The mobilization of metal ions from various insoluble mineral phases has been widely used in biotechnological processes to recover useful metals (White et al., 1998; Donati and Sand, 2007; Khaing et al., 2019). However, the potential of metalliferous steel waste as a circular provider of precious metals for developing technologies has yet to be uncovered. The European Steel Association (EUROFER) states that approximately 278 million tons of steel have been produced in 2020 in Europe (15.2% of worldwide steel production), while generating millions of tons of various waste products (EUROFER, 2020). Many types of hazardous wastes, such as slags, dust, and ash, create space shortages due to their necessary deposition in landfills, contributing to the release of hazardous metalliferous wastes into our environment while leading to a depletion of valuable raw materials (Singh et al., 2013; World Steel Association, 2016).

For this study, a secondary dust waste product of the basic oxygen furnace (BOF) steelmaking process (hereafter referred to as BOF-dust) (Gara and Schrimpf, 1998; voestalpine Stahl Donawitz GmbH, Leoben, Austria) is investigated for microbial metal recovery potential by six thermoacidophilic archaeal strains. The potential of growth, metal solubilization, and the interaction of thermoacidophilic archaea with secondary dust particles was investigated to assess future implementation in high-temperature bioleaching operations. Steel waste products pose a rich resource of metal oxides that need to be recycled or reintroduced into the manufacturing system by natural microbial processes (Pawlowski et al., 1984; Krebs et al., 1997; Barbuta et al., 2015; Waste Disposal Recycling in Steel Industry, 2019). Biomining and bioleaching are well-established operations that are applied in a variety of mineral mining-associated industries and laboratory-scale operations (Bosecker, 1997; Schippers et al., 2013; Jujun et al., 2015; Banerjee et al., 2017; Blazevic et al., 2019). Additionally, microorganisms have been applied to detoxify metal-contaminated compounds derived from industrial processes such as incineration bottom ash, fly ash, slags from smelting processes, heavy metal waste from tanning factories (Batool, 2012; Igiri et al., 2018; Mäkinen et al., 2019; Srichandan et al., 2019), electronic scrap (Brandl et al., 2001), radioactive waste environments (Brim et al., 2003; Manobala et al., 2019), and many more through bioleaching.

Bioleaching is a process based on the ability of microorganisms (archaea, bacteria, fungi, etc.) to convert insoluble metals into a soluble state (Schippers et al., 2013). Usually, this process is performed by acidophilic microorganisms that are genetically equipped to oxidize Fe²⁺ into Fe³⁺ and/or reduced inorganic sulfur compounds (RISCs) at a low pH (acidophilia), preferably by growing in the unity of biofilms and frequently attaching to the mineral material, forming a reaction space (Valdés et al., 2008; Ma et al., 2017; Zhang et al., 2019). Bioleaching at elevated temperatures exploits the potential of thermoacidophilic archaea (~70–80°C), whereas mesophilic bioleaching is performed by bacteria at lower temperatures (~25–40°C) (Norris, 1997). Bioleaching operations executed by thermoacidophilic archaea (e.g., Sulfolobus spp., Metallosphaera spp., and Acidianus spp.) benefit from a more efficient mineral dissolution compared to mesophilic bacterial bioleaching (Marsh and Norris, 1983; Gericke and Pinches, 1999; Wang et al., 2014).

Since bioleaching processes of a large ore mass can generate a significant amount of exothermic heat (Beck, 1967), thermophiles such as Acidianus brierleyi proved themselves inherently suitable for high-temperature operations. A. brierleyi cells propagate chemolithotrophically by S⁰ or Fe²⁺ redox chemistry (Segerer et al., 1986) and provide a higher sphalerite (CuFeS₂) leaching rate compared to a mesophilic bacterium Acidithiobacillus ferrooxidans culture (Konishi et al., 1998; Saitoh et al., 2017). Furthermore, Sulfolobales representatives, such as Sulfolobus metallicus (Huber and Stetter, 1991) and Sulfolobus acidocaldarius (Brock et al., 1972), mobilize copper, zinc, and uranium (Gautier et al., 2007), provide desulfurization strategies for coal (Shivvers and Brock, 1973; Kargi and Robinson, 1985; Tobita et al., 1994), and oxidize arsenite to arsenate in culture (Sehlin and Lindström, 1992). Great potential in industrial heap bioleaching is demonstrated by Metallosphaera hakonensis (Takayanagi et al., 1996) due to high Fe²⁺ oxidation rates when grown on inorganic sulfur compounds (Bromfield et al., 2011; Shiers et al., 2013). Closely related thermoacidophilic Metallosphaera sedula utilizes various metal-containing ores as well as organic carbon sources to feed its respiratory electron transport chain (Huber et al., 1989; Auernik and Kelly, 2010a,b). Besides inherent (chalco-)pyrite bioleaching abilities (Mikkelsen et al., 2007; Ai et al., 2019), M. sedula can oxidize solid triuranium octaoxide (U₃O₈) to U(VI) while feeding own bioenergetic needs (Mukherjee et al., 2012). Furthermore, M. sedula cells are able to biotransform the calcium-tungstate mineral scheelite by leaching mineral-bound tungsten into the surrounding, resulting in tungsten carbide layers on the cell surface (Blazevic et al., 2019). Precious metals from synthetic (Kölbl et al., 2017), as well as genuine extraterrestrial materials such as meteorites (ordinary chondrite NWA 1172; Martian breccia NWA 7034) are also solubilized by M. sedula, which creates possibilities for future asteroid biomining (Milojevic et al., 2019b, 2021). A. manzaensis (Yoshida et al., 2006) is an emerging thermoacidophilic bioleacher, which grows as a facultative anaerobe utilizing molecular hydrogen and elemental sulfur as electron donor under aerobic conditions by using Fe³⁺ as the electron acceptor for anaerobic growth. So far, A. manzaensis has merely been investigated for its chalcopyrite leaching efficiency as well as a possible acid mine drainage (AMD) bioremediation agent (Chang-Li et al., 2012; Liu et al., 2016a,b; Nie et al., 2019; Li et al., 2020). Obviously, current and future challenges in circular economy and waste management demand in-depth analyses of microorganisms with great inherent bioleaching potential.

Recent advances in these technologies (e.g., Coetzee et al., 2020; Giachino et al., 2021; Newsome and Falagán, 2021) may contribute to a great extent to the United Nations Sustainable Development Goals (specifically goal number 12), namely, to achieve sustainable and efficient use of natural resources as well as to implement an environmentally sound management of chemicals and all wastes throughout their life cycle by 2030 (United Nations General Assembly, 2015).

In this study, the growth potential of six thermophilic archaeal strains (S. metallicus, S. acidocaldarius, M. hakonensis, M. sedula, A. brierleyi, and A. manzaensis), their ability to propagate in presence of a multimetallic waste product (BOF-dust), as well as the subsequent solubilization of metals bound to the respective BOF-dust grains is investigated. Furthermore, we focus on the bioleaching potential of A. manzaensis, subjecting cells to different leaching parameters (BOF-dust load, pH, and temperature), while investigating the microbial interactions with dust particles via scanning and transmission electron microscopy (SEM and TEM) coupled to energy-dispersive spectroscopy (EDS) analysis. In our study, we observe efficient metal solubilization from dust particles by A. manzaensis and explore microbial-mineral interactions associated with this case. Our results provide the first insights into laboratory-scale thermoacidophilic bioleaching associated with industrial steelmaking waste products. Integrating not well-studied bioleaching microorganisms into future circular waste management processes might open up a plethora of new applications for environmental biotechnology.

All six thermoacidophilic archaeal strains cultivated for this study were able to grow chemolithotrophically on a high metal-content BOF-dust that is generated during the BOF steel production processes. Our cultivation parameters (strain-specific medium, temperature, acidity) were selected according to commonly applied cultivation parameters for each strain (refer to the “Materials and methods” section). All of the cultivated strains were able to proliferate successfully in course of 17 days. Inherent resistance to a (heavy) metalliferous environment was one of the criteria for strain selection. Previous extensive work with M. sedula in mineral- and metal-containing environments made this archaeon a prime candidate (Auernik and Kelly, 2010b; Blazevic et al., 2019; Milojevic et al., 2019b, 2021), along with its close relative M. hakonensis. Interestingly, a comparison of metal solubilization capacities of M. hakonensis and M. sedula grown on BOF-dust-supplemented medium favors M. sedula, which was able to extract Cr and Pb from BOF-dust grains (Figure 2). Silica content in BOF-dust (Supplementary Table S1) may be solubilized chemically by the acidic pH of the surrounding medium and is subsequently precipitated onto M. hakonensis cells during bioleaching operation (Usher et al., 2009). Under these circumstances, a fusion barrier can be created that consequently slows down the release of metals from dust grains (Usher et al., 2009). This phenomenon has already been observed for M. hakonensis in heap bioleaching operations and might be applicable to other thermoacidophilic strains (Dopson et al., 2008). Both Metallosphaera strains solubilize metals by attaching to their respective substrates as well as by regenerating Fe²⁺ to Fe³⁺ that is vital for chemical attack on minerals (Bromfield et al., 2011; Kölbl et al., 2017). While the growth of S. metallicus is inhibited by a high amount of surrounding Fe²⁺ ions (Shiers et al., 2013), proliferation on a high Fe³⁺ content (as in BOF-dust) does not seem to have significant impact on cellular propagation since S. metallicus cells leach metals by attaching to a mineral surface as well as by providing Fe³⁺ ions as a lixiviant (Gautier et al., 2008). Whereas S. metallicus grows in an obligate chemolithoautotrophic mode, its relative S. acidocaldarius proliferates heterotrophically (Wheaton et al., 2015). This might explain the faster generation time of S. acidocaldarius in course of cultivation. In this study, S. acidocaldarius outperformed S. metallicus in the growth and bioleaching of most elements except for Fe. Contradictory experimental data on lifestyle and substrate utilization (autotrophy vs. heterotrophy; oxidation of S⁰) of S. acidocaldarius (Dworkin et al., 2006) need to be clarified by dedicated experiments in order to determine bioleaching mechanism(s). A. manzaensis is a facultative autotroph capable of utilizing Fe³⁺ as electron acceptor under anaerobic circumstances in contrast to the closely related A. brierleyi species (Yoshida et al., 2006). However, no data exist on how a high concentration of surrounding Fe³⁺ ions can influence the growth rate of A. manzaensis. While Fe³⁺ ions are a vital oxidizing agent in autotrophic bioleaching processes, the generation of protons by acidophilic microorganisms further supports bioleaching and keeps Fe³⁺ ions in solution (Donati and Sand, 2007). Both heterotrophs (e.g., fungi) and autotrophs (e.g., thermoacidophilic iron-oxidizing archaea) are capable of solubilizing metals from ores and minerals using different strategies and bioleaching agents (Schippers et al., 2013). In this study, all cultivated archaeal strains are chemolithotrophs (except S. acidocaldarius) that use a variety of inorganic energy sources, which are in turn closely intertwined with their bioleaching abilities (Donati and Sand, 2007). Chemolithotrophic production of sulfuric acid (protons) by sulfur-oxidizing thermoacidophilic archaea (Metallosphaera spp., Acidianus spp., S. metallicus) might additionally support efficient metal solubilization from BOF-dust grains by maintaining a low pH during bioleaching operations.

All six thermoacidophilic archaea were able to efficiently solubilize various metals from BOF-dust grains due to their inherent bioleaching capacities. Several enzymes involved in bioleaching processes have been identified among the Sulfolobales order. A. manzaensis genome encodes several enzymes involved in sulfur and RISCs oxidation, including a sulfur oxygenase-reductase, thiosulfate:quinone oxidoreductase and a tetrathionate hydrolase (Ma et al., 2017). So far, no genes for iron redox chemistry in A. manzaensis have been annotated, which makes future biochemical characterization on genes responsible for bioleaching capacity necessary. The fox gene cluster responsible for iron oxidation was identified in S. metallicus (Bathe and Norris, 2007) and M. sedula, as well as the genetic presence of rusticyanin homologs (Rus) for iron oxidation and tetrathionate hydrolase (TetH) for sulfur oxidation, appear to distinguish microbes able to perform bioleaching from non-bioleaching microbes (Auernik et al., 2008). Bioleaching (and/or biomining) capacities of M. sedula, M. hakonensis, A. brierleyi, S. metallicus, and S. acidocaldarius have been examined extensively compared to A. manzaensis. Comparative ICP-MS analysis of all thermoacidophiles shows superiority of A. manzaensis in leaching of certain strategically important elements from BOF-dust, such as Cu, Ni, Zn, Cr, Cd, and Mn (Figure 2). Future experiments to determine iron redox (bio)chemistry in A. manzaensis need to be carried out to fully understand its bioleaching potential.

Refinement of the current BOF-bioleaching operation included BOF-dust load, pH, and slight temperature shifts. For most elements (Fe, Zn, Mn, Al, Cu, Ni, Cr, Cd, Co, V, Sr, Ba, Mg), a higher dust load (Figure 3A) equals higher amounts leached into solution. In contrast, elements such as Ti, Pb, Mo, and Al might have formed insoluble precipitates under higher dust load conditions since they seem to be underrepresented in the analyzed bioleachate solution (Figure 3A). Furthermore, a decrease of operation pH from 2.5 to 1.5 increased Cr, Fe, and Ni bioleaching significantly compared to a less acidic pH (Figure 3B). On the contrary, Zn, Mn, Co, Cd, and Cu bioleaching was enhanced at higher pH (2.5). Similar results for A. manzaensis copper bioleaching at different pH-values have been shown by previous experiments, where Cu dissolution increased with a less acidic pH, whereas Fe concentration dropped by pH increase which is in concordance with own measurements (Liang et al., 2014; Liu et al., 2016a). In classic metal sulfide bioleaching, Fe precipitation is usually accounted to the formation of a passivating layer (usually by jarosite), which slows down overall mineral dissolution (Klauber, 2008). Excessive implementation of mesophilic microorganisms such as Acidithiobacillus spp. and Leptospirillum spp. in commercial bioleaching operations of metal sulfides (pyrite, chalcopyrite) shows that biological leaching processes decrease with time and eventually cease due to the formation of a polysulfide (Cu₄Fe₂S₉) passivation layer on the mineral surface (Hackl et al., 1995; Zhao et al., 2019). Thermoacidophilic archaea such as Acidianus spp., Sulfolobus spp., and Metallosphaera spp. are able to significantly accelerate mineral sulfide dissolution, cope with high overall acidity and temperatures (Clark and Norris, 1996; Gericke and Pinches, 1999) and can promote a more efficient dissolution of BOF-dust grains by solubilizing metals into the leachate solution (Figure 2). The formation of unwanted or inhibiting bioleaching by-products formed by these processes needs to be investigated in further studies in order to enhance industrial implementation. Slight changes in bioleaching operation temperature (62°C/64°C/67°C) did not reveal any significant solubilization events; for future investigation, further temperature shifts should be examined in depth.

A. manzaensis was the most efficient strain among all other tested Sulfolobales spp. capable of chemolithotrophic growth on BOF-dust. A. manzaensis broke down the chemical matrix of BOF-dust grains and actively solubilized various metals into the leachate solution. Our SEM observations show the successful colonization of metal-bearing waste particles by A. manzaensis and their subsequent biotransformation (e.g., etched grains surface, Figures 4C–H, Figure 5, Supplementary Figure S3). This is in concordance with previous findings of cooperative contact/non-contact bioleaching mechanisms of A. manzaensis (Zhang et al., 2019). Studies of chalcopyrite bioleaching with A. manzaensis cells also indicate corrosion and break down of the mineral matrix with simultaneous copper solubilization after several days of cultivation (He et al., 2009; Liu et al., 2016b). Furthermore, the formation of a jarosite (KFe₃(SO₄)₂(OH)₆) passivation layer by A. manzaensis was reported from chalcopyrite bioleaching operations (Nie et al., 2019), indicating the formation of by-products during leaching processes. In our study, we observed gypsum crystals (CaSO₄ × 2H₂O) after approximately 7 days of cultivation (Figure 8), which might be accounted for both microbial and chemical-induced processes during cultivation. A high calcium content in the BOF-dust (Supplementary Table S1), as well as the involvement of sulfur derived from the same product, can account for abiotic calcium sulfate formation once these elements are intensively released by A. manzaensis into the leachate solution. Similar to calcium sulfate, nickel and manganese sulfate formation in the leachate solution has been previously reported for M. sedula cultivated on multimetallic extraterrestrial material (Milojevic et al., 2019b). In this case, associated with the potential for asteroid biomining operations, nickel sulfate was proposed as a metabolic biosignature resulting from the microbial leaching activity of meteorite minerals (Milojevic et al., 2019b). In light of terrestrial industrial operations, gypsum is a naturally occurring mineral and is used in various industrial settings. Usually, gypsum is formed as a by-product of sulfide oxidation to produce sulfuric acid and is used excessively in the construction industry (Lutz et al., 2012). In contrast, a study conducted by Vogel et al. shows that biofilms of cyanobacteria and Bacteroidetes species are actively involved in gypsum sedimentation implicating biological involvement in its formation (Vogel et al., 2009). Despite the possibility of biological involvement, a low pH favors the growth of gypsum crystals in inorganic systems (Liu et al., 2015). Furthermore, substrates rich in Fe, Ca, Mg, or Ba combined with continuous CO₂ input in the bioreactor might precipitate as a stable red gypsum mineral through CO₂ mineral carbonation (Rahmani et al., 2016; Rahmani, 2018, 2020). As the origin of gypsum crystals in this study is not resolved, more studies on its influence on bioleaching efficiency and its possible application as a useful by-product need to be executed.

First insights into laboratory-scale thermoacidophilic bioleaching associated with industrial steelmaking waste products are provided in this study. Further genetic analysis and biochemical characterization of metal-oxidizing molecular machinery of A. manzaensis need to be carried out to optimize this strain for future operations. A thorough screening of operation parameters in frames of scale-up processes is highly necessary to implement this technology for upcoming circular processes in order to efficiently recycle/re-feed valuable metals into industrial systems. In addition to microbial analysis on a nanometer-scale, metalliferous waste products need to be sufficiently characterized in order to assess bioleaching efficiency of each tested microorganism.

Six thermoacidophilic archaeal strains (M. sedula, M. hakonensis, S. acidocaldarius, S. metallicus, A. brierleyi, and A. manzaensis) were capable of growing when supplemented with multimetallic BOF-dust derived from a steel production plant. Furthermore, each species was able to leach a set of elements from BOF-dust grains; however, A. manzaensis showed the highest potential in growth and bioleaching of economically relevant elements. Electron microscopy-assisted analysis of BOF-dust grains processed by A. manzaensis reveals a breakdown of the dust-grains mineral matrix in course of cultivation. Additionally, the formation of gypsum crystals was observed during the cultivation process. The obtained results emphasize the utilization of thermoacidophilic archaea for future (industrial) bioleaching operations and the need to conduct more in-depth studies to optimize these processes. So far, bioleaching mechanisms have been described extensively for a handful of acidophilic bacteria (overview by Donati and Sand, 2007). To exploit the full potential of thermoacidophilic archaea, more in-depth studies need to be carried out for the future efficient bioleaching operations and for the development of environmental-friendly circular economies. Further investigations should focus on resolving the microbial-mineral interfaces of the selected thermoacidophiles grown on waste product materials down to nanometer scale. This will help understand the mechanisms of microbial interactions with metal-bearing waste product material and see the relevant cell compartments involved in the bioleaching process.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

DK, AM, MA, MB, HS, and TM performed experiments. DK, AM, and TM planned and executed the cultivation of thermoacidophilic archaea, as well as performance and interpretation, of SEM-EDS experiments. MB performed ICP-MS analyses. MA and TM performed and analyzed STEM-EDS results. All authors made substantial contributions to the acquisition, analysis, and interpretation of the data described in this study. All authors critically reviewed the manuscript and approved the final version of it.

GK, HS, and DW were employed by the company voestalpine Stahl Donawitz GmbH, Leoben, Austria.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.