Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that plays an important role in biogeochemical iron and sulfur cycling and is a member of the consortia used in industrial hydrometallurgical processing of copper. Metal sulfide bioleaching is catalyzed by the regeneration of ferric iron, however, bioleaching of chalcopyrite, the dominant unmined form of copper on Earth, is inhibited by surface passivation. Here, we report the implementation of CRISPR interference (CRISPRi) using the catalytically inactive Cas12a (dCas12a) in A. ferrooxidans to knockdown the expression of genes in the petI and petII operons. These operons encode bc1 complex proteins and knockdown of these genes enabled the manipulation (enhancement or repression) of iron oxidation. The petB2 gene knockdown strain enhanced iron oxidation, leading to enhanced pyrite and chalcopyrite oxidation, which correlated with reduced biofilm formation and decreased surface passivation of the minerals. These findings highlight the utility of CRISPRi/dCas12a technology for engineering A. ferrooxidans while unveiling a new strategy to manipulate and improve bioleaching efficiency.

Biomining using microalgae has emerged as a sustainable option to extract rare earth elements (REEs). This study aims to (i) explore the capability of REEs recovery from bauxite by microalgae, (ii) assess the change of biochemical function affected by bauxite, and (iii) investigate the effects of operating conditions (i.e., aeration rate, pH, hydraulic retention time) to REEs recovery. The results showed that increasing bauxite in microalgae culture increases REEs recovery in biomass and production of biochemical compounds (e.g., pigments and Ca-Mg ATPase enzyme) up to 10 %. The optimum pulp ratio of bauxite in the microalgae culture ranges from 0.2 % to 0.6 %. Chlorella vulgaris was the most promising, with two times higher in REEs recovery in biomass than the other species. REEs accumulated in microalgae biomass decreased with increasing pH in the culture. This study establishes a platform to make the scaling up of REEs biomining by microalgae plausible.

This study aimed to optimise metal extraction from secondary hazardous sources, such as basic oxygen steelmaking dust (BOS-D). Initially, three batch systems approaches, including bioleaching using Acidithiobacillus ferrooxidans, chemical leaching using choline chloride-ethylene glycol (ChCl-EG) and a combined approach were compared. Then, scaling up was evaluated through a semi-continuous bioleaching column system with varied leachate recirculation over 21 days, focusing on Y, Ce, Nd, Li, Co, Cu, Zn, Mn, and Al. Bioleaching outperformed the control experiments within 3 days in the batch, demonstrating the key role of A. ferrooxidans. Chemical leaching conducted with a solid concentration of 12.5 % (w/v) successfully dissolved over 50 % of all metals within 2 h. For rare earth elements (REE), both bioleaching and hybrid leaching outperformed chemical leaching. However, considering factors such as process duration, overall efficiency, and ease of extraction, chemical leaching was the most effective method. Leachate recirculation reached a plateau after 11 days, resulting in extraction efficiency of 39 % when semi-continuous column set-up was used. Interestingly, variations in recirculation rates did not influence the extraction efficiency. Overall, this study emphasizes the considerable potential of bioleaching for metal recovery, but also highlights the need for further studies for enhancing permeability for percolation methods and optimisation, particularly in parameters such as aeration rate, when transitioning to larger scale systems.

Rhodococcus erythropolis bacterium is known for its remarkable resistance characteristics that can be useful in several biotechnological processes, such as bioremediation. However, there is scarce knowledge concerning the behavior of this strain against different metals. This study sought to investigate the behavior of R. erythropolis ATCC 4277 against the residue of chalcopyrite and e-waste to verify both resistive capacities to the metals present in these residues and their potential use for biomining processes. These tests were carried out in a stirred tank bioreactor for 48 h, at 24ºC, pH 7.0, using a total volume of 2.0 L containing 2.5% (v/v) of a bacterial pre-culture. The pulp density of chalcopyrite was 5% (w/w), and agitation and oxygen flow rates were set to 250 rpm and 1.5 LO2 min-1, respectively. On the other hand, we utilized a waste of computer printed circuit board (WPCB) with a pulp density of 10% (w/w), agitation at 400 rpm, and an oxygen flow rate of 3.0 LO2 min-1. Metal concentration analyses post-fermentation showed that R. erythropolis ATCC 4277 was able to leach about 38% of the Cu present in the chalcopyrite residue (in ~ 24 h), and 49.5% of Fe, 42.3% of Ni, 27.4% of Al, and 15% Cu present in WPCB (in ~ 24 h). In addition, the strain survived well in the environment containing such metals, demonstrating the potential of using this bacterium for waste biomining processes as well as in other processes with these metals.

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world\'s attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.

Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.

The process of extracting metals from rock phosphate ore (RPO) by using microorganisms to convert them into soluble compounds is called biomining. Phosphorus is one of the elements proposed to be extracted from RPO. To understand the role of Streptomyces phospholyticus, 12 isolates of Streptomyces were isolated from RPO, their ability to grow on specific phosphate solubilization medium e.g., National Botanical Research Institute\'s phosphate growth agar (NBRIP) was studied, and the best strain with a 3 cm clear zone was selected. Its ability to grow at increasing RPO concentrations from 0.01 to 1 kgl-1 was investigated. This strain showed good growth, with extracellular red pigmentation for all concentrations, but no clear zone. In the modified liquid NBRIP, however, the Streptomyces growth patterns of the two concentrations of 0.25 kg and 1 kgl-1 RPO showed growth of single spherical red colonies with rhizoids on the surface, the colonies somehow grew and became embedded in the fine RPO granules. This ability to grow can resist gamma irradiation with a dose of 32 KGy. Within 3 days of growth, acidic and alkaline phosphatase were 76.2 and 67.1 μg p-nitrophenol g-1 ml-1, respectively. The RPO analysis showed that the %P in the ore was 16.5% at the beginning of the experiment, and after Streptomyces biotreatment, this percentage decreased to 8.4%, with a decomposition rate of 50.7%. This study, to our knowledge, is the first to investigate the efficiency of Streptomyces in mining phosphate rock ore in the laboratory, even at high concentrations, and to examine the role of irradiation as a preservative in increasing this efficiency.

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.

Rare earth elements (REEs) are essential for advanced manufacturing (e.g., renewable energy, military equipment, electric vehicles); hence, the recovery of REEs from low-grade resources has become increasingly important to address their growing demand. Depending on specific mining sites, its geological conditions, and sociodemographic backgrounds, mining waste has been identified as a source of REEs in various concentrations and abundance. Yttrium, cerium, and neodymium are the most common REEs in mining waste streams (50 to 300 μg/L). Biomining has emerged as a viable option for REEs recovery due to its reduced environmental impact, along with reduced capital investment compared to traditional recovery methods. This paper aims to review (i) the characteristics of mining waste as a low-grade REEs resource, (ii) the key operating principles of biomining technologies for REEs recovery, (iii) the effects of operating conditions and matrix on REEs recovery, and (iv) the sustainability of REEs recovery through biomining technologies. Six types of biomining will be examined in this review: bioleaching, bioweathering, biosorption, bioaccumulation, bioprecipitation and bioflotation. Based on a SWOT analyses and techno-economic assessments (TEA), biomining technologies have been found to be effective and efficient in recovering REEs from low-grade sources. Through TEA, coal ash has been shown to return the highest profit amongst mining waste streams.

Extreme acidophiles thrive in acidic environments, confront a multitude of challenges, and demonstrate remarkable adaptability in their metabolism to cope with the ever-changing environmental fluctuations, which encompass variations in temperature, pH levels, and the availability of electron acceptors and donors. The survival and proliferation of members within the Acidithiobacillia class rely on the deployment of transcriptional regulatory systems linked to essential physiological traits. The study of these transcriptional regulatory systems provides valuable insights into critical processes, such as energy metabolism and nutrient assimilation, and how they integrate into major genetic-metabolic circuits. In this study, we examined the transcriptional regulatory repertoires and potential interactions of forty-three Acidithiobacillia complete and draft genomes, encompassing nine species. To investigate the function and diversity of Transcription Factors (TFs) and their DNA Binding Sites (DBSs), we conducted a genome-wide comparative analysis, which allowed us to identify these regulatory elements in representatives of Acidithiobacillia. We classified TFs into gene families and compared their occurrence among all representatives, revealing conservation patterns across the class. The results identified conserved regulators for several pathways, including iron and sulfur oxidation, the main pathways for energy acquisition, providing new evidence for viable regulatory interactions and branch-specific conservation in Acidithiobacillia. The identification of TFs and DBSs not only corroborates existing experimental information for selected species, but also introduces novel candidates for experimental validation. Moreover, these promising candidates have the potential for further extension to new representatives within the class.