Novel insights into the kinetics and mechanism of arsenopyrite bio-dissolution enhanced by pyrite.

Arsenopyrite and pyrite often coexist in metal deposits and tailings, thus simultaneous bioleaching of both sulfides has economic (as well as environmental) significance. Important targets in bio-oxidation operations are high solubilization rates and minimized accumulation of Fe(III)/As-bearing secondary products. This study investigated the role of pyrite bioleaching in the enhancement of arsenopyrite dissolution. At a pyrite to arsenopyrite mass ratio of 1:1, 93.6% of As and 93.0% of Fe were solubilized. The results show that pyrite bio-oxidation can promote arsenopyrite dissolution, enhance S0 bio-oxidation, and inhibit the formation of jarosites, tooeleite, and amorphous ferric arsenate. The dry weight of the pyrite & arsenopyrite residue was reduced by 95.1% after bioleaching, compared to the initial load, while only 5% weight loss was observed when pyrite was absent. A biofilm was formed on the arsenopyrite surface in the presence of pyrite, while a dense passivation layer was observed in the absence of pyrite. As(III) (as As2O3) was a dominant As species in the pyrite & arsenopyrite residue. Novel and detailed findings are presented on arsenopyrite bio-dissolution in the presence of pyrite, and the presented approach could contribute to the development of novel cost-effective extractive bioprocesses. ENVIRONMENTAL IMPLICATION: The oxidation of arsenopyrite presents significant environmental hazards, as it can contribute to acid mine drainage generation and arsenic mobilization from sulfidic mine wastes. Bioleaching is a proven cost-effective and environmentally friendly extractive technology, which has been applied for decades in metal recovery from minerals or tailings. In this work, efficient extraction of arsenic from arsenopyrite bioleaching was presented through coupling the process with bio-oxidation of pyrite, resulting in lowered accumulation of hazardous and metastable Fe(III)/As-bearing secondary phases. The results could help improve current biomining operations and/or contribute to the development of novel cost-effective bioprocesses for metal extraction.

Treating waste with waste: Lignin acting as both an effective bactericide and passivator to prevent acid mine drainage formation at the source.

Acid mine drainage (AMD) induced by pyrite oxidation is a notorious and serious environmental problem, but the management of AMD in an economical and environmentally friendly way remains challenging. Here, lignin, a natural polymer and abundant waste, was employed as both a bactericide and passivator to prevent AMD formation. The addition of lignin to a mimic AMD formation system inoculated with Acidithiobacillus ferrooxidans at a lignin-to-pyrite weight ratio of 2.5: 10 reduced the combined abiotic and biotic oxidation of pyrite by 68.4 % (based on released SO42-). Morphological characterization of Acidithiobacillus ferrooxidans revealed that lignin could act on the cell surface and impair the cell integrity, disrupting its normal growth and preventing biotic oxidation of pyrite accordingly. Moreover, lignin can be used alone as a passivator to form a coating on the pyrite surface, reducing abiotic oxidation by 71.7 % (based on released SO42-). Through multiple technique analysis, it was proposed that the functional groups on lignin may coordinate with iron ions on pyrite, promoting its deposition on the surface. In addition, the inherent antioxidant activity of lignin may also be actively involved in the abatement of pyrite oxidation via the reduction of iron. Overall, this study offered a "treating waste with waste" strategy for preventing AMD formation at the source and opened a new avenue for the management of AMD.

Inhibition of iron oxidation in Acidithiobacillus ferrooxidans by low-molecular-weight organic acids: Evaluation of performance and elucidation of mechanisms.

The catalytic role of Acidithiobacillus ferrooxidans (A. ferrooxidans) in iron biooxidation is pivotal in the formation of Acid Mine Drainage (AMD), which poses a significant threat to the environment. To control AMD generation, treatments with low-molecular-weight organic acids are being studied, yet their exact mechanisms are unclear. In this study, AMD materials, organic acids, and molecular methods were employed to gain a deeper understanding of the inhibitory effects of low-molecular-weight organic acids on the biooxidation of iron by A. ferrooxidans. The inhibition experiments of A. ferrooxidans on the oxidation of Fe2+ showed that to attain a 90 % inhibition efficacy within 72 h, the minimum concentrations required for formic acid, acetic acid, propionic acid, and lactic acid are 0.5, 6, 4, and 10 mmol/L, respectively. Bacterial imaging illustrated the detrimental effects of these organic acids on the cell envelope structure. This includes severe damage to the outer membrane, particularly from formic and acetic acids, which also caused cell wall damage. Coupled with alterations in the types and quantities of protein, carbohydrate, and nucleic acid content in extracellular polymeric substances (EPS), indicate the mechanisms underlying these inhibitory treatments. Transcriptomic analysis revealed interference of these organic acids with crucial metabolic pathways, particularly those related to energy metabolism. These findings establish a comprehensive theoretical basis for understanding the inhibition of A. ferrooxidans' biooxidation by low-molecular-weight organic acids, offering a novel opportunity to effectively mitigate the generation of AMD at its source.

Significant differences in the degree of genomic DNA N6-methyladenine modifications in Acidithiobacillus ferrooxidans with two different culture substrates.

DNA N6-methyladenine (6mA) modification is widespread in organisms and plays an important functional role in the regulation of cellular processes. As a model organism in biohydrometallurgy, Acidithiobacillus ferrooxidans can obtain energy from the oxidation of ferrous iron (Fe2+) and various reduced inorganic sulfides (RISCs) under acidic conditions. To determine the linkage between genomic DNA methylation and the switching between the two oxidative metabolic pathways in A. ferrooxidans, the 6mA landscape in the genome of A. ferrooxidans cultured under different conditions was evaluated by using 6mA-IP-seq. A total of 214 and 47 high-confidence peaks of 6mA were identified under the Fe2+ and RISCs oxidizing conditions, respectively (P<10-5), suggesting that genomic methylation was greater under Fe2+ oxidizing conditions. 6mA experienced a decline at the transcription start site (TSS) and occurs frequently in gene bodies under both oxidizing conditions. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that 7 KEGG pathways were mapped into and most of the differentially methylated genes were enriched in oxidative phosphorylation and metabolic pathways. Fourteen genes were selected for studying the effect of differences in methylation on mRNA expression. Thirteen genes, excluding petA-1, demonstrated a decrease in mRNA expression as methylation levels increased. Overall, the 6mA methylation enrichment patterns are similar under two conditions but show differences in the enriched pathways. The phenomenon of upregulated gene methylation levels coupled with downregulated expression suggests a potential association between the regulation mechanisms of 6mA and the Fe2+ and RISCs oxidation pathways.

Simulating compatible solute biosynthesis using a metabolic flux model of the biomining acidophile, Acidithiobacillus ferrooxidans ATCC 23270.

Halotolerant, acidophilic, bioleaching microorganisms are crucial to biomining operations that utilize saline water. Compatible solutes play an important role in the adaptation of these microorganisms to saline environments. Acidithiobacillus ferrooxidans ATCC 23270, an iron- and sulfur-oxidizing acidophilic bacterium, synthesizes trehalose as its native compatible solute but is still sensitive to salinity. Recently, halotolerant bioleaching bacteria were found to use ectoine as their key compatible solute. Previously, bioleaching bacteria were recalcitrant to genetic manipulation; however, recent advancements in genetic tools and techniques allow successful genetic modification of A. ferrooxidans ATCC 23270. Therefore, this study aimed to test, in silico, the effect of native and synthetic compatible solute biosynthesis by A. ferrooxidans ATCC 23270 on its growth and metabolism. Metabolic network flux modelling was used to provide a computational framework for the prediction of metabolic fluxes during production of native and synthetic compatible solutes by A. ferrooxidans ATCC 23270, in silico. Complete pathways for trehalose biosynthesis by the bacterium are proposed and captured in the updated metabolic model including a newly discovered UDP-dependent trehalose synthesis pathway. Finally, the effect of nitrogen sources on compatible solute production was simulated and showed that using nitrogen gas as the sole nitrogen source enables the ectoine-producing 'engineered' microbe to oxidize up to 20% more ferrous iron in comparison to the native microbe that only produces trehalose. Therefore, the predictive outcomes of the model have the potential to guide the design and optimization of a halotolerant strain of A. ferrooxidans ATCC 23270 for saline bioleaching operations.

Sulfur oxidation kinetics of Acidithiobacillus caldus and its inhibition on exposure to thiocyanate present in cyanidation tailings wastewater.

The sulfur oxidation kinetics of an industrial strain of Acidithiobacillus caldus (At. caldus) cultured on elemental sulfur was explored in batch experiments in the absence and presence of thiocyanate (SCN-), a toxin inherent within cyanidation tailings wastewater. The Contois rate expression accurately described At. caldus sulfate generation (R2 > 0.93) and microbial growth (R2 > 0.87). For a culture maintained at 45 °C a maximum specific growth rate (µmax) of 0.105 h-1, sulfate yield from biomass (Ypx) of 4.8 × 10-9 mg SO42-.cell-1, and Contois affinity coefficient (Kx) of 1.56 × 10-8 mg S.cell-1 were established. The presence of SCN- (0 mg/L - 20 mg/L) in the bulk solution inhibited the microbial system competitively. Moreover, SCN- impeded microbial growth differentially; the rate expression was therefore partitioned with respect to SCN- concentration and inhibition constants (Ki) were determined for each region. Adaptation to discrete concentrations of SCN- (1 mg/L and 20 mg/L) improved SCN- tolerance in At. caldus; however, adapted strains exhibited reduced sulfur oxidation potential when cultured under thiocyanate-free conditions relative to the non-adapted control strain. To describe the adapted systems accurately, the Contois affinity coefficient (Kx) was revised to reflect the suspected metabolic decline. The derived Kx values increased in magnitude and affirmed an innate reduction in microbial substrate affinity or substrate adsorption capacity. Inclusion of these updated Kx constants within the rate equation suitably represented the experimental data for both adapted At. caldus strains with R2 > 0.94. Furthermore, the impact of adaptation on the inhibition kinetics and inhibition mechanism associated with SCN- exposure were reviewed. Thiocyanate inhibited sulfur oxidation non-competitively in the adapted strains, and the shift in inhibition mechanism may be attributed to the compromised metabolic state following adaptation.

Rates of iron(III) reduction coupled to elemental sulfur or tetrathionate oxidation by acidophilic microorganisms and detection of sulfur intermediates.

Bioleaching processes and acid mine drainage (AMD) generation are mainly driven by aerobic microbial iron(II) and inorganic sulfur/compound oxidation. Dissimilatory iron(III) reduction coupled to sulfur/compound oxidation (DIRSO) by acidophilic microorganisms has been described for anaerobic cultures, but iron reduction was observed under aerobic conditions as well. Aim of this study was to explore reaction rates and mechanisms of this process. Cell-specific iron(III) reduction rates for different Acidithiobacillus (At.) strains during batch culture growth or stationary phase with iron(III) (∼40 mM) as electron acceptor and elemental sulfur or tetrathionate as electron donor (1% or 5 mM, respectively) were determined. The rates were highest under anaerobic conditions for the At. ferrooxidans type strain with 6.8 × 106 and 1.1 × 107 reduced iron(III) ions per second per cell for growth on elemental sulfur and tetrathionate, respectively. The iron(III) reduction rates were somehow lower for the anaerobically sulfur grown archaeon Ferroplasma acidiphilum, and lowest for the sulfur grown At. caldus type strain under aerobic conditions (1.7 × 106 and 7.3 × 104 reduced iron(III) ions per second per cell, respectively). The rates for five strains of At. thiooxidans (aerobe) were in between those for At. ferrooxidans (anaerobe) and At. caldus (aerobe). There was no pronounced pH dependence of iron(III) reduction rates in the range of pH 1.0-1.9 for the type strains of all species but rates increased with increasing pH for four other At. thiooxidans strains. Thiosulfate as sulfur intermediate was found for At. ferrooxidans during anaerobic growths on tetrathionate and iron(III) but not during anaerobic growths on elemental sulfur and iron(III), and a small concentration was measured during aerobic growths on tetrathionate without iron(III). For the At. thiooxidans type strain thiosulfate was found with tetrathionate grown cells under aerobic conditions in presence and absence of iron(III), but not with sulfur grown cells. Evidence for hydrogen sulfide production at low pH was found for the At. ferrooxidans as well as the At. thiooxidans type strains during microaerophilic growth on elemental sulfur and for At. ferrooxidans during anaerobic growths on tetrathionate and iron(III). The occurrence of sulfur compound intermediates supports the hypothesis that chemical reduction of iron(III) ions takes place by sulfur compounds released by the microbial cells.

Enhancing column bioleaching of chalcocite by isolated iron metabolism partners Leptospirillum ferriphilum/Acidiphilium sp. coupling with systematically utilizing cellulosic waste.

The iron metabolism partners Leptospirillum ferriphilum and Acidiphilium sp. were screened from industrial bioheap site. An integrated multi-stage strategy was proposed to improve chalcolite column bioleaching coupling with synergistical utilization of cellulosic waste such as acid hydrolysate of aquatic plants. L. ferriphilum was used to accelerate the initial iron metabolism, and Acidithiobacillus caldus maintained a lower pH in the middle stage, while Acidiphilium sp. greatly inhibited jarosite passivation in the later stage. Meanwhile, L. ferriphilum (38.3 %) and Acidiphilium sp. (37.0 %) dominated the middle stage, while the abundance of Acidiphilium sp. reached 63.5 % in the later stage. The ferrous, sulfate ion and biomass were improved and the transcriptional levels of some biofilm and morphology related genes were significantly up-regulated. The final Cu2+ concentration reached 325.5 mg·L-1, improved by 43.8 %. Moreover, Canonical Correlation Analysis (CCA) analysis between bioleaching performance, iron/sulfur metabolism and community verified the important role of iron metabolism partners.

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.

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.

Influence of mobile genetic elements and insertion sequences in long- and short-term adaptive processes of Acidithiobacillus ferrooxidans strains.

The recent revision of the Acidithiobacillia class using genomic taxonomy methods has shown that, in addition to the existence of previously unrecognized genera and species, some species of the class harbor levels of divergence that are congruent with ongoing differentiation processes. In this study, we have performed a subspecies-level analysis of sequenced strains of Acidithiobacillus ferrooxidans to prove the existence of distinct sublineages and identify the discriminant genomic/genetic characteristics linked to these sublineages, and to shed light on the processes driving such differentiation. Differences in the genomic relatedness metrics, levels of synteny, gene content, and both integrated and episomal mobile genetic elements (MGE) repertoires support the existence of two subspecies-level taxa within A. ferrooxidans. While sublineage 2A harbors a small plasmid related to pTF5, this episomal MGE is absent in sublineage 2B strains. Likewise, clear differences in the occurrence, coverage and conservation of integrated MGEs are apparent between sublineages. Differential MGE-associated gene cargo pertained to the functional categories of energy metabolism, ion transport, cell surface modification, and defense mechanisms. Inferred functional differences have the potential to impact long-term adaptive processes and may underpin the basis of the subspecies-level differentiation uncovered within A. ferrooxidans. Genome resequencing of iron- and sulfur-adapted cultures of a selected 2A sublineage strain (CCM 4253) showed that both episomal and large integrated MGEs are conserved over twenty generations in either growth condition. In turn, active insertion sequences profoundly impact short-term adaptive processes. The ISAfe1 element was found to be highly active in sublineage 2A strain CCM 4253. Phenotypic mutations caused by the transposition of ISAfe1 into the pstC2 encoding phosphate-transport system permease protein were detected in sulfur-adapted cultures and shown to impair growth on ferrous iron upon the switch of electron donor. The phenotypic manifestation of the △pstC2 mutation, such as a loss of the ability to oxidize ferrous iron, is likely related to the inability of the mutant to secure the phosphorous availability for electron transport-linked phosphorylation coupled to iron oxidation. Depletion of the transpositional △pstC2 mutation occurred concomitantly with a shortening of the iron-oxidation lag phase at later transfers on a ferrous iron-containing medium. Therefore, the pstII operon appears to play an essential role in A. ferrooxidans when cells oxidize ferrous iron. Results highlight the influence of insertion sequences and both integrated and episomal mobile genetic elements in the short- and long-term adaptive processes of A. ferrooxidans strains under changing growth conditions.

Comparative genomics reveals intraspecific divergence of Acidithiobacillus ferrooxidans: insights from evolutionary adaptation.

Acidithiobacillus ferrooxidans serves as a model chemolithoautotrophic organism in extremely acidic environments, which has attracted much attention due to its unique metabolism and strong adaptability. However, little was known about the divergences along the evolutionary process based on whole genomes. Herein, we isolated six strains of A. ferrooxidans from mining areas in China and Zambia, and used comparative genomics to investigate the intra-species divergences. The results indicated that A. ferrooxidans diverged into three groups from a common ancestor, and the pan-genome is 'open'. The ancestral reconstruction of A. ferrooxidans indicated that genome sizes experienced a trend of increase in the very earliest days before a decreasing tendency during the evolutionary process, suggesting that both gene gain and gene loss played crucial roles in A. ferrooxidans genome flexibility. Meanwhile, 23 single-copy orthologous groups (OGs) were under positive selection. The differences of rusticyanin (Rus) sequences (the key protein in the iron oxidation pathway) and type IV secretion system (T4SS) composition in the A. ferrooxidans were both related to their group divergences, which contributed to their intraspecific diversity. This study improved our understanding of the divergent evolution and environmental adaptation of A. ferrooxidans at the genome level in extreme conditions, which provided theoretical support for the survival mechanism of living creatures at the extreme.

Impact of Fulvic Acid and Acidithiobacillus ferrooxidan Inoculum Amount on the Formation of Secondary Iron Minerals.

The catalytic oxidation of Fe2+ by Acidithiobacillus ferrooxidan (A. ferrooxidans) and the synthesis of iron sulfate-based secondary minerals is considered to be of great significance to the treatment of acid mine drainage (AMD). Along these lines, in this work, the shaker experiment was carried out to study the underlying mechanism of the inoculation amount of fulvic acid (FA) and A. ferrooxidans on the synthesis process of secondary minerals. From the acquired results, it was demonstrated that the oxidation rate of Fe2+ increased with the increase in the concentration of fulvic acid in the range of 0.1-0.2 g/L. On top of that, the concentration of fulvic acid in the range of 0.3-0.5 g/L inhibited the activity of A. ferrooxidans. However, A. ferrooxidans retained its activity, and the complete oxidation time of Fe2+ was delayed. When the concentration of fulvic acid was 0.3 g/L, the TFe (total iron) precipitation efficiency was 30.2%. Interestingly, when 0.2 g/L fulvic acid was added to different inoculum systems, the incorporation of a higher inoculum amount of A. ferrooxidans led to an increased oxidation rate. On the contrary, the lower inoculum amount yielded a more obvious effect of the fulvic acid. From the mineralogical characteristics, it was also revealed that a fulvic acid concentration of 0.2 g/L and different inoculation amounts of A. ferrooxidans did not change the mineral facies, whereas pure schwertmannite was obtained.

Improvement of gold bioleaching extraction from waste telecommunication printed circuit boards using biogenic thiosulfate by Acidithiobacillus thiooxidans.

Cyanide usage in gold processing techniques has become increasingly challenging due to its toxicity and environmental impact. It is possible to develop environmentally friendly technology using thiosulfate because of its nontoxic characteristics. Thiosulfate production requires high temperatures, resulting in high greenhouse gas emissions and energy consumption. The biogenesized thiosulfate is an unstable intermediate product of Acidithiobacillus thiooxidans sulfur oxidation pathway to sulfate. A novel eco-friendly method was presented in this study to treat spent printed circuit boards (STPCBs) using biogenesized thiosulfate (Bio-Thio) obtained from Acidithiobacillus thiooxidans cultured medium. To obtain a preferable concentration of thiosulfate among other metabolites by limiting thiosulfate oxidation, optimal concentrations of inhibitor (NaN3: 3.25 mg/L) and pH adjustments (pH= 6-7) were found to be effective. Selection of the optimal conditions has led to the highest bio-production of thiosulfate (500 mg/L). The impact of STPCBs content, ammonia, ethylenediaminetetraacetic acid (EDTA), and leaching time on Cu bio-dissolution and gold bio-extraction were investigated using enriched-thiosulfate spent medium. The suitable conditions were a pulp density of 5 g/L, an ammonia concentration of 1 M, and a leaching time of 36 h, which led to the highest selective extraction of gold (65 ± 0.78%).

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.

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.

Recombinant expression using the tetrathionate hydrolase promoter in Acidithiobacillus ferrooxidans.

In the iron- and sulfur-oxidizing acidophilic chemolithoautotrophic bacterium, Acidithiobacillus ferrooxidans, tetrathionate hydrolase gene (Af-tth) is highly expressed during tetrathionate growth. The expression levels of Af-tth were specifically determined by quantitative reverse transcription-polymerase chain reaction and the expression ratios of S0/Fe2+ and S4O62-/Fe2+ were found to be 68 ± 21 and 181 ± 5, respectively. The transcriptional start site was identified by primer extension. Promoter regions of Af-tth were cloned into the expression shuttle vector pMPJC and GFP gene was under the direction of the regions. Green fluorescence was observed by UV irradiation in recombinant A. ferrooxidans harboring the plasmid colonies grown on tetrathionate. Furthermore, His-tagged Af-Tth was synthesized in the recombinant cells grown on tetrathionate. Recombinant, His-tagged Af-Tth in an active form, was rapidly purified through metal-affinity column chromatography, although recombinant Af-Tth was synthesized in the inclusion bodies of Escherichia coli and acid-refolding treatment was necessary to recover the activity. The specific activity of purified Af-Tth from recombinant A. ferrooxidans (2.2 ± 0.37 U mg-1) was similar to that of acid-refolded Af-Tth from recombinant E. coli (2.5 ± 0.18 U mg-1). This method can be applied not only to heterologous expression but also to homologous expression of target genes for modification or specific mutation in A. ferrooxidans cells.

Adding organics to enrich mixotrophic sulfur-oxidizing bacteria under extremely acidic conditions-A novel strategy to enhance hydrogen sulfide removal.

Biotreatment of high load hydrogen sulfide (H2S) can lead to rapid acidification of a bioreactor, which greatly challenges the application of bio-desulfurization technology. In this study, the bio-desulfurization performance was improved by enriching acidophilic mixotrophic sulfur-oxidizing bacteria (SOB) by adding organics under extremely acidic conditions (pH < 1.0). A biotrickling filter (BTF) for the removal of H2S was established and operated under pH < 1.0 for 420 days. In the autotrophic period, the maximum H2S elimination capacity (ECmax-H2S) was 135.8 g/m3/h with biofilm mass remaining within 11.1 g/L-BTF. The autotrophic SOB bacterium Acidithiobacillus was dominant (62.1 %). When glucose was added to the BTF system, ECmax-H2S increased by 272 % to 464.3 g/m3/h as biofilm mass increased to 22.3 g/L-BTF. The acidophilic mixotrophic SOB bacteria Mycobacterium (78.4 %) and Alicyclobacillus (20.7 %) were enriched while Acidithiobacillus was gradually eliminated (<0.1 %). Furthermore, the major sulfur metabolism pathways were identified to explore the desulfurization mechanism under extremely acidic conditions. To maintain optimal desulfurization performance and avoid biofilm overgrowth in the BTF system, biofilm mass should be maintained within 20-22 g/L-BTF. This can be achieved by adding 1.0 g/L-BTF glucose every 20 days under a load rate of H2S in 50-90 g/m3/h and a trickling liquid velocity of 1.8 m/h. Extremely acidic conditions eliminated non-aciduric microorganisms so that the addition of organics can increase the abundance of acidophilic mixotrophic SOB (>99 %), thus offering a novel strategy for enhancing H2S removal.

The use of synthetic agonists of quorum sensing N- acyl homoserine lactone pathway improves the bioleaching ability in Acidithiobacillus and Pseudomonas bacteria.

Metal solubilization from discarded electrical material and electronic devices (e-waste) using the bioleaching capabilities of bacterial cells is highly effective. However, gaps in understanding about the microbiological processes involved in the bioleaching reaction leads to less efficient metal solubilization in large-scale e-waste processing. In this study, bacterial species belonging to the genera Acidithiobacillus and Pseudomonas were used to leach copper and gold from discarded printed circuit boards (PCB). Through modulation of the cell-to-cell communication system in these bacteria, phenotypic traits directly involved in the bioleaching reaction were regulated in order to improve the metal solubilization. Addition of the long chain synthetic autoinducer molecule N-acyl homoserine lactone (AHL) of the quorum sensing pathway to the bioleaching reaction resulted in a significant enhancement of metal extraction from PCB. Factors such as: cell attachment to PCB, biofilm formation and hydrogen cyanide (HCN) production were regulated by the quorum sensing system and could be directly related to the improvement of metal bioleaching. Bioleaching reactions using bacterial quorum sensing modulation could represent a valuable tool in overcoming limitations at the industrial level imposed by microbiological traits that lead to inefficient metal bioleaching from e-waste.

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.