Study of Gas-Liquid Transfer of O₂ and CO₂ on
Bioleaching in CSTRs to Recover Critical Raw Materials
from Mine Waste

Douglas Pino-Herrera¹*[0000-0002-9533-2889], Anne-Gwénaëlle Guezennec¹[0000-0003-3259-6366]

Catherine Joulian¹[0000-0003-3834-2551], Mickaël Beaulieu¹, Jérémy Engevin¹,
Mickaël Charron¹, and Françoise Bodénan¹[0000-0001-6518-3336]

¹ BRGM, F-45060 Orléans, France
d.pinoherrera@brgm.fr

**Abstract.** Bioleaching is a promising and feasible option for metal extraction from sulfidic mine wastes. The main objective of this study was to investigate how the gas-liquid mass transfer phenomena affect the bioleaching of sulfidic mining wastes, with the ultimate goal of optimizing the gas supply. Experiments were performed in four continuous stirred tank reactors (CSTRs) arranged in series with a total working volume of 114-L. Three different gas compositions were tested: I) air, II) air enriched with 0.5% v/v CO₂ and III) a mixture of N₂ (69.5% v/v), O₂ (30% v/v), and CO₂ (0.5% v/v) in the first two reactors of the series. Results showed that during Condition I, the CO₂ transfer rate was not sufficient, particularly in the primary reactor resulting in a low sulfide dissolution yield. Enriching air with 0.5% v/v of CO₂ during Condition II increased both sulfide dissolution in the primary reactor and oxygen uptake rate (OUR) in the entire system, which caused oxygen transfer limitations. Sulfide dissolution was further enhanced during Condition III due to increased partial pressure of O₂ in the first and second reactors, achieving a total cumulative sulfide dissolution yield of 81% and the dissolution of 86% of Co and 30% of Sb contained in the MW. This study emphasizes the importance of sufficient gas supply in the bioleaching process and demonstrates that gas-liquid mass transfer considerations are crucial for upscaling and cost optimization.

**Keywords:** Bioleaching, Gas-liquid Mass Transfer, Critical Raw Materials, Mine Wastes.

# 1 Introduction

The current transition to sustainable energies has caused a fast-growing need for critical raw materials (CRMs) in Europe and mining waste (MW) is now considered a potential secondary resource. However, traditional treatments may not be economically or technically feasible for MW, especially when the targeted metals are in low concentrations. Project RAWMINA, funded by the EU's H2020 research program (Grant Agreement N° 958252), proposes an innovative pathway to recover CRMs such as Co, Ge, Sb and W from MW. One of the main technologies included in the
RAWMINA process is bioleaching – as a first step – a promising and feasible option for metal extraction from sulfidic MW. Bioleaching allows the dissolution of sulfidic minerals with the help of microorganisms leading to the dissolution of elements in the solution.

In bioleaching, the consumption of oxygen is directly linked to the sulfide dissolution rate as can be seen in equation (1), which corresponds to the reaction of pyrite dissolution.

$$FeS_2 + \frac{15}{4} O_2 + \frac{1}{2} H_2 O = Fe^{3+} + 2 SO_4^{2-} + H^+ \quad (1)$$

This means that mine wastes containing high amounts of sulfides are expected to have a large oxygen consumption. It is thus crucial to provide enough oxygen to the bioleach pulp to avoid any limitation of the bioleaching kinetics [1]. However, it is also well known that the energy required to inject gas and transfer it from the gas phase to the liquid phase is one of the major operational costs in bioleaching operations. The optimisation of the gas-liquid mass transfer is thus a major challenge in the upscaling procedure of bioleaching processes to achieve fast kinetics on the one hand, and to avoid overconsumption of energy on the other hand [2].

CO₂, which is used as a carbon source for the growth and activity of the bacteria, is also a critical gaseous reagent for bioleaching reactions [3], especially in continuous operations using cascade bioreactors. Indeed, in batch mode, the CO₂ contained in the air may be enough to sustain microbial growth since the microbes are confined in the reactor. In continuous cascade reactors, the residence time of the bacteria in each bioreactor is much shorter and CO₂ must be sufficiently provided to avoid any limitation of microbial growth and activity.

Some CRMs such as Co and Sb are often contained within sulfides in mining waste. Therefore, the availability of O₂ and CO₂ is crucial in the optimization of both sulfur and iron biooxidation, CRM dissolution, and ultimately, CRM recovery. Thus, the main objective of this study was to investigate how the gas-liquid mass transfer phenomena affect the bioleaching of sulfidic mining wastes, with the ultimate goal of optimizing the gas supply.

# 2 Methodology

## 2.1 Mine Wastes, Microbial Consortium and Nutrient Media

Mine wastes used in this study were pyritic materials originating from the Iberian pyrite belt (southern Spain). It was produced as a residue from a copper acid leaching process. Table 1 shows the concentrations of the main elements of interest analysed by AGQ labs (Spain). Estimation of sulfide content was calculated by difference: total S analysed by combustion minus S(SO₄²⁻) analysed by gravimetry, with the hypothesis of elemental S and other S species negligible.

XRD and SEM/EDX analyses showed that Fe was mainly contained in pyrite. A significant amount of metals such as Cu and Co (between 40% of 60% of the total mass) was contained in water-soluble metal sulfates and acid-soluble sulfides. Copper was
mainly associated with pyrite and in the form of complex sulfides: chalcopyrite and tennantite; antimony was mainly linked to tetrahedrite and arsenic was mainly linked to enargite, dufrenosite and tennantite. Cobalt was below the detection limit of SEM/EDX (<0.1%) but associated with pyrite as it was observed in previous pyrite (bio)leaching tests (data not shown).

<table><caption>**Table 1.** Chemical composition of mining wastes</caption><thead><tr><th>S<sub>tot</sub><br>%</th><th>S(SO<sub>4</sub><sup>2-</sup>)<br>%</th><th>S<sup>2-</sup><br>%</th><th>Fe<br>%</th><th>Cu<br>%</th><th>As<br>ppm</th><th>Co<br>ppm</th><th>Sb<br>ppm</th></tr></thead><tbody><tr><td>41.9</td><td>5.5</td><td>37.6</td><td>35.3</td><td>2.25</td><td>4212</td><td>234</td><td>432</td></tr></tbody></table>

The inoculum used in the experiments was the BRGM-KCC consortium maintained on pyrite and originated from the BRGM cultures collection. It consists of a synergic mix of chemolithotrophic, acidophilic and mesophilic bacteria, mainly composed of *Leptospirillum* (L.) *ferriphilum* (exclusively iron-oxidiser), *Acidithiobacillus* (At.) *caldus* (exclusively sulfur-oxidiser) and *Sulfobacillus* (Sb.) spp. (iron- and sulfur oxidiser). They are autotrophs or mixotrophs utilising CO<sub>2</sub> as the main carbon source [2]. Nutrient medium used in these experiments was denoted as 0Km and contained 3.7 g.L<sup>-1</sup> (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 0.81 g.L<sup>-1</sup> H<sub>3</sub>PO<sub>4</sub> 85%, 0.48 g.L<sup>-1</sup> KOH and 0.52 g.L<sup>-1</sup> MgSO<sub>4</sub>.7H<sub>2</sub>O. The pH of the medium was adjusted to pH 2.0 with concentrated H<sub>2</sub>SO<sub>4</sub>.

## 2.2 Methodology

**Experimental Set-up.** The experiments were performed in a 114 L pilot unit, which consisted of a 51 L and three 21 L (working volume) closed stirred tank reactors (STR) connected in series. The tanks were made from 316L stainless steel and had a working height/diameter ratio equal to one. The reactors were equipped with a mechanical agitator including double impellers (axial/radial) mounted on a rotating shaft fixed to a motor. The impellers consisted of a bottom radial turbine with six vertical flat blades and an axial 3-blade propeller (Milton Roy Mixing Europe). The impeller diameter was 145 mm for the 51 L reactor and 110 mm for the 21 L reactors. The reactors were identified according to their position in the cascade (R1, R2, R3 and R4), as depicted in Fig. 1. Each reactor contained four internal baffles (at 90° intervals) fixed to the tank wall. The top of the reactors was connected to a gas cooling system to prevent excessive evaporation. The gas stream was injected beneath the turbine at the bottom of the reactor via a stainless steel pipe. The gas stream was composed of either air from a compressed air system (enriched or not with CO<sub>2</sub>) or a mixture of N<sub>2</sub>, CO<sub>2</sub> and O<sub>2</sub>. Pure gases (99.9%) were supplied by gas cylinders (Air Liquide, France). Gas mixture composition was controlled by valves and mass flow regulators for each gas. Gases went through a gas mixer before injection in the reactor. All reactors had a thermal jacket connected to a cryothermostat (LAUDA RP 245 E) allowing accurate control of the reactor temperature. The pulp transfer between reactors was carried out by overflowing.

<footnote>* Sulfides (S<sup>2-</sup>) calculated as total sulfur (S<sub>tot</sub>) – sulfate sulfur (S(SO<sub>4</sub><sup>2-</sup>))</footnote>
<figure><img src="image_1.png" /><figcaption>Fig. 1. 114 L continuous pilot unit</figcaption></figure>

**Start-up.** Before starting up the experiment, a concentrated pulp at 30% w/w of MW, a concentrated nutrient solution and a calcite suspension at 300 g L⁻¹ for pH control were prepared. The concentrated nutrient solution was prepared so that the concentration of nutrients in the primary reactor (after dilution by the concentrated pulp and the calcite suspension) corresponds to the composition of the 0Km medium [4].

The primary reactor (R1) was started in batch mode at a solid content of 10% (w/w) of MW in 0Km medium and inoculated at 10% v/v with BRGM-KCC active inoculum. The reactor is left under agitation and sufficient aeration. Once a significant bacterial activity was observed (high redox potential and low pH), the reactor was switched to continuous mode by feeding semi-continuously the primary reactor with three streams, i.e., the concentrated pulp, the concentrated nutrient solution and the calcite suspension.

**Operating Conditions.** The total flowrate was set to obtain a pulp with 15% (w/w) of solids and a residence time of 6 days in the entire pilot. The feeding occurred every 45 minutes. Each cycle (32 cycles per day) consisted in three steps: (1) concentrated pulp agitation for 20 minutes in the feeding tank, (2) feeding of the primary tank with concentrated pulp, concentrated nutrient solution and calcite suspension, and (3) a resting period in which the pulp is distributed by overflowing from one reactor to the next. Stirring speed was set to 400 rpm for R1 and 500 rpm for the other reactors. Temperature was set to 42 °C in the four reactors. Three operating conditions linked to gas supply were tested by changing the composition of the gas (Table 2) and the gas flow rate (Table 3). They were fixed according to the needs in gas supply observed during the operation of the bench-scale pilot. It is important to highlight that the first 7 days of the experiment consisted in filling up the secondary reactors by gravity with the pulp overflowing the previous reactor.
<table><caption>**Table 2.** Gas compositions at each operating condition tested</caption><thead><tr><th rowspan="2">Condition</th><th colspan="4">Gas volume fraction (%)</th></tr><tr><th>Air</th><th>N<sub>2</sub></th><th>O<sub>2</sub></th><th>CO<sub>2</sub></th></tr></thead><tbody><tr><td>I</td><td>1.000</td><td>-</td><td>-</td><td>-</td></tr><tr><td>II</td><td>0.995</td><td>-</td><td>-</td><td>0.005</td></tr><tr><td>III*</td><td>-</td><td>0.695</td><td>0.300</td><td>0.005</td></tr></tbody></table>

<table><caption>**Table 3.** Gas flowrates in each reactor at each operating condition tested</caption><thead><tr><th rowspan="2">Condition</th><th colspan="4">Gas flowrate (nm<sup>3</sup> h<sup>-1</sup>)</th></tr><tr><th>R1</th><th>R2</th><th>R3</th><th>R4</th></tr></thead><tbody><tr><td>I</td><td>0.80</td><td>0.50</td><td>0.50</td><td>0.52</td></tr><tr><td>II</td><td>1.3</td><td>0.8 - 1.1</td><td>0.82</td><td>0.83</td></tr><tr><td>III</td><td>0.34</td><td>0.24</td><td>0.82</td><td>0.25</td></tr></tbody></table>

# 3 Results

## 3.1 Oxidation Activity

Fig. 2 and Fig. 3 depict respectively the redox potential (Eh vs SHE) and the pH variations during the 114 L bench-scale pilot operation. During Condition I (using only air to provide O₂ and CO₂ to the reactors), both Eh and pH stabilized after 20 days (roughly 3 times the HRT). Redox potential in the primary reactor reached about 730 mV (vs. SHE), whereas in R4 it reached over 800 mV (vs. SHE). pH stabilized between 1.2 and 0.8 for all reactors. In Condition II, the gas phase (consisting of air) was enriched with 0.5% v/v of CO₂ (after day 23). A notorious increase of the Eh was observed in the days following the change of the operating conditions, as well as a strong decrease in the pH of all reactors. Previous experiments (data not shown) demonstrated that a pH lower than 0.8 had a deleterious impact on the oxidation activity of the microbial consortium. To avoid this, the flow of calcite was increased to enable the pH to stabilize between 0.8 and 1.

A strong increase of oxygen consumption was also observed in R1 and R2, as shown in Fig. 4. This was reflected by a drastic drop in the dissolved oxygen saturation from 90% to 40% in R1 and from 70% to 24% in R2. The concentration of O₂ in both R1 and R2 decreased to values close to and even lower than 2 mg.L⁻¹, which means that not enough oxygen was being transferred from the gas phase to the liquid phase. These results indicate that, during Condition I, the CO₂ transfer rate was not enough to permit the bacteria to grow in the primary reactor (R1). In other words, the growing rate of the bacteria was too slow when R1 was fed only with air and the biological Fe-oxidising activity was low, which explains the low Eh potential reached at this stage.

During Condition II, the supplementation of air with CO₂ enabled it to sustain better biomass growth and to increase the biological Fe and S oxidation activity as shown by

<footnote>* Gas composition change from Condition II to Condition III was done only in R1 and R2. Reactors R3 and R4 kept the gas composition of Condition II.</footnote>
the Eh increase and the pH decrease, respectively. Calcite flow was increased to control the pH at a value close to 1.0, resulting in an increase in the overall flow and a diminution of the total average HRT to 5.6 days.

<figure><img src="image_2.png" /><figcaption>**Fig. 2.** Redox potential (Eh vs SHE) variations during the pilot operation</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>**Fig. 3.** pH variations during the pilot operation</figcaption></figure>

However, as reflected by the low dissolved O₂ concentration measured in R1 (about 1.9 mg.L⁻¹) and R2 (about 1.2 mg.L⁻¹) during Condition II, the O₂ transfer rate was not enough to reach the maximal bio-oxidation kinetics. Thus, airflow was gradually increased in both reactors to increase the oxygen transfer rate to the reactors. The maximum values tested (shown in Table 3) allowed a limited increase in both the oxygen uptake rate and the dissolved oxygen concentration, which continue to be around 2 mg.L⁻¹ in R1 and R2. Moreover, it was noticed that solids were being accumulated in these reactors due to the flooding of the impellers by the high airflow, which caused a decrease in the mixing efficiency of the pulp. The solid accumulation in R1 and R2 was not permitted to reach a steady state for Condition II. A solution to avoid the limitation
in the oxygen transfer and the solid accumulation in the reactor was to increase the oxygen partial pressure in the gas flow.

<figure><img src="image_4.png" /><figcaption>**Fig. 4.** Dissolved oxygen saturation in the liquid phase during the pilot operation (100% corresponds to the saturation at the partial pressure of O₂ in the air)</figcaption></figure>

Therefore, during Condition III, the oxygen partial pressure in the gas flow was increased to increase the O₂ gas-liquid transfer and the gas flow was decreased to avoid the flooding of the impellers and the accumulation of the solid. An increase in the Eh of the reactors was observed, particularly for R4, which confirmed the positive influence of these changes on microbial activity. Moreover, a sharp decrease of the pH was observed at the beginning of the condition in R1 and R2, which can be translated into a higher sulfur oxidation activity. Calcite flow was consequently increased to keep the pH over 0.9. This caused a further decrease of the total HRT of the pilot to 4.9 days.

Unfortunately, the bioleach operation was disturbed by two technical issues during Condition III, which hindered the stability of the reactors in this phase. The first operating problem was a failure of the N₂ flowmeter, which stopped the N₂ flow between days 52 and 55. This translated into a high dissolved oxygen concentration, as high as 16.5 mg.L⁻¹, or about 150% of oxygen saturation with air. The second technical issue consisted of a failure in the feeding of both the concentrated nutrient solution and the alkaline agent for pH control at day 62. Although these problems caused a decrease in the redox potential, as well as pH instabilities in the reactors concerned, in both cases bacteria were able to recover the oxidation activity, without stopping the continuous flow, demonstrating the robustness of the microbial consortium.

## 3.2 Metal and Sulfide Dissolution

Relatively low Fe concentrations in the leachate were observed during Condition I (from 5.7 g L⁻¹ in R1 up to 24.5 g L⁻¹ in R4). They rapidly increased during Condition II, reaching their maximum values between days 30 and 40 (about 42 g L⁻¹ in R1 and 57 g L⁻¹ in R4). However, there is a decrease in the concentration during Condition III,
which ended with 21 g L⁻¹ in R1 and 40 g L⁻¹ in R4. The high concentration values found during Condition II can be explained by the solid accumulation in the reactors, which may have caused a dissociation of the HRT of the leachate and the solid. Higher amounts of sulfides, and thus the metals that they contain, may have then been present in the reactors at this point and this caused a peak in the metal concentrations. This hypothesis is supported by the fact that the final sulfide content in the residue obtained for Condition II were higher than those obtained for Condition III (22% in Condition II vs. 9% in Condition III), meaning that the high metal concentration in the leachate was not due to a higher dissolution yield.

The impact of the low activity observed during Condition I is clearly reflected in the low metal and sulfide dissolution yields reached in each reactor. In the primary reactor only 6% of the sulfides were dissolved during Condition I, whereas 53% of sulfides were dissolved in the same reactor during Condition III. Due to solids accumulation in R1 and R2, it was not possible to perform a mass balance for Condition II. The total sulfide, Co and Sb dissolution yields reached only 54%, 75% and 8%, respectively, for Condition I (with an HRT of 6.2 days). During Condition III, the dissolution yields increased to 81% for sulfides, 86% for Co and 30% for Sb (with an HRT of 4.9 days).

## 3.3 Bacterial Community

The changes in the bacterial diversity, monitored by t-RFLP fingerprinting in time in the pulp of the primary reactor (R1) and the downstream ones (R2, R3 and R4), are presented in Fig. 5. Two bacteria of the BRGM-KCC consortium were found in the pilot, *L. ferriphilum* (iron oxidizer) and *At. caldus* (sulfur oxidizer). In R1 and R2, changes in the community were comparable, with both bacteria retrieved until day 23 and from day 53 until the end. Only *L. ferriphilum* was detected in all samples from days 35 to 49, except in R2 at day 49. In R3, diversity profiles showed a larger relative abundance (> 80%) of *At. caldus* at the beginning of the operation until day 23, and the dominance of *L. ferriphilum* lasted from days 35 to 53. In R4, *At. caldus* was found at the beginning of the operation as in the other reactors, but then *L. ferriphilum* largely dominated the community (83-100%) from day 35 to the end of the run.

Regarding the operating conditions, this test allowed us to confirm that a pH control at about 0.8 was adapted to keep good proportions of *L. ferriphilum*. The period when *L. ferriphilum* was the only bacterium detected in all the reactors corresponds to the times during Condition II (days 35 and 42) when the four reactors were fed with air enriched with CO₂. This result is in line with the fact that this bacterium is an obligate autotroph, requiring CO₂ as a source of carbon for its development [5]. As it was the only iron-oxidizing bacterium detected in the reactors, favouring *L. ferriphilum* in the pilot was anticipated as a means to improve the bioleaching rate. This is consistent with the notorious increase of the Eh, thus of the biological Fe oxidation, at that period (Fig. 2). However, a pH of 0.8 is probably close to the lower pH limit for efficient iron oxidation by *L. ferriphilum*, as partial inhibition has been reported at pH 0.7 for an *L. ferriphilum*-dominated culture at 35 °C [6].
<figure><img src="image_5.png" /><figcaption>**Fig. 5.** Bacterial community fingerprints (t-RF) changes in time in each reactor during the pilot operation</figcaption></figure>

Then, from day 49 until the end of the operation, the oxygen volumetric fraction in the gas phase was augmented to 30% in reactors R1 and R2. In the two reactors from day 53, *At. caldus* showed up again suggesting its development was favoured by increasing dissolved oxygen content in the pulp. More *At. caldus* and less *L. ferriphilum* within the community is consistent with the observed drops of pH and Eh observed in R1 and R2 (Fig. 2 and Fig. 3). The same evolution was observed in R3, although the proportion of *L. ferriphilum* decreased less in this reactor in which the drops in Eh and pH were more moderate. However, in R4, *L. ferriphilum* was dominant throughout the end of the monitoring, in agreement with a high and more stable Eh measured in this reactor.

**3.4 Oxygen and CO₂ consumption**

The pyrite dissolution rate was calculated from the sulfide dissolution yield (assuming that all the sulfides in the mining waste correspond to pyrite) and was compared to the oxygen uptake rate (OUR) in each reactor for Condition I and Condition III. OUR was calculated by a mass balance in the gas phase, assuming that equilibrium in the liquid phase was reached, meaning that the oxygen transfer rate (OTR) equals the OUR in each reactor. Fig. 6 confirms the correlation between these parameters as expected considering Eq. 1**

**\*Error! Source du renvoi introuvable.** During Condition I, the pyrite dissolution rate and the OUR were very low in R1, which was due to the lack of microbial oxidizing activity induced by a limitation in CO₂ gas-liquid transfer rate. It is important to mention that CO₂ was being provided, not only by the natural presence of this gas in the air but also through the pH control system, by the reaction of calcite with the acidic bioleaching medium. However, it seems that, even with this extra source of
CO₂, the transfer rate was not sufficient. Both rates increased then progressively in R2 and R3-R4 (R3 and R4 are cumulated due to an issue encountered during R3 solid sampling). In the contrary, they were very high in R1 during Condition III, reaching around 1200 mg·L⁻¹·h⁻¹.

<figure><img src="image_6.png" /><figcaption>Fig. 6. OUR and pyrite dissolution rate for (A) Condition I and (B) Condition III</figcaption></figure>

This result confirms that the microbial oxidizing activity was very high and enhanced bioleaching rates when O₂ gas-liquid transfer was not limited. The rates then decreased progressively from R2 to R4. The values of OUR and pyrite dissolution rates reached in Condition III are close to the values obtained during industrial bioleach operations [7, 8]. In the contrary, the values obtained in Condition I are unusual, which highlights the importance of providing enough CO₂ and O₂ to sustain the microbial activity.

Fig. 7 shows the CO₂ Uptake Rate (CUR) measured during Condition III. CUR corresponds to the consumption of CO₂ by the bacteria. As OUR, CUR was measured by a mass balance in the gas phase, assuming that the CO₂ transfer rate (CTR) equals the CUR in each reactor

Measures were taken in such a manner that the CO₂ released by the neutralization of the calcite did not influence the balance. CUR was closed to 45 mg L⁻¹·h⁻¹ in R1 and R2 and then decreased in R3 and R4, which is consistent with the results presented above. CUR measured in R1 and R2 is similar to the maximal values obtained in other studies [3]. During Condition I, it was not possible to measure the CUR since the detection limit of the gas analyser was above the CO₂ concentration in the gas streams (inlet and outlet). However, it is possible to calculate the maximal CO₂ flow injected in each reactor. It was 1.2 mg L⁻¹·h⁻¹ in R1 and 1.9 mg L⁻¹·h⁻¹ in R2, R3 and R4. These values are much smaller than the CUR measured during Condition III. The amount of CO₂ injected into the bioreactors during Condition I was thus far below the maximum capability of the bacteria to consume CO₂, probably limiting their growth and activity. These results also confirm the need for a primary reactor with a bigger volume in bioleaching processes with the purpose of allowing better microbial growth and a more efficient gas transfer needed at this point of the process.
<figure><img src="image_7.png" /><figcaption>Fig. 7. Carbon Uptake Rate (CUR) for Condition III</figcaption></figure>

**4 Conclusions**

CO₂ and O₂ are essential gases for the metabolism of autotrophic bioleaching bacteria. An insufficient supply of one or both of these gases results in a limitation in the growth and oxidizing activity of the bacteria. During this study in CSTRs, insufficient CO₂ supply was observed, which resulted in the bacterial growth being shifted from the primary reactor to the secondary reactors. This implied a decrease in the efficiency of the sulfide oxidation capacity of the system. Sufficient CO₂ allowed proper bacterial growth but created a limitation in the O₂ transfer by increasing the O₂ uptake rate (OUR). Increasing the airflow, and hence the O₂ transfer rate, partially helped to solve the problem. However, the limits of the agitation system were reached, since a flooding of the impellers was noticed, which decreased the mixing efficiency and allowed an accumulation of solids in the reactors. Finally, decreasing the airflow, but increasing oxygen volumetric concentration in the gas phase allowed to optimize the sulfide oxidation activity.

This study has demonstrated the importance of studying the gas-liquid mass transfer in continuous mode during the bioleaching process development. Moreover, the methodology proposed allows us to test the limitations of the system and to measure optimal O₂ and CO₂ transfer rates, as well as the gas uptake rates in the reactors. These values can then serve for the design of aeration systems and reactors. Indeed, the optimization of the gas supply is an essential step, since reactor aeration systems represent important investment and operational costs. Nonetheless, the use of air enriched with CO₂ and O₂ must be evaluated carefully, since it may imply both an increase in the bioleaching kinetics (metal and sulfide dissolution rates) and an increase in the costs.

**Acknowledgments.** This work is part of the European collaborative research Project RAWMINA (An integrated, innovative pilot system for recovery of critical raw materials from mine waste), funded by the European Union's Horizon 2020 research
and innovation programme (Grant agreement No 958252). Authors would like to thank AGQ labs (Spain) for the analytical support in the frame of the Project RAWMINA.

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