 1.8, and 3 Fe2+g/L. The compositional analysis showed that the fraction of reducible, oxidizable, and acid-soluble was transferred into the leaching liquor. Therefore, as the alternative to the chemical/physical process, an efficient bioleaching process was proposed to enhance the recovery of vanadium from the vanadium-bearing smelting ash.

Bioleaching of the α-alumina layer of spent three-way catalysts as a pretreatment for the recovery of platinum group metals.

Acid bioleaching of Al by Acidithiobacillus thiooxidans has been explored as an environmentally friendly pretreatment to facilitate the extraction of platinum group metals from spent three-way catalysts (TWC). Biogenic sulfur obtained from desulfurization bioreactors improved the production of acid by A. thiooxidans compared to commercially available elemental sulfur. The lixiviation abilities of bacteria-free biogenic acid and biogenic acid with exponential or stationary phase bacteria were compared against a control batch produced by commercial H2SO4. The maximum Al leaching percentage (54.5%) was achieved using biogenic acids with stationary-phase bacteria at a TWC pulp density of 5% w/v whereas bacteria-free biogenic acid (23.4%), biogenic acid with exponential phase bacteria (21.7%) and commercial H2SO4 (24.7%) showed lower leaching abilities. The effect of different pulp densities of ground TWC (5, 30, and 60% w/v) on Al leaching and bacterial growth was determined. While greater Al leaching yields were obtained at lower TWC pulp density solutions (54.5% at 5% w/v and 2.5% at 60% w/v), higher pulp densities enhanced microbial growth (2.3 × 109 cells/mL at 5% w/v and 9.5 × 1010 cells/mL at 60% w/v). The dissolution of the metal from the solid into the liquid phase triggered the production of biological polymeric substances that were able to absorb traces of both Al (up to 24.80% at 5% w/v) and Pt (up to 0.40% at 60% w/v).

Assessment of the induced effect of selected iron hydroxysulfates biosynthesized using Acidithiobacillus ferrooxidans for biomineralization of acid mine drainage.

Soluble iron and sulfate in acid mine drainage (AMD) can be greatly removed through the formation of minerals facilitated by seed crystals. However, the difference in the effects of jarosite and schwertmannite as endogenous seed crystals to induce AMD mineralization remains unclear. This paper intends to study the effect of Fe2+ oxidation and Fe3+ mineralization in the biosynthesis of minerals using different addition amounts and methods of jarosite or schwertmannite. The results showed that the addition amount and method of different seed crystals had no effect on the Fe2+ bio-oxidation but would change the Fe3+ mineralization efficiency. With the same amount of seed crystals added, jarosite exhibited a higher capacity to promote Fe3+ mineralization than schwertmannite. Adding seed crystals before the initiation of Fe2+ oxidation (0 h) could significantly promote Fe3+ mineralization efficiency. With the increase of seed crystals, jarosite could not only shorten the time required for mineral synthesis but also improve the final mineral yield, whereas schwertmannite could only shorten the time required for mineral synthesis. When Fe2+ was completely oxidized to Fe3+ (48 h), the supplementary of jarosite could still effectively improve Fe3+ mineralization efficiency, but the addition of schwertmannite no longer affected the final mineralization degree.

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.

Complete bioleaching of Co and Ni from spent batteries by a novel silver ion catalyzed process.

In the present work, bioleaching of two valuable metals of cobalt (Co) and nickel (Ni) from spent lithium-ion batteries (LIBs) of laptop by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans through a novel adaptation procedure was investigated. Different bioleaching methods including A. ferrooxidans and A. thiooxidans spent medium, A. ferrooxidans one-step and two-step bioleaching were carried out. The effect of silver ion on the bioleaching of Co and Ni in these methods was evaluated. Moreover, a novel strain adaptation approach to the toxic solid content of the battery powder was chosen, which resulted in a very short adaptation time and bioleaching (2 days). Even though silver ion did not have a significant effect on the spent medium method, it had an increasing effect of 26% and 7%, for Co and Ni recovery, respectively, on two-step bioleaching with silver ion-adapted A. ferrooxidans, in gradual addition of the battery powder. The highest extraction results in the spent medium method were 45.2% and 71.5% for Co and Ni, respectively, and a very high extraction yield of 99.95% for these metals was achieved in a short time of only 3 days by two-step bioleaching with gradual addition of the solid content and in the presence of Ag+. KEY POINTS: • Mixed spent medium of acidophilic bacteria resulted in higher Ni and Co extraction. • Adaptation to Ag+ has enhanced the strain capability for Co and Ni extraction. • With Ag+ presence, Co and Ni extraction reached 99.95% in two-step bioleaching.

EpsRAc is a copper-sensing MarR family transcriptional repressor from Acidithiobacillus caldus.

The MarR family, as multiple antibiotic resistance regulators, is associated with the resistance of organisms to unfavorable conditions. MarR family extracellular polymeric substances (EPS)-associated transcriptional regulator (EpsRAc) was closely associated with copper resistance in Acidithiobacillus caldus (A. caldus). Transcriptional analysis showed high activity of the epsR promoter (PI) in Escherichia coli and differential response to metal ions. The copper content and UV absorption spectrum of the co-purified protein did not increase, but a stoichiometry of 0.667 mol Cu(I) per EpsRAc monomer was observed in vitro in copper titration experiments, suggesting that Cu(II) acts with low affinity in binding to the EpsRAc protein. Electrophoretic mobility shift assays (EMSA) demonstrated that EpsRAc could bind to its own promoter in vitro, and the binding region was the palindrome sequence TGTTCATCGTGTGTGAGCACACA. EpsRAc negatively regulated its own gene expression, whereas Cu(II) mitigates this negative effect. EpsRAc did not bind to other neighboring gene promoters. Finally, we developed a working model to illustrate the regulatory mechanism of A. caldus in response to extreme copper stress. KEY POINTS: • Identification of a MarR family EPS-associated transcriptional regulator, named EpsRAc. • Cu(I) can bind to the EpsRAc protein with low affinity. • EpsRAc negatively regulates the expression of epsR, and Cu(II) can alleviate this negative regulation.

Bioleaching of rare-earth elements from phosphate rock using Acidithiobacillus ferrooxidans.

Phosphate rock containing rare-earth elements (REEs) is considered one of the most promising potential secondary sources of REEs, as evidenced by large tonnages of phosphate rock mined annually. The bioleaching of REEs from phosphate rock using Acidithiobacillus ferrooxidans was done for the first time in this study, and it was found to be greater than abiotic leaching and was more environmentally friendly. The result showed that the total leaching rate of REEs in phosphate rock was 28·46% under the condition of 1% pulp concentration and pH = 2, and the leaching rates of four key rare earths, Y, La, Ce and Nd, were 35·7, 37·03, 27·92 and 32·53% respectively. The bioleaching process was found to be accomplished by bacterial contact and Fe2+ oxidation. The blank control group which contained Fe2+ was able to leach some of the rare earths, indicating that the oxidation of Fe2+ may affect the leaching of rare earths. X-ray diffraction analysis showed that the minerals were significantly altered and the intensity of the diffraction peaks of dolomite and apatite decreased significantly after microbial action compared to the blank control, and it was observed that bacteria adhere to the mineral surface and the minerals become smooth and angular after bioleaching by scanning electron microscope, indicating that bacteria have a further effect on the rock based on Fe2+ oxidation. Finally, Fourier transform infrared spectroscopy and three-dimensional excitation-emission matrix fluorescence spectra analysis showed that extracellular polymeric substances participate in the bioleaching process.

Enhancement Mechanism of Stibnite Dissolution Mediated by Acidithiobacillus ferrooxidans under Extremely Acidic Condition.

Oxidative dissolution of stibnite (Sb2S3), one of the most prevalent geochemical processes for antimony (Sb) release, can be promoted by Sb-oxidizing microbes, which were studied under alkaline and neutral conditions but rarely under acidic conditions. This work is dedicated to unraveling the enhancement mechanism of stibnite dissolution by typical acidophile Acidithiobacillus ferrooxidans under extremely acidic conditions. The results of solution behavior showed that the dissolution of Sb2S3 was significantly enhanced by A. ferrooxidans, with lower pH and higher redox potential values and higher [Sb(III)] and [Sb(V)] than the sterile control. The surface morphology results showed that the cells adsorbed onto the mineral surface and formed biofilms. Much more filamentous secondary minerals were formed for the case with A. ferrooxidans. Further mineral phase compositions and Sb/S speciation transformation analyses showed that more secondary products Sb2O3/SbO2-, Sb2O5/SbO3-, SO42-, as well as intermediates, such as S0, S2O32- were formed for the biotic case, indicating that the dissolution of Sb2S3 and the Sb/S speciation transformation was promoted by A. ferrooxidans. These results were further clarified by the comparative transcriptome analysis. This work demonstrated that through the interaction with Sb2S3, A. ferrooxidans promotes S/Sb oxidation, so as to enhance S/Sb transformation and thus the dissolution of Sb2S3.

Enhanced metal bioleaching mechanisms of extracellular polymeric substance for obsolete LiNixCoyMn1-x-yO2 at high pulp density.

Harmful chemicals present in electric vehicle Li-ion batteries (EV LIBs) can limit the pulp density of bioleaching processes using Acidithiobacillus sp. to 1.0% (w/v) or lower. The strong enhancing mechanisms of extracellular polymeric substances (EPS) on the bioleaching of metals from spent EV LIBs at high pulp density (4% w/v) were studied using bio-chemical, spectroscopic, surface structure imaging and bioleaching kinetic methods. Results demonstrated that the added EPS significantly improved bioleaching efficiency of Ni, Co and Mn improved by 42%, 40% and 44%, respectively. EPS addition boosted the growth of cells under adverse conditions to produce more biogenic H+ while Fe3+ and Fe2+ were adsorbed by the biopolymer. This increased Li extraction by acid dissolution and concentrated the Fe3+/Fe2+ cycle via non-contact mechanisms for the subsequent contact bioleaching of Ni, CO and Mn at the EV LIB-bacteria interface. During the leaching process, added EPS improved adhesion of the bacterial cells to the EV LIBs, and the resultant strong interfacial reactions promoted bioleaching of the target metals. Hence, a combination of non-contact and contact mechanisms initiated by the addition of EPS enhanced the bioleaching of spent EV LIBs at high pulp density.