A partner company provided pre-treated BM from spent NMC-based LIBs. The pre-treatment process involved discharging, dismantling, thermal treatment, crushing, and sorting. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the elemental composition of the BM (Table 1), after acid digestion of the BM using aqua regia (according to ÖNORM EN 13657:2002-12). The particle size of the BM sample was determined according to ISO 13320-1, using a HELOS (Sympatec GmbH, Germany) particle size distribution measurement device, resulting in d₁₀ = 4.4 μm, d₅₀ = 17.2 μm, and d₉₀ = 55.0 μm.

A sediment sample was collected (in sterile 100 mL tubes) in early November 2021 a few centimeters below the water surface in a shallow part of extremely acidic (pH ~ 2.6), metal-rich Lake Hromnice in the Czech Republic (49°51′02.5″N, 13°26′39.3″E) (Hrdinka et al., 2013). The indigenous acidophiles were enriched using a selective liquid medium for isolating acidophiles containing basal salts and trace elements, as described previously (Ňancucheo et al., 2016). Three types of selective media were prepared: (i) 50 mM FeSO₄·7H₂O for culturing Fe²⁺ oxidizers at pH 1.7 (Fe medium), (ii) 1% (w/v) elemental sulfur (S⁰) for culturing RISC oxidizers at pH 3.5 (S medium), and (iii) 50 mM FeSO₄·7H₂O and 1% (w/v) S⁰ for culturing Fe²⁺ and RISC oxidizers at pH 2.0 (FeS medium). The media were sterilized using 0.2 μm Nalgene™ Rapid-Flow™ filters (Thermo Fisher Scientific, United States).

Acidophilic chemolithoautotrophs were enriched in Erlenmeyer flasks (100 mL working volume) containing 5 g lake sediment and Fe, S, or FeS medium. The enrichment in Fe medium was carried out for 14 days, and those in S and FeS media for 21 days at 30°C and agitation (150 rpm). After the incubation, the enriched cultures (10 mL) were inoculated into fresh media and further cultivated. The sediment and residual S⁰ were removed from the remaining 90 mL of the enriched cultures by centrifugation at 1500 g for 1 min, followed by harvesting the cells at 3428 g for 15 min. The cell pellets were stored at −80°C until DNA isolation.

During the enrichment process, 1 mL sample was withdrawn from each flask (daily during week one and alternate days from week two onwards). Each time, 1 mL of fresh medium was added to compensate for the volume loss. Samples were centrifuged at 1500 g for 1 min before the measurement of the optical density at 660 nm (OD₆₆₀), followed by the determinations of pH, oxidation–reduction potential (ORP), SO₄²⁻, and Fe/Fe²⁺ concentrations (prior to the two latter measurements, the samples were filtered through a 0.20 μm filter membrane).

The culture enriched in FeS medium was gradually adapted to increasing concentrations of Li⁺, Co⁺², Ni⁺², Mn⁺², and Cu²⁺. Single-metal stock solutions (1 M) were prepared in ultra-pure water using LiCl, CoCl₂, NiCl₂, MnSO₄, or CuSO₄·5H₂O. The adaptation of acidophiles was done in three subsequent stages in FeS medium containing increasing metal concentrations, corresponding to metal contents of 2.5, 5, and 10 g/L of NMC-based BM (Table 2). The adaptation was performed in Erlenmeyer flasks (100 mL working volume) at 30°C and under agitation (150 rpm). After 14 days, 10 mL of the culture was used as inoculum in the next adaptive stage. The ORP, pH, OD₆₆₀, SO₄²⁻, and Fe/Fe²⁺ values were measured as described in Section 2.6. The adapted culture was then used for two-step bioleaching of BM.

In the first step, 10% (v/v) of the adapted and non-adapted cultures were pre-cultivated in a fresh FeS medium (50 mL working volume, pH = 2.0) for 7 days. In the second step, 1% BM (w/v) was added, and bioleaching of metals was performed for another 7 days at 30°C and 150 rpm. In addition, an abiotic control was run in parallel by mixing 1% (w/v) of the BM with 50 mL of sterile FeS medium. Samples were taken before the addition of BM (day zero) and on days 2, 5, and 7 of metal bioleaching. The ORP, pH, OD, SO₄²⁻, and Fe/Fe²⁺ values were monitored as described in Section 2.6. At the end of the experiment, cells and solid particles were removed by centrifugation at 3428 g for 15 min. Furthermore, the supernatant was filtered through a nylon filter of 0.45 μm pore size. The metal concentrations were determined in the filtrates by inductively coupled plasma optical emission spectroscopy (ICP-OES); see below. The dissolved metal concentrations in samples from day zero were subtracted from those determined in samples collected on days onwards. The solid residues were dried at 60°C for 48 h, ground, and analyzed using energy dispersive X-ray spectroscopy (EDS).

All cultivations were performed in a Multitron Pro shaker (Infors HT, Switzerland). A pH electrode LE422 (Mettler Toledo, Switzerland) and ORP electrode InLab Redox (vs. Ag/AgCl; Mettler Toledo, Switzerland) connected to an S22O pH/ion meter (Mettler Toledo, Switzerland) were used to determine pH and ORP, respectively. A DR3900 spectrophotometer (Hach Lange, Austria) was used for OD₆₆₀ measurement.

Fe²⁺/Fe concentrations were measured at 562 nm using 96-well plates and an Infinite 200 Pro M Plex Microplate Reader (Tecan, Switzerland). Fe²⁺ concentration was determined in 228 μL ferrozine solution mixed with 12 μL sample, and total Fe concentration was measured after a 20 min incubation with 45 μL of HONH₂-HCl and 15 μL of NH₄CH₃CO₂ added to the wells. Seven-point calibration was done over a 0–1 mM FeSO₄·7H₂O concentration range.

The SO₄²⁻ concentration was measured using Dionex ICS-900 ion chromatography (Thermo Fisher Scientific, United States). A mixture of 8 mM Na₂CO₃ and 1 mM NaHCO₃ was used as the eluent, and 60 mM H₂SO₄ was used as the regeneration solution. Before analysis, liquid samples were diluted using the eluent and filtered through a 0.2 μm filter into 0.5 mL vials. Nine-point calibration was done over a 1–300 g/L of SO₄²⁻ (in the form of Na₂SO₄) concentration range.

Metal concentrations in leachate samples were determined using 5,110 ICP-OES with an ICP Expert Autosampler (Agilent, United States). 0.5 mL filtered sample through a 0.2 μm filter was mixed with 200 μL 69% (v/v) HNO₃ and incubated at 60°C for 24 h. After cooling to room temperature (RT), the samples were treated in a Sonorex RK 100H ultrasonic bath (Bandelin, Germany) at 60°C for 30 min. After sonification and cooling to RT, the samples were topped up to 10 mL using ultrapure water (resulting in a 20-fold dilution). The concentrations of selected metals (Li, Co, Ni, Mn, Al, and Fe) were measured at eight wavelengths each, and two wavelengths specific for Ar and one wavelength specific for C were used as internal standards. Multi-metal standard solutions (0.5, 1, 2.5, and 5 ppm) were used for calibration, and 2% HNO₃ was used as blank. A t-test (p < 0.05) was performed to assess the differences between leaching efficiency in the adapted culture, non-adapted culture, and control (abiotic) experiments. The metal content in solid residues was examined at the end of the leaching experiments using a TM 3030 scanning electron microscope (SEM) with an EDS detector (Hitachi, Japan).

The recovery rate of each metal was calculated using the following formula:

where C are metal concentrations in leachate (C_L), in assay before BM addition (C₀), and in BM (C_BM).

Total genomic DNA was extracted using the DNeasy UltraClean Microbial Kit (Qiagen, Netherlands), according to the manufacturer’s instructions. The hypervariable region V4 was amplified with unique barcoded oligonucleotides 515F and 806R, as described previously (Spiess et al., 2021). PCR amplification was performed using Platinum II Taq Hot-Start DNA Polymerase (Thermo Fisher Scientific, United States), as follows: initial DNA denaturation step at 94°C for 3 min, 35 cycles of DNA denaturation at 94°C for 45 s, annealing at 52°C for 60 s with a 50% thermal ramp, and extension at 72°C for 90 s, and a final extension step at 72°C for 10 min. The PCR products were purified using AMPure XP beads (Beckman Coulter, United States) following the manufacturer’s instructions. The Qubit 4.0 fluorometer (Thermo Fisher Scientific, United States) and FragmentAnalyzer (Advanced Analytical Technologies, United States) were then used to determine the library quantity and quality. The library was sequenced using a MiniSeq System (Illumina, United States) with a MiniSeq Mid Output Kit (300 cycles). The raw fastq reads were processed in R software (4.3.1) using the open-source package DADA2 (1.28.0) as described previously (Spiess et al., 2022). A summary of all amplicon sequence variants (ASVs) is shown in Supplementary Table S1. The dataset generated and analyzed in this work is available in the NCBI Sequence Read Archive under BioProject ID: PRJNA1045576.

The highest microbial diversity was observed in the enriched culture in Fe medium (Figure 1A), with the dominant genera being Acidithiobacillus (38%), out of which A. thiooxidans and A. ferrooxidans accounted for 33 and 5%, respectively, and Leptospirillum (32%), out of which L. ferrooxidans accounted for the majority. In addition, other genera such as Ferrithrix (8%), Sulfobacillus (8%), and Acidiphilium (4%) were identified with lower relative abundance (Figure 1B). The enriched culture in S medium was found to be dominated by A. thiooxidans (66%) and Alicyclobacillus (15%), with the majority of the latter being Alicyclobacillus (Acb.) disulfidooxidans. In addition, Acinetobacter (4%), Chryseobacterium (2%), and Staphylococcus (2%) were identified (Figure 1B). The lowest microbial diversity was observed in the enriched culture in FeS medium (Figure 1A), which was highly dominated by A. thiooxidans (95%), followed by Alicyclobacillus (3%), with Acb. disulfidooxidans constituting the majority of the genus abundance (Figure 1B). Mainly, acidophilic Fe²⁺- and RISC-oxidizing chemoautolithotrophs were detected in the enriched cultures, which was consistent with the selective media being of low pH and containing only inorganic electron donors and no organic C source. The extreme acidophile A. thiooxidans can utilize RISCs such as S⁰, thiosulfate, and tetrathionate as sole electron donors but cannot oxidize Fe²⁺. L. ferrooxidans can utilize only Fe²⁺ as an electron donor (Hippe, 2000). On the other hand, A. ferrooxidans and Acb. disulfidooxidans (formerly Sb. thermotolerans) can oxidize both RISCs and Fe²⁺ (Kelly and Wood, 2000; Karavaiko et al., 2005).

The cell growth was most pronounced in the FeS medium (Figure 2A), while pH decreased drastically after day 7 in both S and FeS media, resulting in final pH values of 0.47 and 0.33, respectively (Figure 2B). Correspondingly, the SO₄²⁻ concentration increased in S and FeS media after day 7 (Figure 2C). The above substantial decrease in pH and increase in SO₄² resulted from S⁰ oxidation catalyzed by RISC oxidizers such as A. thiooxidans and Acb. disulfidooxidans (Dopson and Johnson, 2012). A. thiooxidans and Acb. disulfidooxidans have both been detected in industrial bioleaching heaps processing copper sulfides, with a stable abundance of A. thiooxidans throughout different phases of the leaching process (Remonsellez et al., 2009). Thus, these enrichments provided promising species for the following adaptive stages prior to bioleaching. The Fe²⁺ was fully oxidized after day 10 in Fe and FeS media (Figure 2D), which resulted in ORP exceeding +600 mV (Figure 2E), in agreement with Hansford and Vargas (2001). A. ferrooxidans and L. ferrooxidans were mainly responsible for Fe²⁺ oxidation in Fe medium, while Acb. disulfidooxidans oxidized Fe²⁺ in FeS medium. As previously reported (Ishigaki et al., 2005), the metal recovery rate in fly ash bioleaching can be significantly improved by using mixed cultures of Fe²⁺ and RISC oxidizers as opposed to pure cultures. Similarly, the recovery rates of Zn, Co, Cu, and Mn from incineration residues increased from 50% achieved with pure cultures to nearly 100% when a mixed culture of RISC and Fe²⁺ oxidizers was used (Kremser et al., 2021). The Fe present in the sediment was partly solubilized after day 10 in FeS medium which is depicted by the total Fe concentration increasing above 50 mM (Figure 2F). In the present study, the enriched culture in FeS medium exhibited the highest growth and oxidation rates among the three enrichments tested and was thus chosen for further metal adaptation and two-step BM bioleaching. Moreover, this culture showed the lowest microbial diversity, thereby providing species stability during the successive processes.

Contact bioleaching of LIBs can be challenging due to the potential inhibition of acidophiles caused by the acid-consuming nature of LIBs and high dissolved metal concentrations (Roy et al., 2021b). Mixed cultures of Fe²⁺ and RISC oxidizers have been shown to be more resilient than pure cultures (Qiu et al., 2005; Akcil et al., 2007). The metal tolerance of the enriched culture in FeS medium was improved via three subsequent adaptive stages (Figure 3). As shown in Figure 3A, increasing metal concentrations did not hamper the cell growth. On the contrary, the OD₆₆₀ values were higher during all three adaptative stages (until day 12) than those in the non-adapted cultures. The decrease in pH (Figure 3B) and increase in SO₄²⁻ concentration (Figure 3C) confirmed the activity of RISC-oxidizing microorganisms during the adaptive stages. After 2 weeks, all cultures oxidized Fe²⁺ to Fe³⁺, but the culture in the first adaptive stage completely oxidized Fe²⁺ after 1 week, while the elevated metal concentrations in the second and third adaptive stages reduced the Fe²⁺ oxidation rate compared to the first stage but not to the non-adapted culture (Figure 3D). A similar trend was also observed in the ORP values (Figure 3E).

It has been previously shown that contact bioleaching of high loads of LIBs can lower metal recovery rates (Niu et al., 2014). Therefore, two-step bioleaching of metals from 1% (w/v) pulp density BM was investigated in the present study, using the non-adapted and adapted enriched cultures in FeS media described above (Figure 3). As shown in Figure 4A, the adapted cultures showed higher OD₆₆₀ values than those of the non-adapted cultures, indicating a positive effect of the adaptation on the cell growth. In most studies investigating bioleaching of LIBs, pH adjustments were required due to the acid consumption by the alkaline source material. Heydarian et al. (2018) reported that adapted mixed cultures did not grow above 4% (w/v) pulp density until the pH was lowered. However, pH remained below 1.5 in both non-adapted and adapted cultures throughout the whole leaching experiment in this study (Figure 4B), which enhanced the metal release and prevented potential Fe precipitation [Fe³⁺ typically precipitates at pH > 2 (Nurmi et al., 2010)]. The pH in the abiotic control increased to pH > 3 after 2 days (Figure 4B), which was attributed to the alkaline nature of BM (Wood et al., 2020). A gradual adaptation (from 1 to 5% chalcopyrite) improved the resistance of A. ferrooxidans to Cu during bioleaching of the mineral, resulting in a shorter lag phase in an adapted culture compared to that in a non-adapted one (Xia et al., 2008). In this study, the adapted cultures reached higher OD₆₆₀ values (Figure 4A) and SO₄²⁻ concentration (Figure 4C) compared to the non-adapted cultures, indicating that the adaptation to elevated metal concentrations improved the growth and oxidation rates during BM bioleaching. Almost all Fe²⁺ was oxidized to Fe³⁺ at the end of pre-cultivation (day 0), and this was partially reduced back to Fe²⁺ during the BM bioleaching phase on days 2–7 in biotic experiments (Figure 4D). The ORP was around +400 mV in both non-adapted and adapted cultures (Figure 4E), correlating with Fe speciation (Figures 4D,F). In addition, the pH increase resulted in precipitation of Fe, indicated by the decrease in total Fe concentration in solution (Figure 4F). In contrast, no Fe precipitation occurred when bacterial cultures were used; the bacteria maintained a low pH of around 1 and total Fe around 50 mM, further supporting Fe²⁺/Fe³⁺ cycling.

Metal analysis of leachates collected on days 2, 5, and 7 from the (bio)leaching experiments showed that the maximum concentrations of dissolved Li, Co, Ni, Mn, and Al were obtained on day two and did not increase further (Figure 5). During these 2 days, all Fe³⁺ was reduced in non-adapted and adapted cultures (Figure 4D). This indicates that mainly biogenic lixiviants (containing H₂SO₄ and Fe³⁺) produced by the microorganisms during pre-cultivation were involved in the metal leaching. Complete oxidation of Fe²⁺ in abiotic FeS medium was achieved after 7 days (Figure 4D), indicating that chemical oxidation of Fe²⁺ by metal oxides in BM is relatively slow in acidic environments. On the other hand, the Fe²⁺ concentration in the enriched cultures decreased slightly from day 2 to 7, which may imply that besides Fe²⁺ oxidation, either chemically or by Alicyclobacillus, aerobic Fe³⁺ reduction also occurred in the presence of A. thiooxidans. In this case, Fe³⁺ was probably reduced by the RISC intermediates produced during bacterial S⁰ oxidation (Breuker and Schippers, 2023). Thus, iron cycling during BM bioleaching appears to be influenced by both abiotic and biotic reactions.

Significantly higher concentrations of all monitored metals (except Cu) were detected in adapted cultures on day 2 compared to those in non-adapted cultures and abiotic control. Additionally, solid residues were analyzed using SEM/EDS. Figure 6 shows that almost all Co, Ni, Mn, and Al were leached from the BM in non-adapted and adapted cultures. Abiotic control was slightly less efficient in extracting Co and Mn. The high S signals in both non-adapted and adapted cultures can be attributed to the residual electron donor added to the medium (Figure 6). The difference between the two intensities indicates that in the presence of BM, the adapted cultures oxidized S⁰ more effectively than the non-adapted cultures, which is also evident from SO₄²⁻ concentrations (Figure 4C). Similarly, Hosseini et al. (2022) reported enhanced SO₄²⁻ production and Sr. and Ce recovery during bioleaching of gold mine tailings when A. thiooxidans adapted to 4% (w/v) tailings pulp density was used. The Fe precipitation in the abiotic assay due to the increase in pH and the absence of Fe²⁺ and RISC oxidizers was confirmed by the high Fe signal in the solid residue (Figure 6).

The Li leaching is thought to be primarily due to dissolution in H₂SO₄, while the dissolution of other metals such as Co and Ni occurs by a combination of acidolysis and redoxolysis via Fe²⁺/Fe³⁺ cycling (see Eq. 1) (Xin et al., 2009).

Co and Ni are predominantly present in LIBs in the oxidation state +3, which are less soluble than their reduced state +2. Wu et al. (2019) showed that the presence of pyrite and Fe²⁺ enhanced the efficiency of Co and Ni bioleaching, with Fe²⁺ reducing Co³⁺ and Ni³⁺ to Co²⁺ and Ni²⁺. Moreover, the presence of Ag⁺ promoted the dissolution of Co from LiCoO₂ by bioleaching with A. ferrooxidans via formation of AgCoO as an intermediate (Zeng et al., 2013). Zeng et al. (2012) reported an increase in Co extraction from 43.1 to 99.9% during bioleaching in the presence of Cu²⁺ as a catalyst (Eqs 2, 3).

(3) CuCo 2 O 4 + 6 F e 3 + → 6 F e 2 + + C u 2 + + 2 O 2 + 2 C o 2 +

In this study, it is presumed that Li was leached by H₂SO₄ generated by S⁰ oxidation using the enriched culture dominated by A. thiooxidans and Acb. disulfidooxidans, while Co, Ni, and Mn were likely solubilized by the combination of acid dissolution and reduction by Fe²⁺. Still, at low pH, chemical oxidation of Fe²⁺ to Fe³⁺ competes with bacterial oxidation (in this study by Alicyclobacillus). It appears that Fe³⁺ participates in the dissolution of the material along with H₂SO₄, and the Fe²⁺ produced may participate in the reduction of other metals or serve as an electron donor for bacterial oxidation. The resulting re-oxidized Fe³⁺ might dissolve the material further or can be reduced by RISC intermediates during bacterial S⁰ oxidation. Lower pH (< 1.5) and higher ORP (> 400 mV) presented favorable conditions for the leaching of metals such as Co, which is in agreement with a study by Li et al. (2013b) who reported that the dissolution of Co from LiCoO₂ by A. ferrooxidans was highly dependent on ORP with best results obtained at pH 1.5 and ORP > 400 mV measured using Ag/AgCl as reference electrode (which is similar to the electrode used in this study). Furthermore, BM contains a high proportion of Cu, which can dissolve and act as a catalyst via the formation of intermediates such as CuCo₂O₄, which further promotes the dissolution of Co/Ni/Mn.