Metal recovery from spent lithium-ion batteries via two-step bioleaching using adapted chemolithotrophs from an acidic mine pit lake.

The demand for lithium-ion batteries (LIBs) has dramatically increased in recent years due to their application in various electronic devices and electric vehicles (EVs). Great amount of LIB waste is generated, most of which ends up in landfills. LIB wastes contain substantial amounts of critical metals (such as Li, Co, Ni, Mn, and Cu) and can therefore serve as valuable secondary sources of these metals. Metal recovery from the black mass (shredded spent LIBs) can be achieved via bioleaching, a microbiology-based technology that is considered to be environmentally friendly, due to its lower costs and energy consumption compared to conventional pyrometallurgy or hydrometallurgy. However, the growth and metabolism of bioleaching microorganisms can be inhibited by dissolved metals. In this study, the indigenous acidophilic chemolithotrophs in a sediment from a highly acidic and metal-contaminated mine pit lake were enriched in a selective medium containing iron, sulfur, or both electron donors. The enriched culture with the highest growth and oxidation rate and the lowest microbial diversity (dominated by Acidithiobacillus and Alicyclobacillus spp. utilizing both electron donors) was then gradually adapted to increasing concentrations of Li+, Co2+, Ni2+, Mn2+, and Cu2+. Finally, up to 100% recovery rates of Li, Co, Ni, Mn, and Al were achieved via two-step bioleaching using the adapted culture, resulting in more effective metal extraction compared to bioleaching with a non-adapted culture and abiotic control.

Zinc recovery from bioleachate using a microbial electrolysis cell and comparison with selective precipitation.

Metal recycling is essential for strengthening a circular economy. Microbial leaching (bioleaching) is an economical and environmentally friendly technology widely used to extract metals from insoluble ores or secondary resources such as dust, ashes, and slags. On the other hand, microbial electrolysis cells (MECs) would offer an energy-efficient application for recovering valuable metals from an aqueous solution. In this study, we investigated a MEC for Zn recovery from metal-laden bioleachate for the first time by applying a constant potential of -100 mV vs. Ag/AgCl (3 M NaCl) on a synthetic wastewater-treating bioanode. Zn was deposited onto the cathode surface with a recovery efficiency of 41 ± 13% and an energy consumption of 2.55 kWh kg-1. For comparison, Zn recovery from zinc sulfate solution resulted in a Zn recovery efficiency of 100 ± 0% and an energy consumption of 0.70 kWh kg-1. Furthermore, selective metal precipitation of the bioleachate was performed. Individual metals were almost completely precipitated from the bioleachate at pH 5 (Al), pH 7 (Zn and Fe), and pH 9 (Mg and Mn).

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.

Leachability of metals from waste incineration residues by iron- and sulfur-oxidizing bacteria.

Hazardous waste disposal via incineration generates a substantial amount of ashes and slags which pose an environmental risk due to their toxicity. Currently, these residues are deposited in landfills with loss of potentially recyclable raw material. In this study, the use of acidophilic bioleaching bacteria (Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, and Leptospirillum ferrooxidans) as an environmentally friendly, efficient strategy for the recovery of valuable metals from incineration residues was investigated. Zinc, Cobalt, Copper, and Manganese from three different incineration residues were bio-extracted up to 100% using A. ferrooxidans under ferrous iron oxidation. The other metals showed lower leaching efficiencies based on the type of culture used. Sulfur-oxidizing cultures A. ferrooxidans and A. thiooxidans, containing sulfur as the sole substrate, expressed a significantly lower leaching efficiency (up to 50%). According to ICP-MS, ashes and slags contained Fe, Zn, Cu, Mn, Cr, Cd, and Ni in economically attractive concentrations between 0.2 and 75 mg g-1. Compared to conventional hydrometallurgical and pyrometallurgical processes, our biological approach provides many advantages such as: the use of a limited amount of used strong acids (H2SO4 or HCl), recycling operations at lower temperatures (~30 °C) and no emission of toxic gases during combustion (i.e., dioxins and furans).

A Model of Aerobic and Anaerobic Metabolism of Hydrogen in the Extremophile Acidithiobacillus ferrooxidans.

Hydrogen can serve as an electron donor for chemolithotrophic acidophiles, especially in the deep terrestrial subsurface and geothermal ecosystems. Nevertheless, the current knowledge of hydrogen utilization by mesophilic acidophiles is minimal. A multi-omics analysis was applied on Acidithiobacillus ferrooxidans growing on hydrogen, and a respiratory model was proposed. In the model, [NiFe] hydrogenases oxidize hydrogen to two protons and two electrons. The electrons are used to reduce membrane-soluble ubiquinone to ubiquinol. Genetically associated iron-sulfur proteins mediate electron relay from the hydrogenases to the ubiquinone pool. Under aerobic conditions, reduced ubiquinol transfers electrons to either cytochrome aa 3 oxidase via cytochrome bc 1 complex and cytochrome c 4 or the alternate directly to cytochrome bd oxidase, resulting in proton efflux and reduction of oxygen. Under anaerobic conditions, reduced ubiquinol transfers electrons to outer membrane cytochrome c (ferrireductase) via cytochrome bc 1 complex and a cascade of electron transporters (cytochrome c 4, cytochrome c 552, rusticyanin, and high potential iron-sulfur protein), resulting in proton efflux and reduction of ferric iron. The proton gradient generated by hydrogen oxidation maintains the membrane potential and allows the generation of ATP and NADH. These results further clarify the role of extremophiles in biogeochemical processes and their impact on the composition of the deep terrestrial subsurface.

Can Sulfate Be the First Dominant Aqueous Sulfur Species Formed in the Oxidation of Pyrite by Acidithiobacillus ferrooxidans?

According to the literature, pyrite (FeS2) oxidation has been previously determined to involve thiosulfate as the first aqueous intermediate sulfur product, which is further oxidized to sulfate. In the present study, pyrite oxidation by Acidithiobacillus ferrooxidans was studied using electrochemical and metabolic approaches in an effort to extend existing knowledge on the oxidation mechanism. Due to the small surface area, the reaction rate of a compact pyrite electrode in the form of polycrystalline pyrite aggregate in A. ferrooxidans suspension was very slow at a spontaneously formed high redox potential. The slow rate made it possible to investigate the oxidation process in detail over a term of 100 days. Using electrochemical parameters from polarization curves and levels of released iron, the number of exchanged electrons per pyrite molecule was estimated. The values close to 14 and 2 electrons were determined for the oxidation with and without bacteria, respectively. These results indicated that sulfate was the dominant first aqueous sulfur species formed in the presence of bacteria and elemental sulfur was predominantly formed without bacteria. The stoichiometric calculations are consistent with high iron-oxidizing activities of bacteria that continually keep the released iron in the ferric form, resulting in a high redox potential. The sulfur entity of pyrite was oxidized to sulfate by Fe3+ without intermediate thiosulfate under these conditions. Cell attachment on the corroded pyrite electrode surface was documented although pyrite surface corrosion by Fe3+ was evident without bacterial participation. Attached cells may be important in initiating the oxidation of the pyrite surface to release iron from the mineral. During the active phase of oxidation of a pyrite concentrate sample, the ATP levels in attached and planktonic bacteria were consistent with previously established ATP content of iron-oxidizing cells. No significant upregulation of three essential genes involved in energy metabolism of sulfur compounds was observed in the planktonic cells, which represented the dominant biomass in the pyrite culture. The study demonstrated the formation of sulfate as the first dissolved sulfur species with iron-oxidizing bacteria under high redox potential conditions. Minor aqueous sulfur intermediates may be formed but as a result of side reactions.

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.

Are there multiple mechanisms of anaerobic sulfur oxidation with ferric iron in Acidithiobacillus ferrooxidans?

To clarify the pathway of anaerobic sulfur oxidation coupled with dissimilatory ferric iron reduction in Acidithiobacillus ferrooxidans strain CCM 4253 cells, we monitored their energy metabolism gene transcript profiles. Several genes encoding electron transporters involved in aerobic iron and sulfur respiration were induced during anaerobic growth of ferrous iron-grown cells. Most sulfur metabolism genes were either expressed at the basal level or their expression declined. However, transcript levels of genes assumed to be responsible for processing of elemental sulfur and other sulfur intermediates were elevated at the beginning of the growth period. In contrast, genes with predicted functions in formation of hydrogen sulfide and sulfate were significantly repressed. The main proposed mechanism involves: outer membrane protein Cyc2 (assumed to function as a terminal ferric iron reductase); periplasmic electron shuttle rusticyanin; c4-type cytochrome CycA1; the inner membrane cytochrome bc1 complex I; and the quinone pool providing connection to the sulfur metabolism machinery, consisting of heterodisulfide reductase, thiosulfate:quinone oxidoreductase and tetrathionate hydrolase. However, an alternative mechanism seems to involve a high potential iron-sulfur protein Hip, c4-type cytochrome CycA2 and inner membrane cytochrome bc1 complex II. Our results conflict with findings regarding the type strain, indicating strain- or phenotype-dependent pathway variation.

Ferrous iron oxidation by sulfur-oxidizing Acidithiobacillus ferrooxidans and analysis of the process at the levels of transcription and protein synthesis.

In contrast to iron-oxidizing Acidithiobacillus ferrooxidans, A. ferrooxidans from a stationary phase elemental sulfur-oxidizing culture exhibited a lag phase in pyrite oxidation, which is similar to its behaviour during ferrous iron oxidation. The ability of elemental sulfur-oxidizing A. ferrooxidans to immediately oxidize ferrous iron or pyrite without a lag phase was only observed in bacteria obtained from growing cultures with elemental sulfur. However, these cultures that shifted to ferrous iron oxidation showed a low rate of ferrous iron oxidation while no growth was observed. Two-dimensional gel electrophoresis was used for a quantitative proteomic analysis of the adaptation process when bacteria were switched from elemental sulfur to ferrous iron. A comparison of total cell lysates revealed 39 proteins whose increase or decrease in abundance was related to this phenotypic switching. However, only a few proteins were closely related to iron and sulfur metabolism. Reverse-transcription quantitative PCR was used to further characterize the bacterial adaptation process. The expression profiles of selected genes primarily involved in the ferrous iron oxidation indicated that phenotypic switching is a complex process that includes the activation of genes encoding a membrane protein, maturation proteins, electron transport proteins and their regulators.

Stoichiometry of bacterial anaerobic oxidation of elemental sulfur by ferric iron.

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

Kinetics of anaerobic elemental sulfur oxidation by ferric iron in Acidithiobacillus ferrooxidans and protein identification by comparative 2-DE-MS/MS.

Elemental sulfur oxidation by ferric iron in Acidithiobacillus ferrooxidans was investigated. The apparent Michaelis constant for ferric iron was 18.6 mM. An absence of anaerobic ferric iron reduction ability was observed in bacteria maintained on elemental sulfur for an extended period of time. Upon transition from ferrous iron to elemental sulfur medium, the cells exhibited similar kinetic characteristics of ferric iron reduction under anaerobic conditions to those of cells that were originally maintained on ferrous iron. Nevertheless, a total loss of anaerobic ferric iron reduction ability after the sixth passage in elemental sulfur medium was demonstrated. The first proteomic screening of total cell lysates of anaerobically incubated bacteria resulted in the detection of 1599 protein spots in the master two-dimensional electrophoresis gel. A set of 59 more abundant and 49 less abundant protein spots that changed their protein abundances in an anaerobiosis-dependent manner was identified and compared to iron- and sulfur-grown cells, respectively. Proteomic analysis detected a significant increase in abundance under anoxic conditions of electron transporters, such as rusticyanin and cytochrome c(552), involved in the ferrous iron oxidation pathway. Therefore we suggest the incorporation of rus-operon encoded proteins in the anaerobic respiration pathway. Two sulfur metabolism proteins were identified, pyridine nucleotide-disulfide oxidoreductase and sulfide-quinone reductase. The important transcription regulator, ferric uptake regulation protein, was anaerobically more abundant. The anaerobic expression of several proteins involved in cell envelope formation indicated a gradual adaptation to elemental sulfur oxidation.

Kinetic constant variability in bacterial oxidation of elemental sulfur.

Wide ranges of growth yields on sulfur (from 2.4 x 10(10) to 8.1 x 10(11) cells g(-1)) and maximum sulfur oxidation rates (from 0.068 to 1.30 mmol liter(-1) h(-1)) of an Acidithiobacillus ferrooxidans strain (CCM 4253) were observed in 73 batch cultures. No significant correlation between the constants was observed. Changes of the Michaelis constant for sulfur (from 0.46 to 15.5 mM) in resting cells were also noted.

Proteomic and bioinformatic analysis of iron- and sulfur-oxidizing Acidithiobacillus ferrooxidans using immobilized pH gradients and mass spectrometry.

A comparative analysis of the protein composition of Acidithiobacillus ferrooxidans cells grown on elemental sulfur and ferrous iron was performed. A newly developed protocol involving immobilized pH gradients, improved protein reduction, mass spectrometry protein identification and full genome sequence information was applied. This approach resulted in more than 1300 protein spots displayed in broad and basic pH ranges, the best A. ferrooxidans proteome resolution to date. A comparative image analysis revealed that the proteome was significantly influenced by the growth type, and allowed for the detection of many physiologically important proteins. Among them were sulfate adenylyltransferase and sulfide dehydrogenase, which are involved in sulfate assimilation and sulfide metabolism, respectively. Many other proteins were related to important processes like cell attachment and electron transport. Co-migration of phosphate and sulfate transport proteins was also observed.

Use of immobilized cytochrome c as a ligand for affinity chromatography of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans.

Three matrices were used for immobilizing the cytochrome c: Sepharose CL-4B, Silasorb SPH amine and a laboratory-prepared new matrix based on crosslinked triazine (2,4,6-tris(aminoethylamine)-1,3,5-triazine) (TAT). Cytochrome c was immobilized on the matrices by several procedures and the amount of incorporated cytochrome c was determined. Cytochrome c immobilized on Sepharose CL-4B with periodate activation, cytochrome c immobilized on Silasorb-amine with carbodiimide activation and cytochrome c immobilized on crosslinked triazine were suitable for purification of thiosulfate dehydrogenase from Acidithiobacillus ferrooxidans. The yield with all matrices was about 90%. The purification factor of the above matrices was about 15. A new matrix based on TAT with cytochrome c represented a suitable way for thiosulfate dehydrogenase purification.

Kinetic studies on elemental sulfur oxidation by Acidithiobacillus ferrooxidans: sulfur limitation and activity of free and adsorbed bacteria.

The kinetics of sulfur oxidation by Acidithiobacillus ferrooxidans in shaking flasks and a 10-L reactor was studied. The observed linearity of growth and sulfur oxidation was explained by sulfur limitation. Total cell yield was not significantly different for exponential growth as compared to growth during the sulfur-limiting phase. Kinetic studies of sulfur oxidation by growing and nongrowing bacteria indicated that both free and adsorbed bacteria oxidize sulfur. Changes in the number of free bacteria rather than cells adsorbed on sulfur were better predictors of the kinetics of sulfur oxidation, indicating that the free bacteria were performing sulfur oxidation. The active growth phase always followed adsorption of bacteria on sulfur; however, the special metabolic role of adsorbed bacteria was unclear. Their activity in sulfur solubilization was considered.