# Effect of particle size on bioleaching of chalcopyrite at moderate temperature

Adeline Po, Laurent Cassayre, Agathe Hubau, Catherine Joulian, Amine Dakkoune, Solène Touze, Carine Julcour, Florent Bourgeois, Anne-Gwénaëlle Guezennec

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Adeline Po, Laurent Cassayre, Agathe Hubau, Catherine Joulian, Amine Dakkoune, et al.. Effect of particle size on bioleaching of chalcopyrite at moderate temperature. Minerals Engineering, 2026, 236, pp.109885. 10.1016/j.mineng.2025.109885. hal-05418420

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# Effect of particle size on bioleaching of chalcopyrite at moderate temperature

Adeline Po<sup>a,b</sup>, Laurent Cassayre<sup>b</sup>, Agathe Hubau<sup>a,b</sup>, Catherine Joulian<sup>a,b</sup>, Amine Dakkoune<sup>b</sup>, Solène Touze<sup>a,b</sup>, Carine Julcour<sup>b</sup>, Florent Bourgeois<sup>b</sup>, Anne-Gwénaëlle Guezennec<sup>a,* c</sup>

## ARTICLE INFO

**Keywords:**

Bioleaching

Chalcopyrite

Passivation

Particle size

Moderate thermophiles

Mesophiles

Copper

## ABSTRACT

Several methods have been studied to overcome chalcopyrite passivation during bioleaching at atmospheric pressure. One such method is to reduce particle size to increase the reactive surface area of the mineral. However, little is known about the effect of fine particles on the performance of chalcopyrite bioleaching in terms of bacterial growth and activity. In this study, two size fractions were prepared by sieving a raw chalcopyrite concentrate to produce particles between 20 and 100 µm and particles below 20 µm. These three materials were used in batch bioleaching tests performed in 2 L stirred reactors at 42 °C, with a solid concentration of 10 %w/w and the BRGM-KCC microbial consortium. Redox potential and iron and copper concentrations were monitored over time to characterize the reaction progress. With the coarser materials, the redox potential increased to above 750 mV vs. SHE within a day, whereas particles below 20 µm took an additional day to reach this potential. This lag phase permitted faster copper dissolution, resulting in a yield of 35 % after two days. In contrast, a yield of 10–20 % copper was achieved in 15 days using coarser materials. In contrast to previous assumptions, fine particles did not hinder bacterial growth and activity. Rather, they promoted them by increasing the availability of substrates generated by greater sulfide oxidation. Bioleaching chalcopyrite particles < 20 µm produced higher copper yields thanks not only to their larger reactive surface, but also to the delayed increase in redox potential and subsequent chalcopyrite passivation.

## 1. Introduction

Chalcopyrite (CuFeS₂) accounts for 70 % of the Earth's copper (Li et al., 2013) and is the main primary copper resource. Today, 80 % of the worldwide copper production from copper ores is performed by pyrometallurgical processes, and 20 % through hydrometallurgy (International Copper Study Group (ICSG), 2023). Nonetheless, with the decreasing availability of high-grade ores, hydrometallurgical processes are still an interesting alternative to pyrometallurgical processes, since they are more economically viable to treat low grade ores and more complex resources. In that sense, oxidative bioleaching processes are of interest since they use the properties of iron- and sulfur-oxidizing microorganisms to generate or regenerate reagents involved in dissolution reactions of metal-bearing minerals. Furthermore, they are carried out at atmospheric pressure and temperatures lower than 100 °C in aqueous

solution. These processes have been extensively studied to recover copper from chalcopyrite.

To leach metal-bearing sulfides like chalcopyrite, an oxidizing agent is required to oxidize sulfides and liberate metal ions (Li et al., 2013). The oxidant is generally ferric iron (Fe³⁺), and, although chalcopyrite can be oxidized by oxygen (Eq. [1]), it is well established that the mineral is mainly oxidized by ferric iron (Eq. [2]) when both oxidants are present (Zhao et al., 2017). Besides, O₂-driven ferrous iron (Fe²⁺) oxidation rate can be up to five times faster in the presence of microorganisms than under abiotic conditions (Kinnunen and Puhakka, 2005). The main advantage of oxidative bioleaching with regards of conventional leaching is that Fe³⁺ is continuously generated by iron-oxidizing microorganisms (Eq. [3]). Furthermore, sulfur-oxidizing microorganisms also present in the system are able to oxidize the sulfur-intermediates (Eq. [4]) produced by chalcopyrite oxidation reactions

<footnote><sup>a</sup> BRGM (Bureau de recherches géologiques et minières), Raw Materials Department, Orléans, France</footnote>

<footnote><sup>b</sup> Laboratoire de Génie Chimique, Université de Toulouse, CNRS, INP, UPS, Toulouse, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: a.guezennec@brgm.fr (A.-G. Guezennec).</footnote>
(Eq. [1] and [2]).

$$CuFeS_2 + 4H^+ + O_2 \rightarrow Cu^{2+} + Fe^{2+} + 2S + 2H_2O \quad (1)$$

$$CuFeS_2 + 4Fe^{3+} \rightarrow Cu^{2+} + 5Fe^{2+} + 2S \quad (2)$$

$$4Fe^{2+} + 4H^+ + O_2 \xrightarrow{\text{Iron-oxidizing bacteria}} 4Fe^{3+} + 2H_2O \quad (3)$$

$$2S + 3O_2 + 2H_2O \xrightarrow{\text{Sulfur-oxidizing bacteria}} 2SO_4^{2-} + 4H^+ \quad (4)$$

Iron- and sulfur-oxidizing microorganisms used in bioleaching processes are usually bacteria or archaea, which can grow at low pH (acidophiles), on organic carbon (heterotrophs) or inorganic carbon (CO₂) (auto-trophs) or both (mixotrophs). Moreover, some microbial species grow better between 20 °C and 40 °C (mesophiles), others between 45 °C and 55 °C (moderate thermophiles) or at a temperature higher than 50 °C and up to 90 °C (thermophiles). In a bioleaching system, these micro-organisms can be in solution or attached to mineral surfaces thanks to the extracellular polymeric substances (EPS) they secrete. In the literature, it is suggested that EPS create a microenvironment where the oxidation of the sulfide mineral is faster and more efficient than in solution (Rawlings, 2005). In the case of a microbial consortium, the distribution of microorganisms between the liquid and solid phases might be influenced by the nature of the microbial species and the physico-chemical conditions encountered in the bioreactor (Guezennec et al., 2017).

However, copper extraction from chalcopyrite using bioleaching processes is still a challenge since the dissolution of the mineral remains limited at atmospheric pressure and temperature below 100 °C. Until now, there is still no consensus on the links between chalcopyrite dissolution mechanisms and its resistance to leaching, although the formation of a passivation layer on the mineral surface is usually mentioned. According to several studies, this layer could be composed of elemental sulfur, copper intermediate sulfides (covellite (CuS) for example), jarosite or a mixture of these phases (McMillan et al., 1982; Córdoba et al., 2008a; Klauber et al., 2008; Nazari et Asselin, 2009; Sasaki et al., 2009; Li et al., 2013; Khoshkhoo et al., 2017). The composition of this layer could also vary according to the operating conditions (redox potential, pH, temperature, etc.) (Zhu et al., 2011). While elemental sulfur and intermediate sulfides are products of chalcopyrite oxidation (Eq. [1] and [2]), jarosite phases are generated from Fe³⁺ precipitation with sulfates ions (SO₄²⁻) and monocations present in the medium (Eq. [5]).

$$3Fe^{3+} + 2SO_4^{2-} + 6H_2O + M^+ \rightarrow MFe_3(SO_4)_2(OH)_6 + 6H^+ \quad (\text{with } M^+ = K^+, Na^+, NH_4^+, \text{ or } H_3O^+) \quad (5)$$

It is suggested that this layer hinders the diffusion of ions involved in chalcopyrite oxidation and reduces the surface available for this reaction, which would explain the decrease in chalcopyrite dissolution over time. Another explanation suggested in literature involves electronic properties of chalcopyrite. Indeed, as a semiconductor, chalcopyrite possesses a band gap that could hinder electron transfer and thus oxidation reactions, especially as this band gap has an energy range which coincides with the redox potential of the Fe³⁺/Fe²⁺ couple (O' Connor and Eksteen, 2020). This could therefore explain the resistance of the mineral to (bio)leaching. Ren et al. (2022) proposed another explanation combining both previous theories. They hypothesized that a covellite-like layer, with p-type electronic properties, is formed due to preferential dissolution of iron. A p-n junction could then be produced on the chalcopyrite (n-type) that hinders subsequent electrochemical processes. In this paper, the term 'chalcopyrite passivation' will be used to refer to the slowdown of chalcopyrite dissolution during (bio)leaching, regardless of the underlying mechanisms.

Different conditions have been studied to avoid chalcopyrite passivation and enhance mineral (bio)leaching under atmospheric

conditions. They involve selecting operating conditions that favor chalcopyrite dissolution and prevent the formation or accumulation of solid species, in particular jarosite phases. One of them is to control the redox potential value to achieve sufficient yields and to minimize Fe³⁺ precipitation (Ahmadi et al., 2010; Lotfalian et al., 2015). Indeed, it has been demonstrated that chalcopyrite dissolution rate decreases above a potential of around 643 mV vs. SHE (Córdoba et al., 2008b; Gericke et al., 2010). In a system where Fe³⁺ is the main oxidant, redox potential increases with the ratio Fe³⁺/Fe²⁺ in solution. In bioleaching reactors, microbial activity can produce an excess of Fe³⁺, which increases this ratio and therefore the redox potential, leading to chalcopyrite passivation. The excess of Fe³⁺ also favors the formation of jarosite (Eq. [5]), which in turn reduces the amount of oxidant available for chalcopyrite oxidation.

Some other methods that prevent chalcopyrite passivation involve (i) operating at higher temperature (75 to 80 °C) with thermophilic microorganisms (BioCOP® process, Brierley and Brierley, 2013; Batty and Rorke, 2006), (ii) using catalysts or solubilizing agents (Abdollahi et al., 2015; Nazari et al., 2012), (iii) favoring galvanic interaction between chalcopyrite and pyrite (Galvanox® process, Li et al., 2013; Zhao et al., 2019; Esmailbagi et al., 2018), (iv) using microwaves, ultrasounds or glass beads to continuously exfoliate the passivating layer (Kartal et al., 2020; Wen et al., 2020; Dakkoune et al., 2023), and (v) reducing particle size (below 10 µm) by ultrafine grinding (Albion process). In general, fine grinding of ore particles improves the liberation of valuable mineral phases (Mariano et al., 2016) and increases reactive surface area (Granata et al., 2019). Furthermore, the fragmentation energy supplied and transferred to the solid can bring about structural changes in the mineral as well as a partial oxidation (Granata et al., 2019; Onisei et al., 2012; Li et al., 2025). These phenomena, known as mechanical activation, can enhance the reactivity of the initial mineral and thus the leaching efficiency. Higher oxidation rates and yields have been obtained with sulfide minerals when they are finely ground before leaching (Onisei et al., 2012; Bafghi et al., 2013; Tesinský and Baláz, 2017). Bafghi et al. (2013) also demonstrated that under a broad range of mechanical activation conditions, diffusion through protective product layer was the step controlling chalcopyrite leaching rate in sulfuric acid-ferric sulfate media. Granata et al. (2019) observed that fine grinding before leaching resulted in a faster chalcopyrite dissolution rate and a 30 % increase in copper yield. However, their experiments showed that enhancing chalcopyrite reactivity and specific surface area did not necessarily prevent chalcopyrite passivation. The Albion process uses ultrafine grinding to reduce particles to a size small enough for chalcopyrite leaching to reach near completion before passivation occurs, albeit at the cost of an energy penalty (Hourn and Turner, 2012).

In the case of bioleaching, Esmailbagi et al. (2018) and Manafi et al. (2021) found that reducing the particle size (d80) to approximately 10 µm led to a lower copper yield. This may be due to the grinding induced generation of reactive oxygen species (ROS), which are known to cause oxidative stress leading to microbial cell lysis, as well as the agglomeration of fine particles. Furthermore, it has been demonstrated that finer ground particles produce more ROS. This increases the lysis of microorganisms' cells, which can negatively affect sulfide dissolution (Jones et al., 2011). Zhao et al. (2021) also demonstrated that agglomeration and aggregation of fine particles could hinder leaching efficiency by decreasing reactive surface area and microbial attachment to sulfide minerals. Other authors have suggested that fine particles could have a negative effect on microbial viability. Nemati et al. (2000) studied particle size effect on a thermophile archaeon during pyrite (FeS₂) bioleaching and showed that particles with a mean diameter of 6.4 µm (size fraction < 25 µm) damaged microbial cells and inhibited their activity. From this work, it has been suggested that fine particles could have the same detrimental effect on moderate thermophile and mesophile bacteria during bioleaching operations (Olubambi et al., 2009; Manafi et al., 2021). However, other studies have reached different conclusions. During chalcopyrite bioleaching experiments carried out in
Erlenmeyer flasks, Jonglertjunya (2003) observed a shorter lag phase and an increase in the specific growth rate of the mesophilic bacteria *Acidithiobacillus ferrooxidans* when particle size was reduced from more than 100 µm to less than 53 µm. Deveci (2004) also studied the effect of particle size on the viability of mesophilic bacteria, often used in bio-leaching processes. They identified a limit above which decreasing particle size results in more damaged cells and inhibition of bacterial activity, and below which the reverse is observed. The limit is explained by a decrease in the frequency of particle collisions as the particle size increases above the critical size. Conversely, there is a decrease in particle mass and terminal velocity as the particle size decreases below the critical size, whose threshold depends on the agitation system and operating conditions. Their study found a threshold value between 63 and 45 µm. Nevertheless, the tests were carried out with an inert material on which only little bacterial attachment could occur. Therefore, the possible effect on sessile bacteria was not taken into account.

In this context, our study aims at assessing the effect of fine particles on chalcopyrite bioleaching, and especially their impact on bacterial growth, community profile and activity. To this end, two particle size fractions – one with particles between 20 and 100 µm (20–100 µm), and another with particles below 20 µm (< 20 µm) – were prepared by sieving a chalcopyrite concentrate. These fractions and the concentrate were used for bioleaching tests using an acidophilic consortium of mesophilic and moderate thermophilic bacteria. To provide a clear understanding of bacterial behavior and the influence of bacterial activity on the redox potential of the solution, microbiological and chemical analyses, as well as mass balances, were used to monitor the main reactions (dissolution of chalcopyrite, Fe and S bio-oxidation, precipitation of jarosite).

## 2. Materials and methods

### 2.1. Materials

This study was performed using a copper concentrate mainly composed of chalcopyrite and pyrite. Two size fractions were sub-sampled using the method described in Fig. 1. First, the raw concentrate was dry sieved at 100 µm using a 400 mm Roto-Sieve. To minimize material loss, the oversized particles were milled with a millstone-type grinder and then sieved again under the same conditions. The two fractions below 100 µm were mixed together and classified using a hydrocyclone at a cut-size of 20 µm (Mozley, C124 model, internal diameter: 51 mm, vortex finder diameter: 11 mm, spigot: 4.5 mm,

pressure: 2 bar, pulp concentration: between 20 and 25 %w/w). The hydrocyclone underflow and overflow (20–100 µm and 0–20 µm respectively) were dried at 40 °C for one week. Each sample was then homogenized before being used in the experiments.

The mineral phases in the chalcopyrite concentrate and in the two subsamples were identified by XRD analysis (see section 2.5.1) (Fig. 2). Chalcopyrite (CuFeS₂) was the main mineral and the only copper-bearing phase. Pyrite (FeS₂) and molybdenite (MoS₂) were the only two other sulfides detected. The gangue contained albite (Na(AlSi₃O₈)), biotite ((Mg,Fe²⁺)₃(Al,Fe³⁺)Si₃O₁₀(OH,F)₂), clinochlore ((Mg,Fe, Al)₆(Si,Al)₄O₁₀(OH)₈), hornblende (Na₀.9K₀.4Ca₁.6Mg₂.8Fe₁.4Ti₀.₅Al₂.₄Si₆O₂₃(OH)), microcline (KAlSi₃O₈), and quartz (SiO₂).

The chemical composition of the three samples (see section 2.5.2) is summarized in Table 1. Major elements were copper, iron and sulfur (mainly in the form of sulfides). The copper concentrate and 20–100 µm fraction had a similar chemical composition, consistent with the similar diffractograms obtained for these two samples. The < 20 µm fraction however contained more copper, indicating the presence of more chalcopyrite as this was the only identified copper-bearing mineral.

The concentrations of chalcopyrite and pyrite in the raw concentrate and subfractions were determined based on the identified chemical compositions and mineral phases (Table 2). The concentration of chalcopyrite was calculated from the copper concentration. The concentration of molybdenite was calculated from the molybdenum concentration. The concentration of pyrite was calculated from the amount of sulfur corresponding to the sulfides, taking into account the sulfur contained in chalcopyrite and molybdenite.

The < 20 µm fraction contains less pyrite (about 5 %w/w) and more chalcopyrite (about 80 %w/w) than the coarser fractions (10 %w/w pyrite and around 70 %w/w chalcopyrite). This can be attributed to the classification process used to prepare the samples. Nevertheless, chalcopyrite is the dominant phase in each material, while pyrite remains minor, and we assumed that the compositions are sufficiently close to allow comparison between materials. The differences in mineral composition were taken into account when interpreting the results obtained, especially for leaching yield calculations.

Particle size volumetric distribution of the raw chalcopyrite concentrate, the 20–100 µm and < 20 µm fractions (Fig. 3) was measured with a laser diffraction particle size analyzer (see section 2.5.3). The d₅₀ of these materials were 58.1 ± 9.7 µm, 60.9 ± 1.2 µm and 4.9 ± 0.1 µm, respectively.

BET analysis (see section 2.5.4) was carried out to measure the specific surface area of the three materials. The greatest surface area was

<figure><img src="image_4.png" /><figcaption>Fig. 1. Preparation of two size fractions (20–100 µm and < 20 µm) from the chalcopyrite concentrate.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Diffractograms of the raw chalcopyrite concentrate (a) and the 20–100 µm (b) and < 20 µm fractions (c). A: Albite, B: Biotite, Ch: Chalcopyrite, Cl: Clinochlore, H: Hornblende, Mi: Microcline, Mo: Molybdenite, P: Pyrite, Q: Quartz.</figcaption></figure>

<caption>**Table 1**</caption>

Chemical compositions (%w/w) of the raw chalcopyrite concentrate, and the 20–100 µm and < 20 µm fractions. Only concentrations of major elements, sulfur species (sulfides, sulfate and elemental sulfur), K and Mo are indicated. S<sub>SO4</sub>: sulfur from sulfates, S<sub>sulfides</sub>: sulfur from sulfides, S<sub>elemental</sub>: elemental sulfur.

<table><caption>**Table 2**</caption><thead><tr><th>Samples</th><th>Fe</th><th>Cu</th><th>Al</th><th>Si</th><th>K</th><th>Mo</th><th>S</th><th>S<sub>SO4</sub></th><th>S<sub>sulfides</sub></th><th>S<sub>elemental</sub></th></tr></thead><tbody><tr><td>Raw concentrate</td><td>28.4</td><td>24.5</td><td>1.4</td><td>5.2</td><td>0.71</td><td>0.27</td><td>30.6</td><td>0.16</td><td>29.7</td><td>0.01</td></tr><tr><td>20–100 µm</td><td>28.1</td><td>23.5</td><td>1.5</td><td>5.4</td><td>0.77</td><td>0.26</td><td>30.2</td><td>0.13</td><td>29.2</td><td>0.02</td></tr><tr><td>< 20 µm</td><td>28.1</td><td>27.9</td><td>1.0</td><td>3.1</td><td>0.43</td><td>0.22</td><td>30.9</td><td>0.27</td><td>30.7</td><td>0.02</td></tr></tbody></table>

Chalcopyrite and pyrite contents (%w/w) calculated from chemical compositions of the raw chalcopyrite concentrate and the 20–100 µm and < 20 µm fractions.

<table><thead><tr><th>Samples</th><th>Chalcopyrite content</th><th>Pyrite content</th></tr></thead><tbody><tr><td>Raw concentrate</td><td>70.8</td><td>8.94</td></tr><tr><td>20–100 µm</td><td>68.0</td><td>9.78</td></tr><tr><td>20 µm</td><td>80.6</td><td>4.44</td></tr></tbody></table>

obtained with the < 20 µm fraction. It was 8 and 17 times higher than that measured with the raw chalcopyrite concentrate and 20–100 µm fraction, respectively (Table 3). The surface area of the 20–100 µm was also 2 times lower than the surface area determined for the raw chalcopyrite concentrate. These values are consistent with the particle size distributions reported in Fig. 3.

## 2.2. Microorganism consortium and nutrient

The microbial culture, originating from the BRGM-KCC consortium,
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Frequency (a) and cumulative (b) particle size distribution of the raw chalcopyrite concentrate, the 20–100 µm fraction, and the < 20 fraction used for the bioleaching experiments.</figcaption></figure>

<table><caption>**Table 3**

Specific surface of the raw chalcopyrite concentrate, the 20–100 µm fraction and the < 20 µm fraction.</caption><thead><tr><th></th><th>20–100 µm</th><th>raw</th><th>&lt; 20 µm</th></tr></thead><tbody><tr><td>Specific surface (m²·g⁻¹)</td><td>0.2</td><td>0.4</td><td>3.4</td></tr></tbody></table>

was composed of moderate thermophilic and mesophilic bacteria (Guezennec et al., 2017). The predominant microorganisms were *Lep-tospirillum ferriphilum* (Fe-oxidizer), *‘Fervidacidithiobacillus caldus’* (formerly *Acidithiobacillus caldus*, S-oxidizer), and the genus *Sulfobacillus* (Fe- and S-oxidizer). The consortium was first cultured in batch mode, at 42 °C, starting from 100 mL at 3 %w/w solids concentration to 2 L at 10 %w/w solids concentration, using a mixture of copper sulfides (chal-cocite, covellite, bornite and a minor amount of chalcopyrite). These sulfides were easily oxidized in bioleaching environment and proved to be a good substrate for acidophilic Fe- and S-oxidizing microorganisms, while providing an environment rich in copper (Guezennec and d'Hugues, 2015; Guezennec et al., 2018; Hedrich et al., 2018). This methodology enabled to produce an active biomass tolerant to high copper concentrations (up to ~ 17 g·L⁻¹). Afterwards, the culture was maintained by subculturing it every 7 days before being used to inoculate the bioleaching experiments.

All the experiments were performed with the “0Km” nutrient medium (Guezennec et al., 2017) which is composed of 3.7 g·L⁻¹ (NH₄)₂SO₄, 0.48 g·L⁻¹ KOH, 0.52 g·L⁻¹ MgSO₄, 7H₂O, and 0.8 g·L⁻¹ H₃PO₄.

## 2.3. Bioleaching experiment apparatus and operating conditions

Bioleaching experiments were carried out in 2 L baffled stirred tank reactors. A constant temperature was maintained by circulating warm water through the reactor jacket. The temperature was regulated by a

cryothermostat and a thermocouple placed inside the bioreactor. Air enriched with 1 % CO₂ was injected beneath the agitation system comprising a 6-blade Rushton turbine and a 3-blade 45° axial flow impeller to ensure efficient gas dispersion and solid suspension.

The experiments were performed in batch mode at 42 °C. The solid load was 10 %w/w and the inoculation was performed at 10 %v/v. The CO₂ enriched air was injected at a flow rate of 60 L·h⁻¹ and the agitation speed was 700 rpm. Sulfuric acid (H₂SO₄, 96 %) was added daily to maintain the pH below 1.7 until it remained below this value, and water loss by evaporation was compensated by daily addition of the corresponding volume of distilled water.

## 2.4. Bioleaching experiment monitoring

### 2.4.1. Chemical analysis

The pH, redox potential (Eh), temperature, and dissolved copper, iron, potassium and sulfate concentrations were monitored daily (except during the weekends). Measured redox potentials were corrected to the standard hydrogen electrode (SHE). Copper, iron and potassium concentrations were measured from pulp samples filtered at 0.45 µm using microwave plasma atomic emission spectrometer (MP-AES) (4210 MP-AES, Agilent Technologies). Sulfate concentration was measured from the filtered samples using ion chromatography (940 Professional IC Vario, Metrohm) with an anion exchange column (Metrosep A Supp 7—250/4.0, Metrohm). Ferric iron (Fe³⁺) concentration was determined from the total iron concentration and redox potential using Eq. [6] (Yue et al., 2014; Yue et al., 2016):

$$ Eh(mV) = -1 \times 10^{-3} \times [T(K)]^2 + 0.91 \times T(K) \times \frac{2.303R}{nF} \times T(K) \times 10^3 \times \log \frac{C_{\text{ferric.nominal}}}{C_{\text{ferrous.nominal}}} + 492 \quad (6) $$

with C<sub>ferric,nominal</sub> and C<sub>ferrous,nominal</sub> the concentration of Fe³⁺ and Fe²⁺ in solution in g·L⁻¹. Yue et al. (2016) verified the validity of this equation under conditions (temperature, pH and Fe concentration) similar to those applied in this work.

The copper yield was determined using Eq. [7]:

$$ \text{Cuyield}(\%) = \frac{C_{Cu(t)} \times V_{init} + m(Cu)_{\text{sampl}} - C_{Cu\_inoc} \times V_{inoc}}{C_{Cu\_init} \times m_{init}} \quad (7) $$

where C<sub>Cu(t)</sub> (g·L⁻¹) is the copper concentration measured in samples taken at different times during the experiments, V<sub>init</sub> (L) the initial liquid volume, m(Cu)_{sampl} (g) the total mass of copper lost while sampling, C<sub>Cu_inoc</sub> (g·L⁻¹) the copper concentration in the inoculum, V<sub>inoc</sub> (L) the volume of inoculum, C<sub>Cu_init</sub> (%w/w) the concentration of copper in the initial sulfidic solid material, and m<sub>init</sub> (g) the mass of the initial solid material. The amount of solid (sulfides) in the inoculum was negligible.

m(Cu)_{sampl} was calculated as follows:

$$ m(Cu)_{\text{sampl}} = \sum_{i=1}^{n} C_{Cu\_\text{sampl}\_i} \times V_i \quad (8) $$

where C<sub>Cu_sampl,i</sub> (g·L⁻¹) is the concentration of dissolved copper in the sample i, and V<sub>i</sub> (L) is the volume of sample i.

Iron yield is calculated from iron concentration in the same way as copper yield.

The bioleached pulp was filtered at the end of each experiment. The solid residue was washed with acidified distilled water, dried at 40 °C, and then analyzed. The filtrate and washing solutions were also analyzed using MP-AES to measure the concentrations of copper, iron, and potassium. Sulfate concentration was measured by ion chromatography.
### 2.4.2. Microbiological analysis

Biomass concentration was monitored daily (except during the weekends). Pulp samples were left to settle for 5 min before counting the cells using a Thoma cell counting chamber and a phase contrast microscope. The samples were decanted to reduce the amount of fine particles in the supernatant, as these could easily be mistaken for bacteria.

Changes in composition of the microbial community in the pulp and in the liquid and solid phases were also monitored during bioleaching experiments. 2 mL of pulp were sampled for analyses. In addition, 2 mL of pulp sample were centrifuged at 5000 × g for 30 s to separate the supernatant liquid phase from the solid phase. The retrieved supernatant was placed in a clean tube. All the samples (pulp, liquid or solid phases) were then centrifuged at 14500 × g for 10 min and washed with a 10 mM Tris-HCl buffer (pH 8) repeatedly until the supernatant pH reached ca. 7. The resulting pellets were then stored at -20 °C until DNA extraction was performed.

#### DNA extraction and quantification

DNA extraction was carried out using the FastDNA Spin Kit for Soil (MP Biomedicals) according to the manufacturer's protocol. The homogenization step was carried out using a FastPrep® instrument at a speed of 5 m.s⁻¹ for 30 s, followed by centrifugation for 15 min. The concentration of the extracted DNA was then measured using the Quantifluor dsDNA sample kit and the Quantus fluorometer according to the supplier's protocol (Promega).

#### Terminal restriction fragment length polymorphism (t-RFLP).

t-RFLP fingerprinting was used to assess the bacterial community profile and its evolution during the experiments (exponential and stationary phases of bacterial growth). A 900 pb fragment of the 16S rRNA gene contained in the extracted DNA was amplified by PCR from the extracted DNA, using the primers FAM-labelled 8F (5' FAM-AGAGTTTGATCMTGGCTCAG 3') and 907R (5' CCGTCAATTCMTTT-GAGTTT 3'). The reaction mix was composed of 12.2 µL of molecular biology grade water, 4 µL of GoTaq buffer 5x (Promega), 2 µL of MgCl₂ (25 mM), 0.2 µL of dNTP (10 mM), 0.2 µL of each primer (50 µM), 0.2 µL of GoTaq polymerase (Promega), and 1 µL of extracted DNA. The PCR was performed in a C-1000 Touch thermal cycler (Bio-Rad) with the following operating conditions: initial denaturation at 95 °C for 2 min, then 35 cycles of 45 s at 95 °C (denaturation), 45 s at 60 °C (hybridization), 60 s at 72 °C (elongation), and a post-elongation of 10 min at 72 °C.

The quality of the PCR amplification was assessed by electrophoresis of PCR products on an agarose gel (2%) and the amount of PCR products was also measured using a Quantus (Promega). Afterwards, between 27 and 100 ng of PCR product were digested with the restriction endonuclease HaeIII, at 37 °C during 4 h. The digestion was verified by electrophoresis on an agarose gel (3%). Then, 1 µL of the digested product was mixed with 18.7 µL of formamide HiDi and 0.3 µL of Genescan-600 LIZ size standard DNA (Life technologies), and the mixture was heat denatured. The terminal restriction fragments (tRF) were analyzed by capillary electrophoresis with the genetic analyzer SeqStudio (Life technologies). The software Bionumerics (AppliedMaths) was used to determine the size (based on the internal DNA size standard) and calculate the relative abundance (from peak height) of the tRFs. The tRFs were identified by comparison with the tRF of reference bioleaching microorganisms.

#### Quantitative-PCR (qPCR).

Quantitative-PCR was performed with the extracted DNA from the same original samples used for t-RFLP community profiling. This analysis was carried out as a complement to biomass concentration measurements in the liquid phase obtained by microscopic counting. This technique allowed quantifying the amount of bacterial 16S rRNA gene in both the liquid phase and the pulp.

The protocol described in Hedrich et al. (2016) with the primers 341F and 534R was used. The reaction mix was composed of 7.68 µL of water, 10 µL of SSO advanced Supermix 2X (Bio-Rad), 0.4 µM of each

primer and 2 µL of extracted DNA diluted to 1 ng.µL⁻¹ or non-diluted if the concentration was below 1 ng.µL⁻¹. The qPCR was performed in a CFX Connect Real-Time PCR system (Bio-Rad) with the following conditions: an initial denaturation at 95 °C for 3 min, then 35 cycles of 30 s at 95 °C (denaturation), 30 s at 60 °C (hybridization), 30 s at 72 °C (elongation), 30 s at 80 °C (data acquisition), and finally acquisition of melt curves with cycles of 10 s with increasing temperature from 65 to 95 °C at a rate of 0.5 °C per cycle. The number of copies of the 16S rRNA gene was determined with a standard range prepared by serial 10-fold dilutions of a linearized plasmid solution containing known number of copies of the targeted gene. The results are expressed in copies of the 16S rRNA gene per milliliter of sample (liquid phase or pulp).

## 2.5. Initial materials and residues characterization

### 2.5.1. Mineral phase identification

Identification of mineral phases was performed by X-ray diffraction (XRD). The samples were first wet-micronized (RXD Mcrome, Recht) with ethanol (7 mL) at maximum speed for 5 min. Afterwards, the micronized samples were dried at 40 °C, then homogenized before being analyzed in a diffractometer (Bruker D8 Advance Da Vinci) equipped with Cu-Kα radiation (40 kV and 40 mA) and a linear detector LYNXEYE XE-T. The data were collected between 4 and 90θ with a step of 0.03θ with the software Diffrac Start Jobs. Identification of the mineral phases was performed with the database ICDD and the software Diffrac Eva V5.1.

### 2.5.2. Chemical analysis

The content of metals was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) after oxidizing or acid digestion. Total carbon and total sulfur concentrations were determined by infrared analysis after oxidation and heating the carbon and sulfur content. Organic carbon and sulfide concentrations were measured by leaching the sample in warm chloric acid and sodium bicarbonate respectively. The solution obtained was then filtered, and same method was applied as that used to analyze total carbon and total sulfur. Sulfate concentration in chalcopyrite concentrates was determined by ICP-AES after leaching the samples in sodium carbonate, removing insoluble materials by filtering, and acidifying the filtrate. Sulfate concentration in residues was measured by gravimetric analysis after leaching the samples in sodium carbonate, filtering and precipitating the sulfate content as barium sulfate. Elemental sulfur concentration was determined using a carbon disulfide leach followed by evaporation of a portion of the leachate and weighing of the dry residue. Finally, silica concentration was measured using ICP-AES after sodium peroxide fusion. These chemical analyses of solid samples were performed by ALS (Loughrea, Ireland).

From the chemical composition obtained for the initial material and residues, together with mineral phase identification, chalcopyrite and pyrite content was determined as described in section 2.1. Indeed, chalcopyrite was the only identified copper-bearing mineral phase, and chalcopyrite, pyrite and molybdenite the only identified sulfides as shown in Fig. 2 and later in Fig. 7. Chalcopyrite and pyrite dissolution yield were then calculated from the chalcopyrite and pyrite concentrations in the initial materials and in the respective residues (eq. [9] and [10]).

$$ \text{Chalcopyriteyield}(\%) = \frac{C_{\text{CuFeS2_init}} \times m_{\text{init}} - C_{\text{CuFeS2_res}} \times m_{\text{res}}}{C_{\text{CuFeS2_init}} \times m_{\text{init}}} \quad (9) $$

$$ \text{Pyriteyield}(\%) = \frac{C_{\text{FeS2_init}} \times m_{\text{init}} - C_{\text{FeS2_res}} \times m_{\text{res}}}{C_{\text{FeS2_init}} \times m_{\text{init}}} \quad (10) $$

with $C_{\text{CuFeS2_init}}$ (%w/w) the chalcopyrite concentration in the initial material, $C_{\text{FeS2_init}}$ (%w/w) the pyrite concentration in the initial material, $C_{\text{CuFeS2_res}}$ (%w/w) the chalcopyrite concentration in the residue,
$C_{FeS_2\_res}$ (%w/w) the pyrite concentration in the residue, and $m_{res}$ (g) the mass of the residue.

Further details on mass balance calculations, particularly with regard to the evaluation of copper losses during the experiments, can be found in the Supplementary Materials.

### 2.5.3. Particle size distribution

Particle size distribution was measured using a laser diffraction particle size analyzer (Mastersizer S, Malvern). For the initial substrates (raw copper concentrate, 20–100 µm and < 20 µm fractions), a sample was suspended and agitated in water for 4 h. This procedure could not break the agglomerates contained in the bioleaching residues. Therefore, to analyze the bioleaching residues, the samples were suspended in water and agitated for between 30 min and 1 h while ultrasound was applied to break up the agglomerates. This was followed by sieving at 315 µm to prevent damaging the device. The mass of the fraction above 315 µm was always negligible compared to the mass of the whole sample. The fraction below 315 µm was again agitated for 15 min with 10 mL of DISPEX® (10 g.L⁻¹) and 10 mL of sodium hexametaphosphate

(10 g.L⁻¹) while applying ultrasound. The samples were subjected to ultrasound for 1 min inside the analyzer's mixing chamber prior to analysis. The data were collected with the software Mastersizer-Sv2.18F (Malvern).

### 2.5.4. Specific surface area

The specific surface area was measured using the BET method. First, the sample was degassed under vacuum (VacPrep 061, Micromeritics) for one week at room temperature to remove impurities and traces of moisture from the sample surface. The sample was then weighed and placed in the analyzer (ASAP 2050, Micromeritics). The measurements were performed by $N_2$ adsorption. The data acquisition software used was ASAP 2050 V1.01 (Micromeritics).

## 3. Results

### 3.1. Effect of particle size on chalcopyrite dissolution

Chalcopyrite dissolution tests were monitored by measuring the

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Time-evolution of (a) copper and (b) iron dissolution yields, and (c) evolution of dissolved Fe as a function of dissolved Cu, during bioleaching experiments with the 20–100 µm fraction, the raw chalcopyrite concentrate and the < 20 µm fraction. The yields were calculated based on the chemical analysis of the liquid phase from pulp samples.</figcaption></figure>
copper yield, since chalcopyrite was the only identified copper-bearing mineral (Fig. 4a). Copper dissolution kinetics were similar for the chalcopyrite concentrate and the 20–100 µm fraction, with a low dissolution rate (below 0.5 g·L⁻¹·d⁻¹) and almost no dissolution at the end of the experiments. Iron dissolution rate (Fig. 4b) was also low during the first four days (below 1 g·L⁻¹·d⁻¹). From around day 6, dissolved iron concentration stayed constant. On the other hand, copper and iron dissolution rates were significantly higher at the beginning of the experiment with the < 20 µm size fraction (around 8 g·L⁻¹·d⁻¹ between the 1st and 2nd day). After the 2nd day, copper dissolution rate decreased significantly, and no further iron was dissolved. The final yields (Table 4) were calculated from the chalcopyrite and pyrite concentrations in the residues. These concentrations were deduced from the chemical analyses of the residues using the same calculation method applied to determine chalcopyrite and pyrite content in the initial materials (see section 2.1.), since no new sulfide phase was detected in the residues (as will be shown in section 3.2.2). These yields confirm that a higher amount of chalcopyrite was oxidized and all the pyrite was dissolved during the bioleaching of the finer fraction. Chalcopyrite dissolution reached 55 % with < 20 µm particles compared to 11 % and 21 % with the 20–100 µm fraction and the raw concentrate, respectively. These results are consistent with those reported in the literature for bioleaching tests with a pulp density of 10 %w/w, pH between 1.1 and 1.5, at 50 °C (Esmailbagi et al., 2018; Manafi et al., 2021). Although chalcopyrite was the only copper-bearing mineral present, the chalcopyrite dissolution yields calculated from the chemical analysis of the residues are higher than those derived from liquid phase analyses. This discrepancy was explained by copper losses occurring at different stages of the experimental protocol (see Table A and discussion about mass balance in the Supplementary Materials).

The evolution of the redox potential during the bioleaching experiments is reported in Fig. 5. Initially, the redox potential stayed in the range of 550–600 mV vs. SHE. This phase was significantly longer when operating with the finer fraction (2 days) than with the other two materials (0.5 day). This period of 0.5–2 days at low Eh can be related to a specific dissolution pattern. Indeed, as shown in Fig. 4c, the molar ratio of dissolved iron to dissolved copper (Fe/Cu) was close to 1 at the start of the runs, indicating that only chalcopyrite was being dissolved (Fe/Cu = 1 in CuFeS₂). This is consistent with the fact that pyrite, which was the second most abundant sulfidic phase in the concentrate (Table 2), does not oxidize below 650 mV vs. SHE (Liu et al., 2017).

At longer bioleaching times, the redox potential increased exponentially (exponential phase) before stabilizing between 870 and 890 mV vs. SHE. Based on Eq. [6], this increase corresponds to a rise in the Fe³⁺/Fe²⁺ ratio up to 1000 with the raw concentrate and the 20–100 µm fraction, and even up to 2000 with the < 20 µm fraction. From the exponential phase onward, the iron in solution was therefore mostly ferric iron.

Fig. 6 shows that the ferric iron concentration (derived from the redox potential and the total iron concentration – Eq. [6]) increased to 5 g·L⁻¹ during the bioleaching experiments with the coarser materials, and to 10 g·L⁻¹ with the < 20 µm fraction, indicating different bacterial activity (Eq. [3]). As a result, a large excess of Fe³⁺ reactant was available for chalcopyrite oxidation (Eq. [2]). Therefore, in accordance with the literature (Córdoba et al., 2008b; Gericke et al., 2010), the slow chalcopyrite dissolution reported in Fig. 4a can be attributed to

<caption>**Table 4**</caption>

Chalcopyrite and pyrite dissolution yields at the end of the bioleaching experiments with the 20–100 µm fraction, the raw chalcopyrite concentrate and the < 20 µm fraction. The yields were calculated from the chemical analysis of the initial materials and the residues.

<table><thead><tr><th>Experiments</th><th>20–100 µm</th><th>raw</th><th>&lt; 20 µm</th></tr></thead><tbody><tr><td>Chalcopyrite yield (%)</td><td>11</td><td>21</td><td>55</td></tr><tr><td>Pyrite yield (%)</td><td>73</td><td>46</td><td>100</td></tr></tbody></table>

passivation at Eh > 640 mV vs. SHE, and not to a lack of oxidizing reagent.

Our data show that this phenomenon started later for fine particles than for coarser particles. The delayed passivation, together with the higher chalcopyrite concentration (Table 2) and the larger specific surface area (at least ~ 10 times larger in the finer fraction) explains the enhanced copper dissolution measured with the < 20 µm fraction (Fig. 4a). The slightly higher yield obtained with the raw concentrate than with the 20–100 µm fraction is probably because the raw concentrate contains a higher amount of fine particles (Fig. 3).

## 3.2. Characterization of the bioleaching residues

To further characterize bacterial activity during the runs and specifically quantify the total production of Fe³⁺ and SO₄²⁻ (Eq. [3]–[4]), detailed chemical and structural analyses of the solid residues were carried out. Indeed, as can be expected from the evolution of iron concentration in solution and from the literature, it is likely that some ferric and sulfate ions precipitate as jarosite compounds (MFe₃(SO₄)₂(OH)₆, with M = K, NH₄, or H₃O) (Eq. [5]).

### 3.2.1. Particle size distribution

Particle size distribution of the residues recovered at the end of the bioleaching experiments was compared with that of the respective initial materials (Fig. S1). Regardless of the initial particle size, the distribution shifted slightly towards finer particles, which is consistent with the dissolution of the particles as evidenced by liquid phase analyses. It can also be deduced that the precipitates that may have formed did not lead to particles large enough to affect the overall particle size distributions.

### 3.2.2. Main mineral phases

The XRD analyses of the leaching residues are compiled in Fig. 7. In line with the dissolution yields derived from chemical analyses (see Table 4), chalcopyrite was still detected in all the residues, whereas pyrite was only detected in the residue obtained from coarse particles. XRD diffractograms also indicate the presence of jarosite in all the residues and of sulfur in the residue from the fine particles. These phases were not found in the initial samples (Fig. 2). Further characterization and quantification of these neo-formed phases are reported in the next sections.

### 3.2.3. Jarosite formation

The amount of iron precipitated ($Fe_{precip}$) for each experiment can be calculated based on the iron concentration in the residue from equation [11]:

$$Fe_{precip}[mol] = Fe_{res} - Fe_{CuFeS2...res} - Fe_{FeS2...res} \quad (11)$$

with $Fe_{res}$ the amount of iron in the residue (mol), $Fe_{CuFeS2...res}$ the amount of iron from chalcopyrite in the residue (mol), and $Fe_{FeS2...res}$ the amount of iron from pyrite in the residue (mol). The iron in the gangue was negligible.

Chalcopyrite being the only copper-bearing phase, $Fe_{CuFeS2...res}$ is equal to the amount of copper in the residue (mol). The amount of pyrite, and thus $Fe_{FeS2...res}$, is calculated from the concentration in chalcopyrite and molybdenite, which are the only other sulfides in the residue (Eq. [12] and [13]).

$$S_{FeS2} = S_{sulfides\_tot} - S_{CuFeS2} - S_{MoS2} = S_{sulfides\_tot} - Cu_{res} \times 2 - Mo_{res} \times 2 \quad (12)$$

$$Fe_{FeS2...res} = (S_{FeS2}/2) \quad (13)$$

where $S_{sulfides\_tot}$, $S_{FeS2}$, $S_{CuFeS2}$, and $S_{MoS2}$ are the amount of sulfur respectively in all sulfides, in pyrite, in chalcopyrite, and in molybdenite (mol), $Cu_{res}$ is the amount of copper in the residue (mol), $Mo_{res}$ the
<figure><img src="image_8.png" /><figcaption>Fig. 5. Evolution of the redox potential during bioleaching experiments with the 20–100 µm fraction, the raw chalcopyrite concentrate and the < 20 µm fraction. 640 mV vs. SHE is the chalcopyrite passivation threshold according to Córdoba et al. (2008b) and Gericke et al. (2010).</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 6. Evolution of ferric iron concentration (derived from the redox potential and the total iron concentration) during bioleaching experiment with the 20–100 µm fraction, the raw chalcopyrite concentrate and the < 20 µm fraction.</figcaption></figure>

amount of molybdenum in the residue (mol).

The mass balance of the sulphate ions was also calculated to determine the amount of $SO_4^{2-}$ accumulated in the residues during the experiments. By comparison with the amount of precipitated iron (Table 5), our data showed that the Fe/SO$_4$ ratio was about 1.5, which corresponds to the Fe/SO$_4$ ratio in jarosite (Eq. [5]). These mass balance considerations establish that both iron and sulphate mainly precipitated as jarosite during bioleaching tests.

Different types of jarosite can form depending on the monocation involved in the precipitation reaction (Eq. [5]). In the experiments presented here, due to the addition of “0Km” nutrient medium (see section 2.2.), K-jarosite, NH$_4$-jarosite and H$_3$O-jarosite may potentially form. According to Sasaki et al. (2009), K-jarosite is the least soluble, followed by NH$_4$-jarosite and H$_3$O-jarosite. Since the lattice parameters of the individual jarosite phases are very similar, and since these phases

<caption>**Table 5**</caption>

Amount of iron and $SO_4^{2-}$ precipitated during the bioleaching experiments with the 20–100 µm fraction, the raw concentrate and the < 20 µm fraction. n.m.: not measured.

<table><thead><tr><th>Experiments</th><th>20–100 µm</th><th>raw</th><th>&lt; 20 µm</th></tr></thead><tbody><tr><td>Precipitated iron (mol)</td><td>0.043</td><td>n.m.</td><td>0.219</td></tr><tr><td>Precipitated $SO_4^{2-}$ (mol)</td><td>0.029</td><td>0.021</td><td>0.148</td></tr><tr><td>Fe/SO$_4$</td><td>1.48</td><td>—</td><td>1.48</td></tr></tbody></table>

can form solid solutions (Dutrizac and Jambor, 2000), XRD analyses do not provide useful information on the chemical nature of these jarosites.

Fig. 8 summarizes the estimated distribution of the different types of jarosite (K-jarosite on one side, NH$_4$- and H$_3$O-jarosite on the other side) in the residues at the end of the bioleaching experiments, based on the total amount of jarosite in the each residue (Table 5) and their potassium content (Eq. [5]).

The amount of jarosite formed was at least five times higher during bioleaching experiments with the finer fraction than with the two other materials. It accounted for 23 % of the mass of the residue, compared to only 4 % and 3 % for the 20–100 µm fraction and raw concentrate, respectively. The higher concentration of ferric iron in solution during the bioleaching experiment with the < 20 µm fraction (Fig. 6) is consistent with the higher production of jarosite. It can be noted that the amount of K-jarosite was almost the same for all experiments, corresponding to the initial amount of potassium in the medium. This was further evidenced by following the evolution of K concentration over time in the liquid phase (Fig. S2), which confirmed that all available potassium eventually precipitated. A notable amount of NH$_4$- and/or H$_3$O-jarosite was also precipitated with the finer fraction, compared to the other two experiments. These results are in agreement with what Sasaki et al. (2009) and Hubau et al. (2023) observed during bioleaching experiments where K-jarosite formed preferentially over NH$_4$-jarosite when the solution contained $K^+$ and $NH_4^+$.

### 3.2.4. Sulfur formation

In addition to jarosite, substantial amounts of elemental sulfur was found (by specific chemical analysis, see section 2.5.2.) in the bioleached residues (Table 6), whereas it was almost absent in the initial materials (Table 1). All residues contained elemental sulfur, with higher quantities found in the residue obtained from the fine fraction than those obtained from the coarser materials.

The amount of non-oxidized elemental sulfur in the residues was markedly lower (by a factor of 20 to 40) than the quantity of sulfides that was dissolved during the runs. It can be deduced that most of the sulfides were oxidized and generated sulfuric acid according to Eq. [4] and [14]-[15]. This was confirmed by experimental pH monitoring, which showed a decrease from 1.7 to between 1.5 and 1.3 (data not shown).

$$FeS_2 + 6Fe^{3+} + 3H_2O \rightarrow S_2O_3^{2-} + 7Fe^{2+} + 6H^+ \quad (14)$$

$$S_2O_3^{2-} + 8Fe^{3+} + 5H_2O \xrightarrow{\text{Sulfur-oxidizing bacteria}} 2SO_4^{2-} + 8Fe^{2+} + 10H^+ \quad (15)$$
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Diffractogram obtained by XRD analysis of the residues recovered at the end of the bioleaching experiments with the (a) 20–100 µm fraction, (b) the raw concentrate and (c) the < 20 µm fraction. Ch: Chalcopyrite, J: Jarosite, Mo: Molybdenite, P: Pyrite, Q: Quartz, S: elemental Sulfur. Only the main phases are indicated.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Mass quantification of the different jarosites contained in the residues obtained at the end of the bioleaching experiments with the 20–100 µm fraction, the raw concentrate and the < 20 µm fraction.</figcaption></figure>

<caption>**Table 6**</caption>

Amount of elemental sulfur (g) measured in the residues obtained at the end of the bioleaching experiments with the 20–100 µm fraction, the raw concentrate and the < 20 µm fraction.

<table><thead><tr><th>Initial substrate</th><th>20–100 µm</th><th>raw</th><th>&lt; 20 µm</th></tr></thead><tbody><tr><td>Elemental sulfur in the residue (g)</td><td>0.3</td><td>0.5</td><td>1.2</td></tr></tbody></table>

### 3.3. Effect of particle size on the bacterial community

#### 3.3.1. Particle size effect on bacterial growth

To assess the effect of particle size on bacterial growth, the biomass in the reactor was first quantified and monitored by cell counting during the bioleaching experiments (Fig. 9a).

For the bioleaching experiments with the raw concentrate and the 20–100 µm fraction, biomass concentration increased during the first two days before stabilizing around $2 \times 10^{10} \text{ cell.mL}^{-1}$. With the < 20 µm fraction, the biomass concentration hardly increased during the first day
<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Biomass concentration measured by (a) cell counting, as a function of time, and (b) gene copies quantification by qPCR during the exponential and stationary phases of bacterial growth, during bioleaching experiments with the 20–100 µm fraction, the raw concentrate and the < 20 µm fraction. Measurements were carried out with samples of the liquid phase recovered after settling pulp samples for 5 min or after centrifugation of pulp samples for 30 s at 5000 rpm for cell counting and qPCR, respectively. Sampling times for qPCR analysis are indicated by blue arrows.</figcaption></figure>

(lag phase), then increased quickly before stabilizing around 1 x 10¹¹ cell.mL⁻¹. In all three experiments, the increase of biomass concentration was concomitant with the exponential increase in redox potential (Fig. 5). Final biomass concentration was ten times higher with the finer fraction than with the other two materials, indicating that fine particles (< 20 µm) did not hinder bacterial growth. However, distinguishing between cells and fine particles by optical microscope observation can be difficult. Therefore, to verify these results, the biomass was quantified by measuring the copy numbers of the 16S RNA gene, which is present in all bacteria, using qPCR during the bioleaching experiments (Fig. 9b). Since cell counting was performed on the liquid phase of a pulp sample that had been left to settle for 5 min (see section 2.4.2), qPCR was performed on DNA recovered from samples of the liquid phase obtained after centrifugation of a pulp sample. Gene quantification results confirmed that the finer fraction contained one order of magnitude more bacteria. The slight decrease in biomass concentration between the exponential and stationary phases of bacterial growth might be due to more fragile cells, which could be more easily lysed during sample preparation before DNA extraction, resulting in less DNA being recovered. Gene copies quantification by qPCR performed on pulp samples also showed a higher biomass concentration with the < 20 µm fraction (Fig. S3).

These analyses prove that despite a longer lag phase, fine particles (< 20 µm) did not inhibit bacterial growth; rather, they promoted it, resulting in a higher growth rate.

### 3.3.2. Particle size effect on bacterial community profile

To assess if particle size impacts the bacterial community profile, the relative abundance of the strains was monitored during the bioleaching

experiments. Fig. 10 compares the proportion of *L. ferriphilum*, *F. caldus* and *Sulfobacillus* spp. in the inoculum and in the pulp sampled at two different times during the bioleaching experiments (during and after the exponential phase).

The community profile of the inoculum comprised ‘*F. caldus*’ (36 %) (sulfur-oxidizing), *L. ferriphilum* (42 %) (iron-oxidizing) and the iron- and sulfur-oxidizing species from the genus *Sulfobacillus* (22 %) (probably *S. thermosulfidooxidans* and *S. benefaciens* which were identified in the initial BRGM-KCC consortium). Their relative abundance varied slightly between the start and the end of the experiments. Relative abundance of *Sulfobacillus* increased with time, while the relative abundance of ‘*F. caldus*’ decreased substantially. The proportion of *L. ferriphilum* remained quite stable over the experiments. *Sulfobacillus* is a mixotroph and can grow on inorganic (CO₂) or organic carbon, while ‘*F. caldus*’ and *L. ferriphilum* are strict autotrophs, which can only use CO₂ as a source of carbon. *Sulfobacillus* is known to use cellular debris and other organic components produced by other microorganisms as substrate, which can promote its growth as cells are being lysed with time during the experiments. *Sulfobacillus* was not detected during the exponential phase of the bioleaching experiments with the raw concentrate. As t-RFLP resolution can only identify the most abundant bacteria species, the relative abundance of *Sulfobacillus* was therefore too low compared to the other two strains for it to be detected. The evolution of the bacterial community profile is consistent with that of previous reports on this type of consortium (Hedrich et al., 2016). As the bacterial profiles of the three experiments exhibited similar evolution, it can be concluded that particle size had little effect on the bacterial community.

Relative abundance was also monitored in the biomass attached to the solid particles and in the biomass present in the liquid phase (see Fig. S4 and Fig. S5, respectively). *L. ferriphilum* was predominant in the solid phase, except for the finer fraction during the exponential phase. In the liquid phase, the bacterial community was mainly composed of the species ‘*F. caldus*’ at the beginning (exponential phase), then was co-dominated by the three bacteria identified in the inoculum. The dominance of ‘*F. caldus*’ in the liquid phase has already been observed in the literature (Guezennec et al., 2017). Our results show that location of bacterial species between the solid and liquid phases was not affected by particle size, and thus the attachment capacity of each species to the particle surface was not significantly modified.

### 3.3.3. Particle size effect on bacterial activity

The increase in Fe³⁺ concentration over time during the bioleaching experiments (Fig. 6) indicates that iron-oxidizing bacteria were active. To compare the bacterial activity in the three experiments, the bio-oxidation kinetics of Fe²⁺ and sulfur species (reactions [3] and [4]) were derived from mass balance considerations. Indeed, the kinetics of Fe and S bio-oxidation cannot be determined solely from the evolution of dissolved Fe³⁺ and SO₄²⁻ during the bioleaching experiments. This is because Fe³⁺ is consumed by the oxidation of chalcopyrite and pyrite (Eq. [2], [14], [15]), as well as by the precipitation as jarosite, which also consumes SO₄²⁻ (Eq. [5]).

The calculation protocol implemented to quantify the cumulative amount of Fe³⁺ and SO₄²⁻ produced is described in the **supplementary materials**. The amounts of precipitated Fe³⁺ and SO₄²⁻ could not be measured during the bioleaching experiments, only at the end when the residue was analyzed. Therefore, they were initially not included in the calculations. Fig. 11 shows the kinetics of cumulated Fe³⁺ production by bio-oxidation (Eq. [A] in **supplementary materials**).

Overall, the cumulated Fe³⁺ production was much higher than the Fe³⁺ concentration in the liquid phase (Fig. 6). With the particles below 20 µm, the maximum cumulated production of Fe³⁺ reached around 60 g.L⁻¹ whereas the maximum dissolved Fe³⁺ concentration did not exceed 10 g.L⁻¹. Regardless of the initial particle size, oxidation kinetics of Fe²⁺ to Fe³⁺ can be divided into two phases. First, Fe²⁺ was quickly oxidized. Then the production of Fe³⁺ slowed down.
<figure><img src="image_13.png" /><figcaption>Fig. 10. Relative abundance of strains in (a) the inoculum and during the exponential and stationary phases of bacterial growth, during bioleaching experiments with (b) the 20–100 µm fraction, (c) the raw concentrate and (d) the < 20 µm fraction. The results were obtained from pulp samples.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>Fig. 11. Cumulated Fe³⁺ produced (Fe³⁺prod) during bioleaching experiments with the 20–100 µm fraction, the raw concentrate and the < 20 µm fraction. The amounts of Fe³⁺ produced were calculated according to equation [A] (described in supplementary materials), which does not take into account Fe³⁺ precipitation as jarosite.</figcaption></figure>

Fe²⁺ oxidation kinetics was much slower with the 20–100 µm and the raw concentrate than with the finer fraction. The maximum oxidation rates were respectively 7 gFe.L⁻¹.d⁻¹ and 22 gFe.L⁻¹.d⁻¹. The latter is of the same order of magnitude as the maximum Fe²⁺ bio-oxidation rate measured by Hubau (2019) with the same consortium in continuous mode (21 gFe.L⁻¹.d⁻¹). In Hubau's (2019) study, microorganisms were used to oxidize dissolved Fe²⁺ sulfate salts in a bubble column containing a neutral solid support to which bacteria could attach. The bacterial cells were not subjected to hydrodynamic stress caused by solid particles or mechanical agitation. Therefore, the maximum oxidation rate of Fe²⁺ with fine particles (22 gFe.L⁻¹.d⁻¹) was similar to that

obtained under conditions more favorable to the growth and activity of iron-oxidizing bacteria (21 gFe.L⁻¹.d⁻¹).

The cumulated production of SO₄²⁻ (Fig. 12) was evaluated according to equation [C] in [supplementary materials]. This evaluation did not take into account SO₄²⁻ precipitation in order to assess sulfur oxidation kinetics.

During bioleaching of the 20–100 µm fraction and the raw concentrate, sulfur oxidation kinetics were similar to those observed for Fe²⁺ oxidation (Fig. 11). Sulfur was oxidized faster during the first week. Then, from the 7th day onward, only a small amount of SO₄²⁻ was produced. With the < 20 µm fraction, the sulfur oxidation rate was first equal to the one observed initially with the other two materials, then it increased before decreasing quickly. This decrease in sulfur oxidation rate was consistent with the copper and iron dissolution rates, or, in

<figure><img src="image_15.png" /><figcaption>Fig. 12. Cumulated SO₄²⁻ produced (SO₄²⁻prod) during bioleaching experiments with the 20–100 µm fraction, the raw concentrate and the < 20 µm fraction.</figcaption></figure>
other words, with the sulfide oxidation rate.

The maximum sulfur oxidation rate was 7 times higher with the finer fraction (7.2 g.L⁻¹.d⁻¹) than with the 20–100 µm fraction and the raw concentrate (0.9 g.L⁻¹.d⁻¹). It was also faster than the ones measured in literature for elemental sulfur oxidation at 45 °C, at pH 1.5 (0.55 g.L⁻¹.d⁻¹) and at pH 2 (0.75 g.L⁻¹.d⁻¹) (Plumb et al., 2008) or at 40 °C at pH 2.5 (0.83 g.L⁻¹.d⁻¹) (Basitere et al., 2012) using *F. caldus*. On the other hand, the maximum sulfur oxidation rates obtained with the 20–100 µm fraction and the raw concentrate were comparable with those reported in the literature, where the operating conditions promoted growth and activity of sulfur-oxidizing bacteria. Therefore, microbial sulfur oxidation with < 20 µm particles was likely enhanced due to higher sulfur production from chalcopyrite oxidation.

The production of Fe³⁺ calculated with equation [A] was underestimated since Fe³⁺ precipitation was not taken into account. The production of jarosite could only be determined at the end of the bioleaching tests. Therefore, to assess the previous results, the total amount of Fe³⁺ produced at the end of the experiment was calculated from the composition of the initial material and the residues, which accounts for Fe³⁺ precipitation (Eq. [D] in supplementary materials). The amount of precipitated SO₄²⁻ was also only determined at the end of the experiments. Therefore, the final cumulative amount of SO₄²⁻ produced at the end of the experiments was recalculated, taking into account SO₄²⁻ precipitation (Table 7).

Table 6 shows that 14 % to 30 % of the final amount of Fe³⁺ produced was overlooked when Fe³⁺ precipitation was not considered. Nevertheless, the finer < 20 µm fraction produced 1.6 times more Fe³⁺ than the other two materials. Because the amount of chalcopyrite and pyrite oxidized with the < 20 µm fraction was much higher than with the 20–100 µm fraction and the raw concentrate, a much higher amount of Fe³⁺ produced could have been expected. However, pyrite concentration in the < 20 µm was half that of the other materials (Table 2). Pyrite oxidation consumed more Fe³⁺ than chalcopyrite oxidation (equations [14] and [15]), which can explain the obtained results. Mineral composition of the initial material is therefore an important factor in understanding Fe³⁺ consumption.

On the other hand, the amount of precipitated SO₄²⁻ was low compared to the amount of precipitated Fe³⁺. The highest amount of precipitated SO₄²⁻ was obtained with the finer fraction. At the end of this experiment, the amount of precipitated SO₄²⁻ was 7,1 g.L⁻¹, which accounted for 13 % of the total SO₄²⁻ produced (Table 7). Even if it slightly underestimates the data, equation [C] is therefore considered as adequate way to evaluate SO₄²⁻ production (sulfur oxidation) kinetics.

It can finally be deduced that fine particles (< 20 µm) did not hinder bacterial growth. Rather, they promoted faster oxidation of sulfides, providing more Fe²⁺ and sulfur substrates for bacterial activity, which in turn increased. Nevertheless, the enhanced production of Fe³⁺ and SO₄²⁻ can promote jarosite precipitation and, eventually, chalcopyrite passivation.

## 4. Discussion

The work carried out in this study provides further insight into the effect of particle size, particularly that of fine particles, on chalcopyrite

<table><caption>**Table 7**
Fe³⁺ and SO₄²⁻ produced (g.L⁻¹) at the end of bioleaching tests carried out with the 100–20 µm fraction, the raw concentrate and the < 20 µm fraction according to equation [A], equation [C] or equation [D] described in supplementary materials.</caption><thead><tr><th>Amount of Fe³⁺ and SO₄²⁻ (g.L⁻¹)</th><th>20–100 µm</th><th>raw</th><th>&lt; 20 µm</th></tr></thead><tbody><tr><td>Fe³⁺ (Eq. [A])</td><td>54.4</td><td>46.8</td><td>74.6</td></tr><tr><td>Fe³⁺ with precipitation (Eq. [D])</td><td>67.6</td><td>54.5</td><td>107.1</td></tr><tr><td>SO₄²⁻ (Eq. [C] without precipitation)</td><td>23.9</td><td>23.2</td><td>49.6</td></tr><tr><td>SO₄²⁻ (Eq. [C] with precipitation)</td><td>25.3</td><td>24.2</td><td>56.7</td></tr></tbody></table>

bioleaching.

First, increasing the amount of particles finer than 20 µm resulted in faster chalcopyrite oxidation and hence higher yields during the bioleaching process. This phenomenon can be attributed to the higher specific surface area, which is 8 to 17 times greater. High reactive surfaces accelerate mineral dissolution and increase the number of sites available for bacterial attachment (Jonglertjunya, 2003). A more efficient chalcopyrite oxidation with fine particles has often been reported in literature in the case of chemical leaching (Onisei et al., 2012; Tešinský and Baláž, 2017; Granata et al., 2019; Cao et al., 2020) and bioleaching (Nemati et al., 2000; Jonglertjunya, 2003; Esmailbagi et al., 2018).

We also evidenced that the evolution of the redox potential over time was affected by the amount of fine particles in the substrate. Redox potential was directly related to the Fe³⁺/Fe²⁺ ratio (Eq. [6]). Consequently, the evolution of the redox potential was related to Fe³⁺ consumption and production rates, or, in other words, sulfides and Fe²⁺ oxidation rates (Eq. [2], [14] and [15], and equation [3] respectively).

During the bioleaching experiments carried out in this study, the redox potential was first below 600 mV vs. SHE and the Fe³⁺/Fe²⁺ ratio below 0.05 (i.e. iron in solution was mostly as Fe²⁺), regardless of particle size. Then, chalcopyrite and pyrite dissolution started (increase of Fe²⁺ in solution) (Fig. 4) together with an increase in biomass concentration over time (Fig. 9), which promoted the oxidation of Fe²⁺ into Fe³⁺. Then, the Fe²⁺ bio-oxidation rate increased and exceeded the Fe³⁺ consumption rate. This resulted in a sharp rise in the Fe³⁺/Fe²⁺ ratio that changed the redox potential (Fig. 5). The rise in redox potential (up to 600–750 mV vs. SHE) led to a strong drop in chalcopyrite dissolution rate. These results are consistent with the ones obtained by Córdoba et al. (2008b) and Gericke et al. (2010). Indeed, Gericke et al. (2010) identified a threshold between 633 and 643 mV vs. SHE (at 45 °C, 1 atm) above which chalcopyrite dissolution yield decreases due to the mineral's slower oxidation. A similar passivation limit, around 643 mV vs. SHE, was observed by Córdoba et al. (2008b) during chemical leaching of chalcopyrite. They showed that fast oxidation of Fe²⁺ into Fe³⁺ can promote chalcopyrite oxidation only if the Fe³⁺ produced is rapidly consumed, keeping the redox potential (or Fe³⁺/Fe²⁺ ratio) below the passivation threshold. This phenomenon was observed at the beginning of the bioleaching experiment with the < 20 µm fraction: the chalcopyrite oxidation kinetics was probably faster than with coarser particles due to higher reactive surface area and faster than Fe²⁺ bio-oxidation kinetics. The result was a delay in the increase of the Fe³⁺/Fe²⁺ ratio, which maintained the redox potential below the passivating threshold for a longer time. Using fine particles (< 20 µm) provided not only larger reactive surface for oxidation of the mineral and bacterial attachment, but also delayed chalcopyrite passivation.

This study provides new insights into the effects of fine particles (< 20 µm) on acidophilic bacteria used in bioleaching. In literature, there is very little data on the impact of particle size on these microorganisms. As previously mentioned, Nemati et al. (2000) proposed an explanation for the lower yields obtained when decreasing particle size. They suggested that fine particles damage cells and inhibit the activity of archaea. Based on this proposition, other studies assumed that fine particles could have similar negative impact on sulfur- and iron-oxidizing bacteria (Olubambi et al., 2009; Manafi et al., 2021). However, thermophilic archaea are known to have a cell wall that is less resistant to shear stress than bacteria. Our work, which relies on a consortium mainly composed of moderate thermophilic and mesophilic bacteria, which are commonly used in bioleaching operations, provides new insights on their resistance to shear stress and on the impact of particle size on their growth and activity. We demonstrated that, during bioleaching experiments with the < 20 µm fraction, the biomass concentration was one order of magnitude higher than in experiments with coarser size fractions. The same was observed for the maximal growth rate (6 x 10¹⁰ cells.mL⁻¹.d⁻¹ vs. 6 x 10⁹ cells.mL⁻¹.d⁻¹). In addition to biomass growth, the experiments with the fine particles also improved
microbial activity, which is linked to bio-oxidation kinetics. The oxidizing rates of sulfur and Fe²⁺ were respectively 7.2 gS.L⁻¹.d⁻¹ and 22 gFe.L⁻¹.h⁻¹, whereas they reached only 0.04 gS.L⁻¹.h⁻¹ and 0.29 gFe.L⁻¹. h⁻¹ with coarser particles. Furthermore, the maximum oxidation rates of Fe²⁺ and sulfur were similar to or higher than those measured under conditions specifically chosen to promote the growth of either iron- or sulfur-oxidizing bacteria (no solid particles, weak shear stress, etc.). Contrary to what has been suggested in the literature, our results demonstrate that fine particles (less than 20 µm, $d_{80} = 10$ µm) do not hinder the growth or activity of the moderate thermophilic and mesophilic bacteria used in this study, unlike archaea, which have been shown to be inhibited under such conditions.

It can be noted that most of the studies investigating the effect of particle size during bioleaching of metal-bearing sulfides use a freshly ground material (Esmailbagi et al., 2018; Manafi et al., 2021). These studies suggest that microbial viability might be affected by ROS generated during the grinding of these substrates. According to Jones et al. (2011), the amount of ROS produced increases with grinding intensity, and thus, with particle size reduction. It also depends on the nature of the material. For example, less ROS is produced with chalcopyrite than with pyrite. In our case, the raw chalcopyrite concentrate contained 70.8 %w/w chalcopyrite and 8.9 %w/w of pyrite only, while the initial material used by Manafi et al. (2021) contained 44 %w/w chalcopyrite and 25 %w/w pyrite. Furthermore, in our study, the chalcopyrite used was obtained from an industrial copper concentrate, which was finely ground several months before being prepared as described in Fig. 1. Therefore, the substrates used for the bioleaching experiments probably did not contain ROS, unlike other studies that used freshly ground materials. This highlights the importance of the mineralogy of the material to be leached and the process used to prepare it.

Furthermore, at the end of the bioleaching experiments, measurement of biomass concentration in the pulp was also carried out by measuring gene copy numbers using qPCR (Fig. S3). This measurement was then compared to the measurement taken in the liquid phase. Gene abundance was almost always higher in the pulp than in the liquid phase. The difference was attributed to the biomass attached to particles (in the solid phase), which was six times greater with the < 20 µm fraction than with the 20–100 µm fraction or the raw concentrate. This might be due to the increased solid surface area, which promoted bacterial attachment and the oxidation of sulfide minerals. However, irrespective of particle size, the dominant species in the solid phase was *L. ferriphilum* (except at the start of the test with the fraction < 20 µm). This species is known to be the first to colonize particles when co-cultured with *F. caldus* (Noël et al., 2010). Furthermore, the relative abundance of *L. ferriphilum* in the pulp was similar from one experiment to another. Since the biomass amount was greater with the < 20 µm fraction, this means that the number of bacteria from this species was greater with this fraction. *L. ferriphilum* being dominant in the solid phase, reducing the particle size (i.e increasing the solid surface area) could promote the growth of the *L. ferriphilum* cells attached to the surface of the sulfide particles, where it can regenerate the Fe³⁺ consumed, promoting sulfide oxidation.

## 5. Conclusion

Bioleaching experiments carried out in our study showed that fine particles (< 20 µm) allow for the rapid oxidation of chalcopyrite and, consequently, the rapid consumption of Fe³⁺. These two phenomena combined delayed the passivation of the mineral by keeping the Fe³⁺/Fe²⁺ ratio (or redox potential) below the passivation threshold. This enhanced the copper dissolution yield by two to five times.

Contrary to what is often suggested in the literature, fine particles (less than 20 µm) did not hinder or inhibit the growth or activity of moderate thermophilic and mesophilic iron- or sulfur-oxidizing bacteria, but rather promoted them. Greater sulfide oxidation allowed for

more Fe²⁺ and sulfur to be available for bacterial growth. Consequently, the biomass was more abundant and more active. Furthermore, the presence of fine particles did not affect the evolution of the bacterial community profile over time.

Although decreasing particle size enabled greater oxidation of chalcopyrite, passivation of the mineral was only delayed as long as Fe²⁺ oxidation rate was faster than chalcopyrite oxidation. In our study only half of the chalcopyrite was dissolved after 15 days with particles finer than 20 µm. To further enhance chalcopyrite dissolution, bioleaching of fine particles must be coupled with another technique that prevents or remediates to chalcopyrite passivation, such as redox potential control or the removal of the passivating layer by attrition-leaching (Dakkoune et al., 2023).

## CRediT authorship contribution statement

**Adeline Po:** Writing – original draft, Visualization, Validation, Methodology, Investigation. **Laurent Cassayre:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization. **Agathe Hubau:** Writing – review & editing. **Catherine Joulian:** Writing – review & editing, Validation, Methodology, Investigation. **Amine Dakkoune:** Writing – review & editing. **Solène Touze:** Writing – review & editing, Methodology. **Carine Julcour:** Writing – review & editing, Methodology. **Florent Bourgeois:** Writing – review & editing, Methodology. **Anne-Gwénaëlle Guezennec:** Writing – review & editing, Validation, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This work was part of the project BIOMECALIX funded by the French National Research Agency under Grant n° ANR-18-CE07-0031-01. Boliden is greatly acknowledged for the supply of chalcopyrite concentrate. The authors thank the technical support of Pascal Auger, Mickaël Beaulieu and Jérôme Bodin for their help with the solid preparation and characterization, as well as Hafida Tris and Guylian Laurent for their help with the biomolecular analyses.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mineng.2025.109885.

## Data availability

Data will be made available on request.

## References

* Abdollahi, H., Noaparast, M., Shafaei, S.Z., Manafi, M., Muñoz, J.A., Tuovinen, O.H., 2015. Silver-Catalyzed Bioleaching of copper, molybdenum and rhenium from a chalcopyrite-molybdenite concentrate. Int. Biodeter. Biodegr. 104 (October), 194–200. https://doi.org/10.1016/j.ibiod.2015.05.025.

* Ahmadi, A., Schaffie, M., Manafi, Z., Ranjbar, M., 2010. Electrochemical bioleaching of high grade chalcopyrite flotation concentrates in a stirred bioreactor. Hydrometall. 104, 99–105. https://doi.org/10.1016/j.hydromet.2010.05.001.

* Bafghi, M.S., Emami, A.H., Zakeri, A., 2013. Effect of specific Surface area of a mechanically activated chalcopyrite on its rate of leaching in sulfuric Acid-Ferric Sulfate Media. Metall. Mater. Trans. B 44, 1166–1172. https://doi.org/10.1007/s11663-013-9890-0.

* Basitere, M., Ntwampe, S.K.O., Sheldon, M.S., 2012. Optimal sulphuric acid production using Acidithiobacillus caldus (DSM 8584): Bioprocess design for application in ion-
exchange. Afr. J. Biotechnol. 11, 14946-14955. https://doi.org/10.4314/ajb.v11i83.

Batty, J.D., Rorke, G.V., 2006. Development and commercial demonstration of the BioCOPTM thermophile process. Hydrometall. 83, 83-89. https://doi.org/10.1016/j.hydromet.2006.03.049.

Brierley, C.L., Brierley, J.A., 2013. Progress in Bioleaching: Part B: applications of Microbial Processes by the Minerals Industries. Appl. Microbiol. Biotechnol. 97, 7543-7552. https://doi.org/10.1007/s00253-013-5095-3.

Cao, S.-T., Zheng, X.-F., Nie, Z.-Y., Zhou, Y.-H., Liu, H.-C., Chen, J.-H., Yang, H.-Y., Xia, J.-L., 2020. Mechanical activation on bioleaching of chalcopyrite: a new insight. Minerals 10, 788. https://doi.org/10.3390/min10090788.

Córdoba, E.M., Muñoz, J.A., Blázquez, M.L., González, F., Ballester, A., 2008a. Leaching of chalcopyrite with ferric iron. Part i: General Aspects. Hydrometallurgy 93, 81-87. https://doi.org/10.1016/j.hydromet.2008.04.015.

Córdoba, E.M., Muñoz, J.A., Blázquez, M.L., González, F., Ballester, A., 2008b. Leaching of chalcopyrite with ferric iron. Part II: effect of redox potential. Hydrometall. 93, 88-96. https://doi.org/10.1016/j.hydromet.2008.04.016.

Dakkoune, A., Bourgeois, F., Po, A., Joulian, C., Hubau, A., Touzé, S., Julcour, C., Guezennec, A.-G., Cassayre, L., 2023. Hydrometallurgical Processing of Chalcopyrite by Attrition-aided Leaching. ACS Eng. Au 3, 195-209. https://doi.org/10.1021/acsengineeringau.2c00051.

Deveci, H., 2004. Effect of particle size and shape of solids on the viability of acidophilic bacteria during mixing in stirred tank reactors. Hydrometall. 71, 385-396. https://doi.org/10.1016/S0304-386X(03)00112-9.

Dutrizac, J.E., Jambor, J.L., 2000. Jarosites and their Application in Hydrometallurgy. Rev. Mineral. Geochem. 40, 405-452. https://doi.org/10.2138/rmg.2000.40.8.

Esmailbagi, M.R., Schaffie, M., Kamaybi, A., Ranjbar, M., 2018. Microbial assisted galvanic leaching of chalcopyrite concentrate in continuously stirred bioreactors. Hydrometall. 180, 139-143. https://doi.org/10.1016/j.hydromet.2018.07.020.

Gericke, M., Govender, Y., Pinches, A., 2010. Tank bioleaching of low-grade chalcopyrite concentrates using redox control. Hydrometall. 104, 414-419. https://doi.org/10.1016/j.hydromet.2010.02.024.

Granata, G., Takahashi, K., Kato, T., Tokoro, C., 2019. Mechanochemical activation of chalcopyrite: Relationship between activation mechanism and leaching enhancement. Miner. Eng. 131, 280-285. https://doi.org/10.1016/j.mineng.2018.11.027.

Guezennec, A.-G., d'Hugues, P., 2015. Bioprocessing low grade copper ores - a promising alternative In 13th SGA Bienn. Meet, Georesources Nancy, Nancy, France https://hal.brgm.archives-ouvertures.fr/hal-01154886.

Guezennec, A.-G., Joulian, C., Jacob, J., Archane, A., Ibarra, D., de Buyer, R., Bodénan, F., d'Hugues, P., 2017. Influence of dissolved oxygen on the bioleaching efficiency under oxygen enriched atmosphere. Miner. Eng. 106, 64-70. https://doi.org/10.1016/j.mineng.2016.10.016.

Guezennec, A.-G., Joulian, C., Delort, C., Bodénan, F., Hedrich, S., d'Hugues, P., 2018. CO₂ mass transfer in bioleaching reactors: CO₂ enrichment applied to a complex copper concentrate. Hydrometall. 180, 277-286. https://doi.org/10.1016/j.hydromet.2018.08.006.

Hedrich, S., Guézennec, A.-G., Charron, M., Schippers, A., Joulian, C., 2016. Quantitative monitoring of Microbial Species during Bioleaching of a Copper Concentrate. Front. Microbiol. 07 (December). https://doi.org/10.3389/fmicb.2016.02044.

Hedrich, S., Joulian, C., Graupner, T., Schippers, A., Guézennec, A.-G., 2018. Enhanced chalcopyrite dissolution in stirred tank reactors by temperature increase during bioleaching. Hydrometall. 179, 125-131. https://doi.org/10.1016/j.hydromet.2018.05.018.

Hourn, M., et Turner, D. W., 2012. Commercialisation of the Albion process. In: Proceedings of the ALTA 2012 gold conference (Vol. 19).

Hubau, A., 2019. Conception d'un procédé de biolivivation pour la valorisation des métaux contenus dans les déchets de cartes électroniques. Université Paris sciences et lettres, Thèse.

Hubau, A., Pino-Herrera, D.O., Falagán, C., Hudson-Edwards, K., Joulian, C., Guezennec, A.-G., 2023. Influence of the nutrient medium composition during the bioleaching of polymetallic sulfidic mining residues. Waste Biomass Valoriz. https://doi.org/10.1007/s12649-023-02090-y.

International Copper Study Group (ICSG). 2023. The World Copper Factbook 2023. International Copper Study Group. https://icsg.org/copper-factbook/.

Jones, G.C., Corin, K.C., van Hille, R.P., Harrison, S.T.L., 2011. The generation of toxic reactive oxygen species (ROS) from mechanically activated sulphide concentrates and its effect on thermophilic bioleaching. Miner. Eng. 24, 1198-1208. https://doi.org/10.1016/j.mineng.2011.05.016.

Jonglertjunya, W., 2003. Bioleaching of chalcopyrite. The University of Birmingham, These.

Kartal, M., Xia, F., Ralph, D., Rickard, W. D.A., Renard, F., Li, W., 2020. "Enhancing Chalcopyrite Leaching by Tetrachloroethylene-Assisted Removal of Sulphur Passivation and the Mechanism of Jarosite Formation". Hydrometallurgy 191 (January): 105192. doi: 10.1016/j.hydromet.2019.105192.

Khoshkhoo, M., Dopson, M., Engström, F., Sandström, Å., 2017. New insights into the influence of redox potential on chalcopyrite leaching behaviour. Miner. Eng. 100, 9-16. https://doi.org/10.1016/j.mineng.2016.10.003.

Kinnunen, P.-H.M., Puhakka, J.A., 2005. High-rate iron oxidation at below pH 1 and at elevated iron and copper concentrations by a *Leptospirillum ferriphilum* dominated biofilm. Process Biochem. 40, 3536-3541. https://doi.org/10.1016/j.procbio.2005.03.050.

Klauber, C., 2008. A critical review of the surface chemistry of acidic ferric sulphate dissolution of chalcopyrite with regards to hindered dissolution. Int. J. Miner. Process. 86, 1-17. https://doi.org/10.1016/j.minpro.2007.09.003.

Li, Y., Kawashima, N., Li, J., Chandra, A.P., Gerson, A.R., 2013. A review of the structure, and fundamental mechanisms and kinetics of the leaching of chalcopyrite. Adv. Colloid Interface Sci. 197-198, 1-32. https://doi.org/10.1016/j.cis.2013.03.004.

Li, Y., Tian, Z., Wang, X., Wen, J., Mao, Q., Yang, C., 2025. Enhanced chalcopyrite bioleaching with mechanical activation and redox potential regulation. Miner. Eng. 227, 109292. https://doi.org/10.1016/j.mineng.2025.109292.

Liu, C., Jia, Y., Sun, H., Tan, Q., Niu, X., Leng, X., Ruan, R., 2017. Limited role of sessile acidophiles in pyrite oxidation below redox potential of 650 mV. Sci. Rep. 7, 5032. https://doi.org/10.1038/s41598-017-04420-2.

Lotfalian, M., Ranjbar, M., Fazaelipoor, M.H., Schaffie, M., Manafi, Z., 2015. The effect of redox control on the continuous bioleaching of chalcopyrite concentrate. Miner. Eng. 81, 52-57. https://doi.org/10.1016/j.mineng.2015.07.006.

Manafi, Z., Kargar, M., Kafilzadeh, F., 2021. Tank bioleaching of a copper concentrate using the moderately thermophilic microorganisms Sulfobacillus thermosulfidoxidans KMM3 and Sulfobacillus acidophilus KMM26. Rev. Metal. 57 (4), e207.

Mariano, R.A., Evans, C.L., Manlapig, E., 2016. Definition of random and non-random breakage in mineral liberation - a review. Miner. Eng. 94, 51-60. https://doi.org/10.1016/j.mineng.2016.05.005.

McMillan, R.S., MacKinnon, D.J., Dutrizac, J.E., 1982. Anodic dissolution of n-type and p-type chalcopyrite. J. Appl. Electrochem. 12, 743-757. https://doi.org/10.1007/BF00617495.

Nazari, G., Asselin, E., 2009. Morphology of chalcopyrite leaching in acidic ferric sulfate media. Hydrometall. 96, 183-188. https://doi.org/10.1016/j.hydromet.2008.09.004.

Nazari, G., Dixon, D.G., Dreisinger, D.B., 2012. The Role of silver-enhanced pyrite in enhancing the electrical conductivity of sulfur product layer during chalcopyrite leaching in the Galvanox TM Process. Hydrometall. 113-114 (February), 177-184. https://doi.org/10.1016/j.hydromet.2011.12.024.

Nemati, M., Lowenadler, J., Harrison, S.T.L., 2000. Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC). Appl. Microbiol. Biotechnol. 53, 173-179. https://doi.org/10.1007/s0025300050005.

Noël, N., Florian, B., Sand, W., 2010. AFM & EFM study on attachment of acidophilic leaching organisms. Hydrometall. 104, 370-375. https://doi.org/10.1016/j.hydromet.2010.02.021.

O'Connor, G.M., Eksteen, J.J., 2020. A critical review of the passivation and semiconductor mechanisms of chalcopyrite leaching. Miner. Eng. 154, 106401. https://doi.org/10.1016/j.mineng.2020.106401.

Olubambi, P.A., Potgieter, J.H., Ndlovu, S., Borode, J.O., 2009. Electrochemical studies on interplay of mineralogical variation and particle size on bioleaching low grade complex sulphide ores. Trans. Nonferrous Met. Soc. Chin. 19, 1312-1325. https://doi.org/10.1016/S1003-6326(08)60443-4.

Onisei, S., Badilita, V., Stoiciu, F., Velea, T., Predica, V., Lupu, C., Butu, M., Moldovan, P., 2012. Mechanistic Activation of Copper Concentrate and the effect on Oxidation of Metal Sulphides. Rev. Chim. 6, 591-597.

Plumb, J.J., Muddle, R., Franzmann, P.D., 2008. Effect of pH on rates of iron and sulfur oxidation by bioleaching organisms. Miner. Eng. 21, 76-82. https://doi.org/10.1016/j.mineng.2007.08.018.

Rawlings, D.E., 2005. Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microb. Cell Fact. 4, 13. https://doi.org/10.1186/1475-2859-4-13.

Ren, Z., Chao, C., Krishnamoorthy, P., Asselin, E., Dixon, D.G., Mora, N., 2022. The overlooked mechanism of chalcopyrite passivation. Acta Mater. 236, 118111. https://doi.org/10.1016/j.actamat.2022.118111.

Sasaki, K., Nakamura, Y., Hirajima, T., Tuovinen, O.H., 2009. Raman characterization of secondary minerals formed during chalcopyrite leaching with Acidithiobacillus ferrooxidans. Hydrometall. 95, 153-158. https://doi.org/10.1016/j.hydromet.2008.05.009.

Tesinsky, M., Baláž, P., 2017. Copper Leaching from Chalcopyrite: Mechanochemical Approac. Journal of the Polish Mineral Engineering Society. https://doi.org/10.29227/IM-2017-01-01.

Wen, T., Zhao, Y., Ma, Q., Xiao, Q., Zhang, T., Chen, J., Shaoxian Song, S., 2020. Microwave improving Copper Extraction from Chalcopyrite through Modifying the Surface Structure. J. Mater. Res. Technol. 9 (1), 263-270. https://doi.org/10.1016/j.jmrt.2019.10.054.

Yue, G., Zhao, L., Olvera, O.G., Asselin, E., 2014. Speciation of the H2SO4-Fe2SO4 3-FeSO4-H2O system and development of an expression to predict the redox potential of the Fe3+/Fe2+ couple up to 150°C. Hydrometall. 147-148, 196-209. https://doi.org/10.1016/j.hydromet.2014.05.008.

Yue, G., Guezennec, A.-G., Asselin, E., 2016. Extended validation of an expression to predict ORP and iron chemistry: Application to complex solutions generated during the acidic leaching or bioleaching of PCBs. Hydrometall. 164, 334-342. https://doi.org/10.1016/j.hydromet.2016.06.027.

Zhao, H., Wang, J., Tao, L., Cao, P., Yang, C., Qin, W., Qiu, G., 2017. Roles of oxidants and reductants in bioleaching system of chalcopyrite at normal atmospheric pressure and 45 °C. Int. J. Miner. Process. 162, 81-91. https://doi.org/10.1016/j.minpro.2017.04.002.

Zhao, H., Zhang, Y., Zhang, X., Qian, L., Sun, M., Yang, Y., Zhang, Y., Wang, J., Kim, H., Qiu, G., 2019. The dissolution and passivation mechanism of chalcopyrite in bioleaching: an overview. Miner. Eng. 136, 140-154. https://doi.org/10.1016/j.mineng.2019.03.014.

Zhao, S., Wang, G., Yang, H., Chen, G., Qiu, X., 2021. Agglomeration-aggregation and leaching properties of mechanically activated chalcopyrite. Trans. Nonferrous Met. Soc. Chin. 31, 1465-1474. https://doi.org/10.1016/S1003-6326(21)65590-5.

Zhu, W., Xia, J., Yang, Y., Nie, Z., Zheng, L., Ma, C., Zhang, R., Peng, A., Tang, L., Qiu, G., 2011. Sulfur oxidation activities of pure and mixed thermophiles and sulfur
speciation in bioleaching of chalcopyrite. *Bioresour. Technol.* 102, 3877–3882.

https://doi.org/10.1016/j.biortech.2010.11.090.