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Bioleaching of metal sulfides is performed by diverse microorganisms. The dissolution of metal sulfides occurs via two chemical pathways, either the thiosulfate or the polysulfide pathway. These are determined by the metal sulfides’ mineralogy and their acid solubility. The microbial cell enables metal sulfide dissolution via oxidation of iron(II) ions and inorganic sulfur compounds. Thereby, the metal sulfide attacking agents iron(III) ions and protons are generated. Cells are active either in a planktonic state or attached to the mineral surface, forming biofilms. This review, as an update of the previous one (Vera et al., 2013a), summarizes some recent discoveries relevant to bioleaching microorganisms, contributing to a better understanding of their lifestyle. These comprise phylogeny, chemical pathways, surface science, biochemistry of iron and sulfur metabolism, anaerobic metabolism, cell–cell communication, molecular biology, and biofilm lifestyle. Recent advances from genetic engineering applied to bioleaching microorganisms will allow in the future to better understand important aspects of their physiology, as well as to open new possibilities for synthetic biology applications of leaching microbial consortia. Key points • Leaching of metal sulfides is strongly enhanced by microorganisms • Biofilm formation and extracellular polymer production influences bioleaching • Cell interactions in mixed bioleaching cultures are key for process optimization

Bioleaching is an established process for sulfidic ores and is increasingly applied to the recycling of industrial residues. However, unlike ores, many residues like sludge contain inhibitory elements, among which fluoride poses a major challenge due to its toxicity toward acidophilic microorganisms even at low concentrations. This study systematically investigated fluoride tolerance in pure and mixed cultures of various acidophilic sulfur- and iron-oxidizing bacteria commonly used for bioleaching, including Acidithiobacillus spp., Leptospirillum spp., and Sulfobacillus thermosulfidooxidans . Fluoride toxicity was found to be substrate-dependent. During sulfur oxidation, A. thiooxidans displayed the highest fluoride tolerance (0.5 mM F⁻), whereas S. thermosulfidooxidans showed complete inhibition. In contrast, iron-oxidizing bacteria demonstrated increased fluoride tolerance, with S. thermosulfidooxidans remaining active at 1.5 mM F⁻ when grown on ferrous iron. Mixed cultures showed enhanced fluoride tolerance during sulfur oxidation but reduced tolerance during iron oxidation. pH was identified as a critical factor influencing fluoride toxicity due to increased formation of undissociated HF at low pH. To mitigate fluoride inhibition, fluoride complexation with ferric iron or aluminum was evaluated. For A. ferrooxidans , iron oxidation resumed at Fe 3 ⁺:F⁻ ratios of 7.5:1, while other cultures required ratios of at least 10:1. Aluminum complexation required Al:F⁻ ratios between 1:1 and 2:1, depending on the culture and growth conditions. Overall, fluoride inhibition during bioleaching is influenced by multiple factors, including pH, ferric iron concentration, and the fluoride dissolution rate. Early addition of aluminum is recommended to prevent microbial inhibition and ensure stable bioleaching performance. Key points • Higher fluoride tolerance was observed during iron oxidation. • S. thermosulfidooxidans remained active up to 1.5 mM F⁻. • Fluoride toxicity is strongly pH dependent due to increased HF formation at low pH. • Effective fluoride complexation requires higher Fe 3+ :F⁻ ratios (> 7.5:1) than Al3⁺:F⁻ ratios (> 1:1) Graphical Abstract

C-type cytochromes fulfil many essential roles in both aerobic and anaerobic respiration. Their characterization requires large quantities of protein which can be obtained through heterologous production. Heterologous production of c-type cytochromes in Escherichia coli is hindered since the ccmABCDEFGH genes necessary for incorporation of heme c are only expressed under anaerobic conditions. Different strategies were devised to bypass this obstacle, such as co-expressing the ccm genes from the pEC86 vector. However, co-expression methods restrict the choice of expression host and vector. Here we describe the first use of Vibrio natriegens V max X2 for the recombinant production of difficult-to-express redox proteins from the extreme acidophile Acidithiobacillus ferrooxidans CCM4253, including three c-type cytochromes. Co-expression of the ccm genes was not required to produce holo-c-type cytochromes in V max X2. E. coli T7 Express only produced holo-c-type cytochromes during co-expression of the ccm genes and was not able to produce the inner membrane cytochrome CycA. Additionally, V max X2 cell extracts contained higher portions of recombinant holo-proteins than T7 Express cell extracts. All redox proteins were translocated to the intended cell compartment in both hosts. In conclusion, V. natriegens represents a promising alternative for the production of c-type cytochromes and difficult-to-express redox proteins.

The research and education mine “Reiche Zeche” in Freiberg (Saxony, Germany) represents one of the most famous mining facilities reminiscent to the century-long history of silver production in the Ore Mountains. The mine was set up at the end of the fourteenth century and became part of the “Bergakademie Freiberg” in 1919. Galena, pyrite, sphalerite, arsenopyrite, and chalcopyrite are the most common minerals found in the mine. As acid mine drainage is generated from the dissolution of sulfidic ores, the microbial habitats within the adits and galleries are characterized by low pH and high concentrations of metal(loid)s. The community composition was investigated at locations characterized by biofilm formation and iron-rich bottom pools. Amplicon libraries were sequenced on a MiSeq instrument. The taxonomic survey yielded an unexpected diversity of 25 bacterial phyla including ten genera of iron-oxidizing taxa. The community composition in the snottites and biofilms only slightly differed from the communities found in acidic bottom pools regarding the diversity of iron oxidizers, the key players in most investigated habitats. Sequences of the Candidate Phyla Radiation as, e.g., Dojkabacteria and Eremiobacterota were found in almost all samples. Archaea of the classes Thermoplasmata and Nitrososphaeria were detected in some biofilm communities.

Iron redox reactions shape nutrient turnover, contaminant mobility, and primary productivity, yet the microbes driving these processes are often studied in isolation. By integrating decades of data into a trait-based guild framework, we reveal the ecophysiological diversity and niche differentiation of microbial iron redox cycling taxa across environments. Our synthesis exposes major gaps, such as limited trait data for >80% of dual-capacity Fe oxidizing/reducing species and highlights the need for functional trait surveys to complement metagenomics and cultivation efforts. The guild framework presented here advances predictive microbial ecology by linking metabolic traits with environmental gradients, offering a robust foundation for incorporating iron cycling into ecosystem models and biogeochemical forecasts.

A comprehensive physiological and phylogenetic characterisation was carried out of “ Thiobacillus ferrooxidans ” m-1, an acidophilic iron-oxidizing bacterium first described over 25 years ago. Phylogenetically, strain m-1 is a gammaproteobacterium, most closely related to alkaliphilic Ectothiorhodospira spp. and only distantly to iron-oxidizing acidithiobacilli. Physiological examination confirmed that strain m-1 can grow autotrophically not only by ferrous iron oxidation but also, in contrast to previous reports, by oxidation of elemental sulfur, sulfide and tetrathionate, using either oxygen or ferric iron as terminal electron acceptor. The bacterium was also found to be thermo-tolerant, growing optimally at 38°C and up to a maximum of 47°C. Growth in liquid media required an external osmotic potential of >2 bar, and was optimal at ~5 bar, though no growth occurred where the medium osmotic potential was close to that of sea water (~26 bar). From this, it was concluded that strain m-1 is a moderate osmophile. Strain m-1 was also shown to be diazotrophic and tolerant of elevated concentrations of many metals typically found in mine-impacted environments. On the basis of these data, m-1 is proposed as the type strain of a new genus and species of bacteria, Acidiferrobacter thiooxydans (DSM 2392, JCM 17358).

ABSTRACT The geothermal Copahue-Caviahue (GCC) system (Argentina) is an extreme acidic environment, dominated by the activity of Copahue volcano. Environments characterised by low pH values, such as volcanic areas, are of particular interest for the search of acidophilic microorganisms with application in biotechnological processes. In this work, sulfate-reducing microorganisms were investigated in geothermal acidic, anaerobic zones from GCC system. Sediment samples from Agua del Limón (AL1), Las Máquinas (LMa2), Las Maquinitas (LMi) and Baño 9 (B9–2, B9–3) were found to be acidic (pH values 2.1–3.0) to moderate acidic (5.1–5.2), containing small total organic carbon values, and ferric iron precipitates. The organic electron donor added to the enrichment was completely oxidised to CO2. Bacteria related to ‘Desulfobacillus acidavidus’ strain CL4 were found to be dominant (67–83% of the total number of clones) in the enrichment cultures, and their presence was confirmed by their isolation on overlay plates. Other bacteria were also detected with lower abundance (6–20% of the total number of clones), with representatives of the genera Acidithiobacillus, Sulfobacillus, Alicyclobacillus and Athalassotoga/Mesoaciditoga. These enrichment and isolates found at low pH confirm the presence of anaerobic activities in the acidic sediments from the geothermal Copahue-Caviahue system. This study explores, for the first time, the anaerobic environments at Copahue volcano focusing on the enrichment and isolation of acidophilic/acid-tolerant sulfate-reducing bacteria and their characterization by 16S rRNA gene sequencing.

Growth media have been developed to facilitate the enrichment and isolation of acidophilic and acid-tolerant sulfate-reducing bacteria (aSRB) from environmental and industrial samples, and to allow their cultivation in vitro. The main features of the ‘standard’ solid and liquid devised media are as follows: (i) use of glycerol rather than an aliphatic acid as electron donor; (ii) inclusion of stoichiometric concentrations of zinc ions to both buffer pH and to convert potentially harmful hydrogen sulphide produced by the aSRB to insoluble zinc sulphide; (iii) inclusion of Acidocella aromatica (an heterotrophic acidophile that does not metabolize glycerol or yeast extract) in the gel underlayer of double layered (overlay) solid media, to remove acetic acid produced by aSRB that incompletely oxidize glycerol and also aliphatic acids (mostly pyruvic) released by acid hydrolysis of the gelling agent used (agarose). Colonies of aSRB are readily distinguished from those of other anaerobes due to their deposition and accumulation of metal sulphide precipitates. Data presented illustrate the effectiveness of the overlay solid media described for isolating aSRB from acidic anaerobic sediments and low pH sulfidogenic bioreactors. The paper describes how bacteria that live in acidic environments, and that form hydrogen sulphide from sulfate, may be isolated and grown in the laboratory. Graphical Abstract Figure. The paper describes how bacteria that live in acidic environments, and that form hydrogen sulphide from sulfate, may be isolated and grown in the laboratory.