Influence of Native Leptospirillum ferriphilum Strains on Ferric Iron and Leached Copper Recovery from Chalcopyrite to Mesophilic Temperature Under Laboratory Conditions

Abstract

Bioleaching represents a sustainable and economically viable method for recovering metals from sulfide ores. This study evaluates the bioleaching potential of eight native mesophilic strains isolated from chalcopyrite ore sourced from the Toquepala mine in Peru. The strains were molecularly and phylogenetically identified as Leptospirillum ferriphilum based on 16S rRNA gene sequencing. Bioleaching process was evaluated over 648 h in triplicate flask assays at 30 °C and 120 rpm. All strains demonstrated leaching activity, achieving an average copper recovery above 30%. Notably, strains M1D and M3E demonstrated superior performance, with iron oxidation reaching approximately 50%, copper recoveries of 34.78% and 32.61%, respectively, and peak cell counts of up to 2.45 × 10⁹ cells/mL. Specific growth rates were determined as 0.03469 h⁻¹ for M1D and 0.03651 h⁻¹ for M3E. A positive correlation was observed among microbial growth, iron oxidation, and copper recovery. These results confirm the potential of native mesophilic L. ferriphilum strains as efficient agents for chalcopyrite bioleaching at moderate temperatures, supporting their application in biotechnological metal recovery processes.

1. Introduction

Chalcopyrite (CuFeS₂) is the most abundant copper-bearing mineral; its low cost is fundamental to copper production profitability. Current research focuses on optimizing its bioleaching process [1], which primarily relies on microorganisms that oxidize sulfide minerals in both mesophilic and thermophilic ranges [2]. Genomic, metagenomic, and transcriptomic studies have provided key insights into the microbial communities and functional genes responsible for iron and sulfur oxidation under the extremely acidic conditions characteristic of mining environments [3]. Leptospirillum ferriphilum is a key bacterium in copper bioleaching, thriving under moderately thermophilic (37–40 °C) and extremely acidic (pH < 1.5) conditions. Its bioleaching efficiency derives from its high iron-oxidizing capacity during chalcopyrite dissolution. This Gram-negative bacterium measures 0.3–0.5 µm in width and 0.9–4.0 µm in length, with a generation time of 12–15 h [4,5,6,7]. Furthermore, the production of extracellular polymeric substances (EPS) confers it resistance to elevated concentrations of heavy metals, such as Ni, Co, Cd, Cu, and Zn. Genomic analyses have further identified genes involved in metal resistance, nitrogen fixation, and CO₂ assimilation, granting L. ferriphilum ecological advantages over other bioleaching microorganisms [8,9].

Beyond this role, L. ferriphilum also promotes the proliferation of other acidophilic microorganisms such as Acidiphilium spp. and Ferroplasma acidiphilum, thereby enhancing community resilience. These synergistic associations improve system stability and operational efficiency by accelerating the ferric iron cycle, degrading inhibitory organic compounds, and maintaining functionality under extremely acidic and metal-rich conditions [10,11]. Moreover, recent findings reveal that interspecies quorum sensing mechanisms mediate microbial communication, regulating biofilm formation and the expression of genes involved in iron oxidation and heavy-metal tolerance [12].

The ecological and industrial relevance of L. ferriphilum is emphasized by its strong osmotic tolerance, which allows it to withstand the high sulfate concentrations of chalcopyrite ores [10]. In contrast, other mesophilic acidophiles like Acidithiobacillus ferrooxidans (optimal at ~30 °C and pH ~2.0) exhibit lower tolerance to elevated metal ion concentrations, which can constrain their leaching performance under extreme conditions [1,2]. By comparison, Sulfobacillus spp. grows optimally at higher temperatures (45 °C), which requires increased energy input for heating [1]. Comparatively, L. ferriphilum thrives under moderately thermophilic (37–40 °C) and extreme acidic (pH < 1.5) conditions, eliminating the need for external heating, and thereby reducing energy consumption and the carbon footprint of industrial bioleaching operations [7,9]. Evidence from experimental, kinetic, and genomic studies confirms that L. ferriphilum withstands extreme acidity and high-sulfate concentrations while efficiently oxidizing Fe²⁺ under these conditions [6,7,9,13]. As evidenced by a 2023 study by Vardanyan et al., L. ferriphilum oxidized 91.4% of Fe (II) in 17 days, achieving 44.1% copper extraction from chalcopyrite [4].

The sequential inoculation of L. ferriphilum, Acidithiobacillus caldus, and Acidiphilium sp. improved the Fe²⁺/Fe³⁺ redox cycle and copper dissolution efficiency during chalcocite bioleaching. L. ferriphilum and A. caldus enhanced iron oxidation and microbial synergy, while Acidiphilium sp. maintained redox stability through Fe³⁺ reduction. The integration of cellulosic residues further stimulated microbial activity, resulting in superior bioleaching performance [10].

These attributes position L. ferriphilum as a prime candidate for sustainable industrial bioleaching, outperforming other widely used thermoacidophilic species and facilitating enhanced metal recovery with minimal environmental impact [3,4,5,8,9]. Subsequently, this study aims to assess the growth kinetics of native L. ferriphilum strains at 30 °C to determinate their viability for initiating chalcopyrite bioleaching under lower mesophilic conditions. We hypothesize that these strains can effectively bioleach chalcopyrite under the tested conditions.

2. Materials and Methods

2.1. Isolation of Bioleaching Bacterial Strains

Bacterial strains were isolated from chalcopyrite ore samples collected from a mineral pile at the Toquepala Copper Leaching plant (LESDE zone: Leaching, Solvent Extraction, and Electrowinning), operated by Southern Copper Corporation in Tacna, Peru (2982 m elevation) [14]. Samples of active leachable material were collected from a depth of 20 cm, transported to the laboratory, and crushed to obtain a uniform particle size. The crushed ore was used to inoculate a modified version of the 9K liquid medium originally formulated by Amaro et al. [15]. The medium was prepared from three separate solutions. Solution A contained MgSO₄·7H₂O (29.21 mM) dissolved in 200 mL of distilled water. Solution B contained (NH₄)₂SO₄ (0.908 mM) and KH₂PO₄ (0.353 mM) dissolved in 3000 mL of distilled water. Solution C contained FeSO₄·7H₂O (1078 mM) dissolved in 400 mL of distilled water. Each solution was prepared separately and mixed aseptically prior to inoculation. The medium was adjusted to pH 1.8 and incubated for 15 days at 30 °C with shaking at 100 rpm. Following this initial enrichment, a 0.1 mL aliquot of the culture was plated on solid FeTSB medium [16], yielding a microbial count of 4 × 10⁷ CFU/mL. For purification, individual colonies were transferred to 100 mL of fresh liquid 9K medium and incubated at 30 °C and 150 rpm until the medium exhibited a reddish coloration, indicative of Fe (III) production. To obtain pure isolates, a 0.1 mL aliquot from this enriched culture was subsequently spread-plated onto solid 9K medium [17] and incubated under identical conditions. The isolates were then characterized macroscopically and microscopically. Pure cultures were preserved in the culture collection of the Microbial Biotechnology Research Laboratory at Jorge Basadre Grohmann National University in a modified liquid 9K medium at 30 °C.

2.2. Morphology of Leptospirillum ferriphilum by SEM

For morphological analysis, Leptospirillum ferriphilum cells were prepared for scanning electron microscopy (SEM) following the protocol described by Castillo et al. [14]. The strain was initially cultivated in a modified 9K medium formulated by Amaro (1987) for 72 h. Cells were harvested by centrifugation to obtain the cell pellet, which was then dried at 70 °C for 48 h. Subsequently, the dried pellet was transferred to microtubes, flash-frozen in liquid nitrogen using a cryogenic storage tank (MVE-Chart SC/8.5, Chart Industries, Garfield Heights, OH, USA), and freeze-dried.

The prepared samples were examined using a Thermo Scientific Quattro-S Field Emission Scanning Electron Microscope (FE-SEM) (Thermo Fisher Scientific Inc., Waltham, MA, USA). Imaging was performed at 24,000× magnification with an accelerating voltage of 10 kV, a working distance of 10 mm, and under low-vacuum conditions.

2.3. Phylogenetic Analysis of Leptospirillum ferriphillum Strains

DNA extraction was performed using the Soil DNA Isolation Plus Kit (Cat. No. 64000) (Norgen Biotek Corp., Thorold, ON, Canada), in accordance with the manufacturer instructions. Amplification of the 16S rRNA gene was conducted by polymerase chain reaction (PCR) using the primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). The amplified products were purified and sequenced by Macrogen Inc. (Seoul, Republic of Korea) using the Sanger sequencing method. The phylogeny was then reconstructed using 16S rRNA gene sequences. Closely related sequences were identified via a BLASTn (Basic Local Alignment Search Tool, https://blast.ncbi.nlm.nih.gov/, accessed on 8 April 2025) search against the NCBI database [18,19] (accessed on 8 April 2025). Accession numbers for all sequences utilized are listed in Table S1, and reference sequences were retrieved from GenBank [20,21] (accessed on 8 April 2025).

Sequences were aligned using MUSCLE v5.3 [22], and the resulting alignment was curated with BMGE v1.12_1 under default parameters, as implemented on the NGPhylogeny.fr platform (https://ngphylogeny.fr/, accessed on 8 April 2025) [23,24] (accessed on 8 April 2025). The alignment was manually edited in Mesquite v3.81 [25] (accessed on 8 April 2025) to remove ambiguously aligned regions corresponding to positions 1–22, 157–171, 184–196, 436–459, 824–825, 1116–1117, 1118–1123, and 1439–1482. The final alignment was exported in both Phylip [26] and Nexus [27] formats.

Phylogenetic analyses were conducted on the CIPRES Science Gateway platform [28] (https://www.phylo.org/, accessed on 8 April 2025). The best-fit nucleotide substitution model was selected using jModelTest2 v2.1.10 [29] based on the Akaike Information Criterion (AIC).Maximum Likelihood (ML) inference was performed using IQ-TREE v2.4.0 [30,31,32], with branch support values calculated from 5000 ultrafast bootstrap replicates implemented in the same software.

Bayesian Inference (BI) was performed using MrBayes v3.2.7a [33]. Two indepen-dent runs were carried out, each comprising four chains (three heated, one cold), for 10 million generations, with trees sampled every 500 generations. The first 25% of sampled trees were discarded as burn-in, and posterior probabilities were calculated from the remaining 75%. Convergence was assessed by analyzing log-likelihood trace files in Tracer v1.7.2 [34]. Finally, the resulting phylogenetic trees were visualized in FigTree v1.4.4 [35] and prepared for publication using Inkscape v1.2.2 [36] (accessed on 8 April 2025).

2.4. Reactivation of the Strains

The strains were reactivated by transferring 7 mL of the preservation medium into 63 mL (10%) of modified and buffered 9K culture medium. Incubations were conducted at 30 °C with shaking at 120 rpm until a cell concentration of 10⁸ cells/mL was achieved, determined by direct counting in a Petroff-Hauser counting chamber (Hauser Scientific, Model 3900, Horsham, PA, USA) with bright field microscopy Leica DM 750 (Leica Microsystems, Wetzlar, Germany). The reactivation procedure was repeated through five successive subcultures to ensure metabolic recovery and growth stability of both strains.

2.5. Recovery of Copper from Chalcopyrite ore Using Bioleaching with Leptospirillum ferriphilum Strains

The strains were reactivated by inoculating 7 mL of the preservation medium into 63 mL of a modified and buffered 9K medium, resulting in a 10% (v/v) inoculum [37]

Cultures were incubated at 30 °C with shaking at 120 rpm. Growth was monitored by direct cell counting using a Petroff-Hausser chamber under a bright-field microscope until a density of approximately 10⁸ cells/mL was reached. To ensure full metabolic recovery and growth stability, both strains were subcultured five consecutive times under these conditions.

2.6. Bioleaching Kinetics Applying M1D and M3E Strains

Triplicate 250 mL Erlenmeyer flasks was made of borosilicate glass (SCHOTT DURAN, Wertheim, Germany), each containing 180 mL of modified 9K medium, were inoculated with 20 mL of the reactivated culture for each strain. The medium was supplemented with elemental sulfur (10 g/L) and 20 g of chalcopyrite ore, previously sieved to a particle size of <150 µm (Tyler mesh 100). The ore, sourced from the Toquepala mine (Tacna, Peru), contained 0.23% copper (46 mg) and 5.38% iron (1.076 g). An abiotic control was prepared under identical conditions, substituting the microbial inoculum with 20 mL of sterile 9K medium.

Each experimental unit, including controls, had a total iron content of 2.416 g (1.340 g from the medium and 1.076 g from the ore) and a total copper content of 46 mg (sourced solely from the ore). Prior to use, the chalcopyrite was sterilized by tyndallization—autoclaved at 100 °C for 15 min throughout three consecutive cycles with 24 h intervals—to eliminate contaminants without altering its mineralogical structure.

Flasks (SCHOTT DURAN, Wertheim, Germany) were incubated at 30 °C with constant agitation at 120 rpm for 27 days (648 h). Samples were collected every 72 h to monitor the process. Population dynamics were assessed by direct cell counting using a Petroff-Hausser chamber under bright-field microscopy.

Ferrous iron (Fe²⁺) and total iron concentrations were determined spectrophotometrically using the 1,10-phenanthroline method, with the ferric iron (Fe³⁺) concentration calculated by difference. Dissolved copper concentration was quantified using atomic absorption spectrophotometry (AAS) with a Shimadzu 6701F spectrophotometer (Shimadzu, Kyoto, Japan).

2.7. Statistical Analysis and Data Processing

Experimental data were processed and analyzed using Microsoft Excel 2019 (MSO 16.0, 64-bit) (Microsoft Corp., Redmond, WA, USA) and GraphPad Prism v8.0. These software tools were employed to generate growth curves and bar graphs representing ferric iron and copper production, expressed in both absolute (g) and relative (%) units, as well as their respective productivities. The specific microbial growth rate (k) was estimated by fitting the data to the sigmoidal Gompertz model [38], as adapted for microbiology by Zwietering et al. [39].

For statistical analysis, one-way analysis of variance (ANOVA) was performed, followed by Fisher’s Least Significant Difference (LSD) test for multiple mean comparisons. All statistical analyses were conducted at a 99% confidence level. The analysis focused on key variables, including growth rate, ferric iron production, and copper leaching, comparing the performance of the different microbial strains against an abiotic control.

The percentage of metal production was calculated by dividing the final quantity of metal produced by the total amount available at the beginning of the experiment, averaging the replicates for each experimental condition, and multiplying the result by 100. Productivity was defined as the amount of ferric iron or copper produced per unit of time, calculated by dividing the total output by the experiment’s duration (648 h).

The influence of the microbial strains on iron biooxidation and copper solubilization was assessed by comparing the maximum production and productivity values across treatments to determine significant differences. Additionally, correlations between cell concentration, ferric iron production, and dissolved copper concentration were analyzed.

3. Results

3.1. Characterization of Leptospirillum ferriphilum Isolated from Chalcopyrite Mineral

Colonies isolated from the chalcopyrite mineral on FeTSB culture medium were circular with smooth edges, a slightly convex and smooth surface, and exhibited an orange-red, creamy texture (Figure 1A). Scanning electron micrograph (SEM) was acquired at 10 kV with a working distance of 10 mm (Figure 1B); the image revealed a biofilm (a) adhered to the mineral surface, with bacteria within the biofilm exhibiting a curved bacillary morphology (b).

Phylogenetic analysis was conducted using 33 sequences representing 18 different strains obtained from GenBank. Acidithiobacillus ferrooxidans BRGM1 and Thiomonas intermedia ATCC 15466 were used as an outgroup. After manual editing, the alignment matrix contained 1354 characters. The optimal phylogenetic tree was generated using IQ-TREE, yielding a maximum likelihood (ML) value of −8789.119. The best-fit substitution model, determined by jModelTest2, was TIM1 + I + G, with a proportion of invariant sites (I) of 0.2270 and a gamma distribution shape parameter (α) of 0.5510. Subsequently, IQ-TREE optimized this model to TIM + F + I + G4. Base frequencies were A = 0.2470, C = 0.2275, G = 0.3280, and T = 0.1976; estimated substitution rates were AC = 1.0000, GA = 2.0037, TA = 0.7818, CG = 0.7818, CT = 4.0230, and GT = 1.0000.

Bayesian inference (BI) analysis was conducted with the TIM1 + I + G model (I = 0.2270; α = 0.5510), with estimated base frequencies A = 0.2458, C = 0.2278, G = 0.3283, and T = 0.1981; relative substitution rates were AC = 1.0000, GA = 1.9831, TA = 0.7906, CG = 0.7906, CT = 3.8078, and GT = 1.0000. The phylogenetic tree resulting from ML analysis showed topology congruent with that obtained by BI. The isolates M1E, M3D, M1A, M3F, M6B, M7A, and TSB clustered closely with Leptospirillum ferriphilum strains, with strong statistical support (MLBP/BIPP = 100/1.00), confirming their assignment to this species (Figure 1C). In contrast, Leptospirillum ferrooxidans and Leptospirillum ferrodiazotrophum formed clearly distinct clades, confirming significant phylogenetic separation from L. ferriphilum.

3.2. Comparative Analysis of Eight Leptospirillum ferriphilum Strains Isolated from Chalcopyrite

Eight isolated strains of Leptospirillum ferriphilum were sub-cultured in modified 9K medium before prior to inoculation into sterilized, ground chalcopyrite. Copper recovery efficiency was assessed by atomic absorption spectrophotometry. All strains achieved copper leaching percentages greater than 30% after 30 days of incubation at 30 °C. Statistical analysis revealed no significant differences in copper recovery among the strains. This finding was supported by one-way analysis of variance (ANOVA) at a 99% confidence level and further confirmed by a post-hoc Fisher’s Least Significant Difference (LSD) test (Figure 2).

3.3. Growth Kinetics of Leptospirillum ferriphilum Strains M1D and M3E

The growth kinetics of two Leptospirillum ferriphilum strains, M1D and M3E (Figure 3), were evaluated in sterilized chalcopyrite mineral (Figure 3). Both strains exhibited similar growth patterns, achieving significantly higher cell densities compared to the control group throughout the experiment. At 360 h, strains M1D and M3E reached peak cell densities of 2.45 × 10⁹ cells/mL and 2.42 × 10⁹ cells/mL, respectively. In contrast, the control group reached a maximum of only 8.50 × 10⁷ cells/mL at this time point. By the end of the experiment (648 h), cell counts slightly decreased to 1.27 × 10⁹ cells/mL for strain M1D and 1.22 × 10⁹ cells/mL for strain M3E, while the control group reached 9.75 × 10⁸ cells/mL. The specific growth rates for the microbial strains were notably higher at 0.03469 h⁻¹ (M1D) and 0.03651 h⁻¹ (M3E), compared to the control’s rate of 0.00642 h⁻¹. The control group exhibited a delayed onset of growth followed by a rapid increase in biomass during the final stage of the experiment, a distinct pattern from that of the inoculated cultures.

3.4. Ferric Iron Production

Ferric iron production was significantly higher in the inoculated treatments compared to the control group throughout the experiment. At 72 h, strains M1D and M3E produced 1.07 g and 1.06 g of ferric iron, respectively, while the control produced a negligible amount (0.00 g). The ferric iron production curves (Figure 4A) revealed minimal differences in the kinetic profiles of both strains. Strain M1D reached its maximum value of 1.24 g (51.09% of theoretical yield) at 648 h, whereas strain M3E peaked at 1.21 g (50.00%) at 576 h. By 648 h, ferric iron production for M1D and M3E was 1.24 g (51.09%) and 1.21 g (50.00%), respectively (Figure 4B,C), with corresponding productivities of 1.91 mg/h and 1.86 mg/h (Figure 4D). In contrast, the control group produced only 0.08 g (3.16% yield), with a productivity of 0.12 mg/h. Statistical analysis using one-way ANOVA followed by an LSD post-hoc test confirmed no significant differences between the strains, but both were significantly higher than the control (Figure 4B–D).

3.5. Copper Bioleaching Kinetics and Production

Copper production was significantly higher in the microbial treatments compared to the control group throughout the experiment (Figure 5). At 72 h, copper concentrations reached 6.52 mg and 6.66 mg for the M1D and M3E strains, respectively, while the control group yielded only 4.13 mg (Figure 5A). By 648 h, the strains had produced 16 mg of copper (34.78% recovery) and 15 mg (32.61% recovery) (Figure 5B,C), with productivities of 0.025 mg/h and 0.023 mg/h, respectively (Figure 5D). In contrast, the control group’s copper production was significantly lower at 12.80 mg (27.80% recovery), with a productivity of 0.020 mg/h. One-way analysis of variance (ANOVA) and post-hoc Fisher’s LSD test confirmed no statistically significant differences in copper production or productivity between the microbial strains, but both were significantly greater than the control (Figure 5B–D). The copper production curves for both M1D and M3E strains (Figure 4A) showed similar trends with a consistent positive slope throughout the experiment.

Correlations of microbial growth in strains M1D and M3E with ferric iron (r = 0.6633 and r = 0.5752) (Figure 6A,C), and ferric iron with leachate copper (r = 0.8062 and r = 0.8302) (Figure 6B,D), demonstrate a positive correlation of the M1D and M3 inoculate with copper and ferric iron production.

4. Discussion

The isolated strains of Leptospirillum ferriphilum formed small (0.2–0.5 cm), hard colonies with a reddish-brown color, resulting directly from iron precipitation (Figure 1A). Colony development was observed after 15 days of incubation. Morphologically, these bacteria are curved, spiral bacilli measuring 0.3–0.5 µm in width and 0.9–4.0 µm in length. They are Gram-negative, obligate acidophilic, aerobic, motile, and classified as chemolithoautotrophs, deriving energy from the oxidation of Fe²⁺ ions rather than sulfur compounds [4,13,17].

A key characteristic of these bacteria is their ability to adhere to mineral surfaces and form biofilms, a mechanism well-documented in various studies [8,40,41]. This adhesion is primarily regulated by quorum sensing [42,43,44,45], which controls the expression of genes involved in the synthesis of exopolysaccharides, including c-di-GMP and various adhesins [46]. Biofilm formation is critical for enhancing both bacterial adhesion and the bioleaching process itself [9,46], while also facilitating intra- and interspecific microbial associations [47].

The 16S rRNA gene remains a key molecular marker in microbiology due to its utility for the primary identification and phylogenetic assignment of acidophilic bacteria such as L. ferriphilum [4,13]. The gene’s value stems from the coexistence of highly conserved regions, which facilitate the alignment of distant sequences, and hypervariable domains that provide discriminatory resolution at the intra-genus and species levels. Within the context of bioleaching, this marker has been instrumental in delineating Leptospirillum clades and repeatedly documenting the dominance of L. ferriphilum in industrial biooxidation systems and acid mine drainage sites [3,7].

The practical taxonomic validity of the 16S gene has been further reinforced by omics studies (metagenomes and metatranscriptomes), which have confirmed that populations classified by 16S analysis as L. ferriphilum share a consistent functional repertoire. This includes the ability to oxidize Fe^2+, high tolerance to heavy metals, and sustained metabolic activity under extreme pH conditions [3,9,13,14]. These findings strengthen the conclusion that the 16S gene not only offers diagnostic value but also accurately reflects the functional ecology of acidophilic communities.

However, as a single marker, 16S has intrinsic limitations in addressing cryptic lineages or intraspecific microdiversity. Therefore, its combination with statistically robust phylogenetic methods and complementary genomic evidence is essential. The reconstruction of phylogenies using Maximum Likelihood (ML) and Bayesian Inference (BI), supported by refined matrices and optimized evolutionary models, mitigates ambiguities and ensures the topological stability of the resulting phylogenetic trees [30,33].

The bioinformatics pipeline employed in this study integrated several state-of-the-art tools to ensure methodological rigor. Multiple alignment with MUSCLE5 provided accuracy in variable regions, while subsequent refinement with BMGE eliminated ambiguous positions while preserving informative sites [22,25]. Model selection by AIC (using jModelTest2) and the evaluation of nodal support using UFBoot2 with 5000 replicates provided rigorous statistical control [30,32]. The use of IQ-TREE2 efficiently integrated these components into a reproducible framework optimized for ribosomal sequences [32].

The phylogenetic results demonstrate that our eight isolates form a monophyletic clade with reference strains of L. ferriphilum, including the ATCC type strains 49881. This clade received strong statistical support, with bootstrap (ML) values approaching 100% and posterior probabilities ≥ 0.95 at critical nodes (Figure 1C). This topology is consistent with patterns previously reported in industrial bioleaching studies, particularly for Leptospirillum group II clades isolated from biooxidation tanks at 40 °C. The low intra-clade divergence (short patristic distances between our isolates and the type strains) aligns with the selective pressures of the studied environment (high concentrations of Fe and sulfates) and corresponds to the intrinsic genetic stability documented for this species by Vardanyan et al. [4].

In the comparative analysis of the eight isolated strains, all achieved copper recoveries greater than 30% within 30 days, with no statistically significant differences observed between treatments (ANOVA, p > 0.01) (Figure 2). These findings align with previous reports on the mesophilic bioleaching of chalcopyrite, where slow kinetics and low copper recovery are attributed to the formation of passivating surface layers composed of copper polysulfides, elemental sulfur, and iron hydroxides [48]. While L. ferriphilum can grow at 30 °C, its optimal bioleaching activity is reported at 40 °C, achieving 44.1% copper recovery and 91.4% iron recovery in just 17 days. [4].

The M1D and M3E strains exhibited active growth over 27 days at 30 °C (Figure 3), reaching maximum cell densities of approximately 2.4 × 10⁹ cells/mL after 360 h. The specific growth rates were calculated as k ≈ 0.03469–0.03651 h⁻¹ for M1D and M3E, which were substantially higher than that of the abiotic control (k ≈ 0.00642 h⁻¹). The growth of L. ferriphilum on chalcopyrite was considerably slower than the maximum specific growth rate (μmax = 0.09 h⁻¹) previously reported in synthetic medium containing 400 mM Fe [49].

During the experiment (Figure 4), L. ferriphilum oxidized 50% of the iron in the chalcopyrite within 30 days. This efficiency is lower than the nearly 100% iron oxidation reported for this microorganism in pyrite and synthetic 9K media, highlighting the inherent challenges of bioleaching recalcitrant sulfide minerals like chalcopyrite, which often require specific conditions for optimal microbial activity. The optimum iron concentration for oxidation by L. ferriphilum is between 5 and 40 g/L, at a pH of 1.05 to 1.8, suggesting our experimental conditions may not have been fully optimized for maximum iron oxidation [50,51,52].

In Figure 5, initial copper bioleaching showed a slight early advantage over the abiotic control, indicating a contribution from chemical leaching. Throughout the 648-h monitoring period, the two strains exhibited comparable performance, with no significant differences observed. Both strains achieved higher copper recovery percentages than reported in the initial experiment, with M1E reaching 34.78% and M1D reaching 32.61%. These results demonstrate that L. ferriphilum can adapt and biooxidize iron under mesophilic conditions of 30 °C, contributing to copper leaching from the early stages of the process. This is further supported by the positive correlations observed in Figure 6 between microbial growth, Fe oxidation, and Cu recovery.

Previous studies indicate that the optimal temperature for chalcopyrite bioleaching is between 35 and 40 °C. However, in natural environments, this process also involves sulfur-oxidizing microorganisms such as Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. The application of microbial consortia has been shown to enhance the process, with some studies reporting copper recoveries up to 39.7% in just 80 h [53]. Interestingly, real-time PCR studies on inoculated microbial consortia oxidizing sulfur and iron have shown that L. ferriphilum predominates in the later weeks of bioleaching, favoring the growth of other acidophiles like Acidithiobacillus caldus, A. thiooxidans, Acidiphilum spp., and Ferroplasma. While L. ferriphilum competes with Sulfobacillus thermosulfidooxidans, its prior colonization can be unfavorable for the latter, whereas simultaneous inoculation improves pyrite bioleaching [50,51].

In late-stage biooxidation, characterized by very low pH and high concentrations of metal ions (Fe³⁺, Cu²⁺), L. ferriphilum remains a major contributor of ferric ions, even when the activity of A. ferrooxidans is inhibited. The increase in temperature to 35 °C, often a result of the exothermic nature of the chemical reactions, further favors the growth of L. ferriphilum, ensuring continued iron oxidation [54,55,56]. Multi-omics studies have elucidated key adaptive mechanisms that allow L. ferriphilum to maintain metabolic activity on chalcopyrite, including the production of reducing power, CO₂ fixation, pH homeostasis, and resistance to high metal concentrations. Furthermore, the presence of nif genes has been reported in some strains, suggesting a potential for nitrogen fixation [9].

Sustainable bioleaching of low-grade minerals, such as chalcopyrite, hinges on the effective utilization of microorganisms. In industrial practice, a direct correlation is observed between higher biomass and increased metal extraction. For example, inoculating a column with 1.5 × 10⁸ cells/mL (compared to 1 × 10⁷ cells/mL) increased copper extraction from 32% to 44% in a 0.45 m column [57,58]. These data confirm the positive correlation between biomass and leaching efficiency (Figure 6): a greater number of available cells leads to enhanced Fe²⁺ oxidation and, consequently, greater copper extraction. While our flask-level study yielded a moderate 32% recovery with a thermophilic consortium, this result underscores the potential for improved performance at the column and bioreactor scale through process optimization. Ultimately, managing environmentally sustainable bioleaching processes requires a comprehensive understanding of microbial population dynamics through integrated physiological, genomic, metagenomic, and metatranscriptomic studies. As reported by Zhang and Schippers [59], a mesophilic consortium at 30 °C comprising A. thiooxidans, A. ferrooxidans, Leptospirillum ferrooxidans, and Ferroplasma acidiphilum, together with a moderately thermophilic consortium consisting of A. caldus, L. ferriphilum, S. thermosulfidooxidans, Sulfobacillus benefaciens, and F. acidiphilum, resulted in 38% cobalt and 86% copper extraction from mining tailings in 2-L stirred-tank reactor (STR) experiments with 10% (w/v) solids.

In addition to bacteria, fungi have also shown remarkable potential in the bioleaching of metals from various minerals. In particular, Aspergillus niger achieved up to 46% scandium recovery from bauxite residue through the secretion of organic acids such as citric and oxalic, which promote metal solubilization [60]. These findings demonstrate the metabolic versatility of microorganisms in biometallurgical applications and support the expansion of bioleaching strategies beyond traditional sulfide minerals.

L. ferriphilum exhibited marked efficiency in copper bioleaching and iron oxidation at 30 °C, underscoring its role as a key functional species within mesophilic bioleaching systems. Prior studies have demonstrated its capacity to tolerate elevated sulfur concentrations while sustaining copper-leaching activity, a physiological trait that enhances its applicability in large-scale operations [13]. Moreover, Zhang and Schippers (2022) reported that a mesophilic consortium at 30 °C—comprising A. thiooxidans, A. ferrooxidans, L. ferrooxidans, and F. acidiphilum in combination with a moderately thermophilic consortium consisting of A. caldus, L. ferriphilum, S. thermosulfidooxidans, S. benefaciens, and F. acidiphilum—yielded 38% cobalt and 86% copper extraction from mining tailings in 2-L stirred-tank reactor (STR) experiments with 10% (w/v) solids [59]. Taken together, these findings highlight the ecological resilience and industrial significance of L. ferriphilum, reinforcing its position as a keystone microorganism in biohydrometallurgical applications.

5. Conclusions

The isolated strains, molecularly and phylogenetically identified as Leptospirillum ferriphilum, exhibit low genetic divergence, suggesting a coherent functional repertoire and effective adaptation to the environmental conditions of the Toquepala mine. These native strains of L. ferriphilum successfully adapt and function at a mesophilic temperature of 30 °C, demonstrating their capacity to oxidize ferrous iron and promote copper bio-leaching from the early stages of the process. Strains M1D and M3E showed a high bioleaching kinetic rate, achieving iron oxidation close to 50% and copper recoveries over 30% in 648 h. These results highlight their potential as effective bioleaching agents for this type of ore. A positive correlation exists between the cell concentration of the L. ferriphilum strains, ferric iron production, and copper recovery, confirming microbial activity as a key driver of the bioleaching process. The use of native L. ferriphilum strains represents a viable and sustainable alternative for bioleaching of low-grade chalcopyrite, particularly under mesophilic conditions, thereby reducing the need for high-temperature processes and their associated energy costs. Future research should focus on optimizing process parameters such as pH, aeration, and nutrient supply at larger scales (columns and bioreactors) to maximize copper recovery and confirm the industrial applicability of these strains.

6. Patents

Section 2.1 and Section 2.4 are in the patent process called “Inoculation Process of the Leptospirillum ferriphilum Strain with Application in Bioleaching Columns”, File No. 002980-2024.