Causes of inhibition of bioleaching by Cu are also thermodynamic

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is the peer reviewed version of the following article: Mazuelos, A., García- Tinajero, C.J., Romero, R., Iglesias-González, N. and Carranza, F. (2019), Causes of inhibition of bioleaching by Cu are also thermodynamic. J. Chem. Technol. Biotechnol., 94: 185-194, which has been published in final form at https://doi.org/10.1002/jctb.5761. This article may be used for noncommercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited." 1 CAUSES OF INHIBITION OF BIOLEACHING BY Cu ARE ALSO THERMODYNAMIC A. Mazuelos*, C. J. García-Tinajero, R. Romero, N. Iglesias-González, F. Carranza Address: Department of Chemical Engineering of the University of Seville Faculty of Chemistry of the University of Seville c/ Profesor García González, Edf. Química 41012 Seville Spain * Corresponding author: A. Mazuelos e-mail: [email protected] fax: +34 954 55 64 41 A.Mazuelos: Alfonso Mazuelos Rojas; e-mail: [email protected] C.J. García-Tinajero: Carlos Jesús García-Tinajero; email: [email protected] R. Romero: Rafael Romero Aleta: email: alet[email protected] N. Iglesias: Nieves Iglesias-González: [email protected] F. Carranza: Fancisco Carranza Mora: [email protected] 2 CAUSES OF INHIBITION OF BIOLEACHING BY Cu ARE ALSO THERMODYNAMIC 1 2 A. Mazuelos*, C. J. García-Tinajero, R. Romero, N. Iglesias-González, F. Carranza 3 4 Abstract 5 BACKGROUND. Cu is an indispensable natural resource for society, as its use is widespread and 6 essential for many technologies. Consequently, worldwide production is around 18·106 tn/year, and 7 close to 25% is produced by bioleaching. Bioleaching enables Cu extraction from minerals in 8 atmospheric conditions by virtue of microorganisms’ catalytic activity. Microbial catalysis consists 9 mainly in Fe3+ production as a leaching agent, a process known as Fe2+ bio-oxidation. However, 10 bio-oxidation is inhibited by Cu2+ which limits the use of bioleaching with Cu mineral ores. 11 RESULTS. This paper, for the first time, examines the effect of Cu2+ on continuous Fe2+ bio- 12 oxidation using a packed bed bioreactor with supported cells. Bio-oxidation was possible in the 13 presence of 20 g/L Cu2+, with a less than 25% reduction in rate. About 15% of this drop in rate is 14 due to biological inhibition and the rest to a reduction in oxygen solubility because of salting-out. 15 The biotic effect is reversed when Cu2+ is removed from the input, and the salting-out effect can be 16 overcome by improving aeration conditions. 17 USEFULLNESS. These results, apart from having important ramifications for designing Cu ore 18 bioleaching facilities, contribute to a more versatile and competitive idea of this clean technology. 19 20 Keywords: 21 Bioleaching, Bioreactor, Heavy Metals, Hydrometallurgy, Mass Transfer 22 23 3 Highlights: 1 - Influence of Cu2+ on continuous Fe2+ bio-oxidation is studied for the first time. 2 - Fe2+ bio-oxidation slowdown because of Cu2+ presence is caused both by biotic and physical 3 phenomena. 4 - Adequately modifying bio-reactor aeration would reduce the negative effect associated with 5 Cu2+ by 35%. 6 - Adaptation of cells to Cu2+ is possible in continuous bio-reactors. 7 - The inhibitory effect of Cu2+ is reversible. 8 4 Symbols: a: gas-liquid specific interfacial area (m-1) CT: complete oxidation time (h) CL: oxygen concentration in the liquid (mg/L) CL*: saturation concentration for oxygen (mg/L) CLo : saturation concentration for oxygen in water at working temperature and pressure (mg/L) CLS: concentration of oxygen on the surface of the packing material (mg/L) [Cu2+]: copper concentration (g/L) E(t): residence time distribution function F(t): cumulative residence time distribution function [Fe2+]: ferrous iron concentration (g/L) [Fe2+]0: initial ferrous iron concentration (g/L) [Fe2+]t: ferrous iron concentration at time t (g/L) kL: gas-liquid mass transfer coefficient for oxygen (min-1) kS: mass transfer coefficient for oxygen transfer across the boundary layer surrounding the packing material (min-1) m: mass of ferrous iron inside the bioreactor (g) MBR: maximum bio-oxidation rate (g/Lh) Me: mass flow rate of ferrous iron entering the bioreactor (g/h) MOTR: maximum oxygen transfer rate (g/Lh) MOUR: maximum oxygen uptake rate (g/Lh) Ms: mass flow rate of ferrous iron leaving the bioreactor (g/h) QO: gas-liquid oxygen transfer rate (g/Lh) QFe: instantaneous bio-oxidation rate (g/Lh) s: specific surface area of the packing material (m-1) V: liquid volume inside the bioreactor (L) 1 5 INTRODUCTION 1 The strategic importance Cu has in industrialised societies is well known; distribution of water, 2 energy and communications are to a great extent influenced by the use we make of this 3 natural resource. Currently, the main raw materials from which Cu is obtained are 4 mineralised rocks containing mineralogical species in which Cu is part of sulphide-like 5 chemical compounds, the most abundant being chalcopyrite (CuFeS2), chalcocite (Cu2S) 6 and covellite (CuS).1 7 In recent decades, biotechnological applications used for producing commercially valuable 8 metals, classified within the field of biohydrometallurgy, have garnered increasing approval 9 in industrial contexts. This is owing to the technical simplicity, low operational costs and 10 moderate environmental impact inherent to them.2 As a result, between 20 and 25% of 11 global Cu production is obtained by these methods; this amounts to over 4.5 million tons 12 per year.3 Considering that the price of Cu over the last five years averages at 6600 13 US$/tn,4 this gives us an idea of the economic impact associated with Cu 14 biohydrometallurgy. 15 Biohydrometallurgical technologies for producing metals from sulphide minerals are based 16 on bioleaching.2,3,5,6 Bioleaching is a process in which raw minerals are subjected to the 17 action of microorganisms so as to free the metal from the sulphide crystal lattice by 18 oxidising it.7 This way, the metal dissolves as ions and can be purified by solvent 19 extraction and electrolysis, reaching the specifications demanded by the market for its 20 commercialisation. Various mechanisms have been described to explain bioleaching,2,8,9 21 among which the most widely accepted — constituting the base for other models — is 22 known as indirect bioleaching.10 According to this mechanism, metal sulphides (MeS) are 23 indirectly oxidised by O2 in the air when they are in contact with microorganisms in an 24 acidic medium. 25 6 MeS + ½ O2 + 2H+ S0+ Me2+ + H2O (1) 1 O2 and MeS do not physically interact, and electron transfer between them happens 2 through an electronic intermediary, the Fe2+/Fe3+ electrochemical couple. This model 3 postulates that the bioleaching process happens in two simultaneous stages: 4 - A chemical leaching stage, in which Fe3+ takes electrons from the sulphide, oxidising it to 5 Sº, and modifies its oxidation state to Fe2+. 6 MeS + 2Fe3+ Sº + Me2+ + 2Fe2+ (2) 7 - A biological stage, in which the electrons taken by Fe2+ are finally transferred to O2, and, 8 in doing so, Fe3+ is regenerated so it is once more available as an oxidising agent. The 9 cells use Fe2+ as an energy substrate and act as a catalyst to the process, which is known 10 as bio-oxidation.11 Cells capable of metabolising Fe2+ are known as iron-oxidising 11 microorganisms.12,13 12 2Fe2+ + ½ O2 + 2H+ 2Fe3+ + H2O (3) 13 Due to this mechanism, bioleaching applications happen in atmospheric conditions, and no 14 reagents are consumed in the process. These facts, derived mainly from the microbial 15 activity in Fe2+ bio-oxidation, place bioleaching at an advantage when compared with high 16 pressure leaching technologies, which require far greater pressures and temperatures in 17 order to regenerate the leaching agent.14 18 Among biohydrometallurgical technologies based on bioleaching, the most widely used for 19 producing Cu are those known as heap bioleaching and dump bioleaching.2,3,5,6 20 Conceptually, these technologies consist in wetting — with a leaching solution — mineral 21 particles piled into heaps or dumps that act as a packed bed, the process of metal 22 extraction being catalysed by cells adhered to the mineral surface. 23 7 It is possible to accelerate bioleaching by carrying out the chemical leaching and biological 1 Fe2+ oxidation stages in separate locations, allowing to optimise each independently.15-17 2 Bio-oxidation can be improved using reactors designed specifically for this purpose. 3 Among the tested designs, the one which has shown the greatest Fe3+ productivities is the 4 flooded packed bed reactor.18,19 In these reactors, cells adhere to the particles of the 5 packed bed forming a bio-film, and they are fed with liquors containing Fe2+ and air, which 6 are introduced through the bottom of the reactor and flow upward. 7 A special trait of iron-oxidising microorganisms is their ability to tolerate high 8 concentrations of heavy metals as a result of them almost always being present in their 9 natural environment.20-23 However, there is literature reporting bioleaching inhibition when 10 cells interact with the heavy metal ions freed from the sulphide, the negative impact of 11 which depends on the microbial species, the metal and its concentration.11 12 Regarding the interaction between Cu and iron-oxidising microorganisms, most of the 13 consulted papers concur that cellular metabolism is inhibited when Cu concentration 14 reaches 5 g/L, and that the biological tolerance limit is about 20 g/L.24-31 However, when it 15 comes to quantifying kinetic variations caused by Cu2+, little has been published and with 16 varying results, despite using the same system; that is batch culture trials. Difficulty in 17 processing data and interpreting results is inherent to this methodology, partly because 18 cells are amidst a medium the composition of which is ever changing.32 On the other hand, 19 literature always attributes the effect of Cu exclusively to biotic factors. 20 Another point cited authors have in common is the lack of attention payed to the supply of 21 oxygen to cells, offering no guarantee that, under the tested conditions, there was no bio- 22 oxidation limitation due to lack of oxygen, a necessary condition for [Cu2+] to have been 23 the sensitive variable in these tests. 24 8 It is common in aerobic systems for the supply of oxygen to cells to limit their growth and 1 functionality. The limiting stage of this transfer is located in the gas-liquid interphase. In 2 such a case, the rate of transfer, QO, is given by the equation: 3 QO = kL · a · (CL* - CL) (4) 4 Where kL is the gas-liquid mass transfer coefficient, a is the gas-liquid specific interfacial 5 area, CL* is the saturation concentration for oxygen, and CL is the concentration of oxygen 6 in the liquid phase. It is possible to estimate CL* in mg/L for typical bioleaching liquors, if 7 pH, and [Fe2+], [Fe3+] and [Cu2+] in g/L are known, using the equation from Mazuelos et 8 al.33 9 CL*= CLo- 12.698.10-pH - 0.0555 [Fe2+] - 0.0290 [Fe3+] - 0.0265 [Cu2+] (5) 10 Where CLo is oxygen solubility in water at working temperature and pressure. 11 According to equation 5, the greater [Cu2+], the lower CL* will be, and the lower the driving 12 force for oxygen transfer. 13 The consulted literature only mentions the negative effect of Cu2+ on microbial activity and 14 overlooks the simultaneous — and synergic — effect Cu2+ has as a chemical catalyst on 15 Fe2+ oxidation.14 16 Therefore, nowadays the available information about the influence of Cu2+ on bioleaching, 17 which is necessary for the design of plants for the treatment of Cu ores, is very limited. 18 This paper shows the results obtained from studying the effect of Cu2+ on Fe2+ bio- 19 oxidation, with the novelty of experiments being conducted in continuous operation of a 20 packed bed bioreactor with immobilised cells. Using this type of bioreactor, as well as 21 obtaining results about the influence of Cu2+ on bio-oxidation in a device closer to 22 industrial reality, it is possible to attain steady state conditions for the microbial population 23 that it holds.18,19 Like this, it is possible to maintain cells’ response to variations in [Cu2+] in 24 15 With these caveats in mind, there is some research with results similar to those reached in 1 this paper. For example, in the paper by Hong-Mei Li and Jia-Jun Ke30, time until complete 2 Fe2+ bio-oxidation with 5 g/L of Cu2+ was 70 hours, compared to the 69 hours used for the 3 same purpose in this research project. In the paper by Brahmaprakash et al26, complete 4 Fe2+ oxidation was reached after 84 hours with 5 g/L of Cu2+ and, with 10 g/L of Cu2+, it 5 took 8 days to fully oxidise the 9K medium, compared to the 7 days needed in this paper 6 for the same purpose. However, other authors report very different findings to the ones in 7 this paper, like those in the paper by Das et al27, in which adaptation led to far more 8 effective bio-oxidation. In this regard, other differences between the experiments carried 9 out for this paper and those found in literature must be mentioned, namely initial pH and 10 non-limiting oxygen conditions. 11 To use 9K medium, with a pH value above 2 — as do most cited papers24-31 — means to 12 accept that part of the Fe3+ generated during bio-oxidation will precipitate; Fe3+ 13 precipitation is to be avoided in bioleaching applications as Fe3+ only acts as a leaching 14 agent when it is dissolve. Modified 9K medium with lower pH by adding H2SO4 was chosen 15 for this paper in order to avoid Fe3+ precipitation and obtain results that are closer to 16 industrial situations. 17 The oxygen requirement of the cells is an essential operational variable to implement 18 when the microbial population being studied is aerobic.32 If limiting aeration conditions had 19 occurred, the effect of Cu2+ on Fe2+ bio-oxidation could have been masked. In relation to 20 this, it must be mentioned that oxygen solubility decreases as Cu2+ concentration 21 increases because of the salting-out effect. 33 22 Experiments were conducted to characterise oxygen transfer under conditions analogous 23 to those used for the cultures. In addition, other stirring rates were assayed. Figure 3 24 16 shows measured transfer rates as a function of stirring rate across the full operational 1 range of the orbital shaker employed (0-200 r.p.m.). 2 As figure 3 shows, oxygen transfer rate is directly proportional to stirring rate for most of 3 the studied range. At the stirring rate assayed in the cultures, that is 180 rpm, oxygen 4 transfer rate was 0.25 g/Lh. Taking into account that the average oxygen solubility under 5 the studied conditions is 6.44 mg/L,34 the value of kL·a calculated with the equation (4) is 6 0.63 min-1. 7 Using this value for kL·a, the maximum oxygen transfer rate MOTR was calculated for each 8 of the bio-oxidation assays, having previously calculated CL* using the equation (5), and 9 considering that MOTR implies that CL=0. The results reached for CL* and MOTR are 10 shown in table 2. 11 From the maximum Fe2+ bio-oxidation rates (MBR), the maximum oxygen uptake rate 12 (MOUR) was determined using the reaction stoichiometry (equation 3). Table 2 shows the 13 results calculated for MOTR and MOUR, revealing that MOTR was far greater than MOUR 14 in all experiments. It can therefore be stated that in this preliminary study limiting oxygen 15 conditions did not exist. 16 17 Continuous bio-oxidation. 18 Continuous bio-oxidation assays were carried out in a packed bed bioreactor with adhered 19 biofilm. 20 21 Working volume and flow model. 22 17 Prior to continuous operation, the packed bed column was geometrically characterised. Its 1 total volume was 824 mL, before adding the particles of packing material and air. This 2 volume dropped 314 mL when the packing material was added. Finally, air was pumped at 3 a flow of 750 mL/min; the bubbles occupied 31.7 mL. In other words, the volume taken up 4 by liquid when operation started was 478.3 mL. 5 After continuous operation concluded, the bioreactor was drained, leaving a drained 6 volume of 184.2 mL. It was then placed in an oven at 40ºC. After 600 hours its weight 7 remained constant, leaving an evaporated volume of 178.5 mL. This means that, after 8 continuous operation, the volume taken up by liquid was 362.7 mL. 9 The difference in volume occupied by liquid in the bioreactor between the start and the end 10 of its continuous operation (115.6 mL) is attributed to the biofilm. The biofilm was made up 11 of cells and accumulated ferric precipitates forming a continuous three dimensional 12 structure.35 (See in complementary data the evolution of the pH throughout the continuous 13 operation) 14 Stirring of the liquid inside the bioreactor is caused by the bubbles of air, although the 15 packing material limits this induced movement. In order to obtain information about the flow, 16 assays were carried out to determine residence time distribution. Figure 4 shows the 17 cumulative residence time distribution curve F(t). This curve’s appearance suggests that flow 18 inside the bioreactor might resemble the perfect mix model.36 For this reason a least squares 19 adjustment was performed. It can be seen in figure 4 that the experimental results fit the 20 model, the calculated correlation coefficient being R2 = 0.996, and mean residence time 21 being 90.51 min. 22 23 Rate of bio-oxidation at different concentrations of Cu2+. 24 18 After inoculation the bioreactor was operated by 700 hours. It was fed with an acidic 1 solution of FeSO4 and CuSO4, the latter increasing its concentration stepwise19. Taking 2 into account the results obtained in the batch bio-oxidation tests, the selected range of 3 [Cu2+] was 0 to 20 g/L. Figure 5 shows the mean bio-oxidation rate plotted against [Cu2+]. 4 For each [Cu2+] studied steady state was reached; the operation was considered in steady 5 state if the [Fe2+] in the outlet stream varies less than 5% with respect to the mean value 6 for a time higher than mean residence time multiply by 50. 7 As can be seen in figure 5, Cu2+ also exerts a negative effect on continuous bio-oxidation. 8 However, in relative terms, the influence of this effect is considerably smaller in continuous 9 operation than in batch cultures (figure 5, table 2). Fe2+ bio-oxidation was not only possible 10 when [Cu2+] = 20 g/L, but also the bio-oxidation rate suffered a less than 25% drop in this 11 situation, while the maximum bio-oxidation rate dropped by about 50% in batch cultures 12 with only 5 g/L of Cu2+. It seems reasonable to state that cells inside the bioreactor have 13 been adapted to Cu2+ concentration. Cell adaptation to Cu2+ is a phenomenon 14 corroborated by several authors. 26, 27, 31 15 16 Oxygen transfer 17 Figure 6 shows the values of CL measured in the bioreactor and the values of CL* 18 calculated with equation 5. 19 It can be seen in figure 6 that CL never was lower than 45% of CL*. For this reason, it might 20 be thought that the supply of oxygen to the bioreactor was sufficient to cover cellular 21 demand. However, the cells are not suspended in the liquid medium, but rather adhered to 22 the packing material, forming a biofilm. O2 concentration on the surface of this biofilm 23 could be lower than measured in the liquid phase. 24 19 After 526 and 651 hours of continuous operation, experiments were carried out in which 1 the current of air was replaced with oxygen, [Cu2+] being 15 and 20 g/L respectively. 2 Figure 7 shows how Fe2+ concentration and instantaneous bio-oxidation rate QFe evolved 3 over time during the transient state, as calculated by the corresponding substrate 4 continuity equation for Fe2+: 5 dt dm QMM Fese  (8) 6 Where: 7 dt dm is the rate of accumulation of Fe2+ inside the reactor (accumulation term) calculated 8 with the following formula: 9 dt VIIFed dt dm salida )]([  (9) 10 where V is the volume of liquid. 11 Me and Ms are the mass flow rates of Fe2+ entering and leaving the bioreactor. 12 It can be observed in figure 7 that, at both the tested [Cu2+], the instantaneous bio- 13 oxidation rate rose suddenly when oxygen replaced air. Subsequently, the bio-oxidation 14 rate gradually dropped, which can be attributed to gradual Fe2+ depletion as this substrate 15 becomes limiting, dropping below 1 g/L. 16 According to Henry’s law, the bioreactor’s yield should have grown by a factor of 5 if there 17 were sufficient active biomass inside it. For [Cu2+] of 15 and 20 g/L in the feed, the 18 average yield during continuous operation with air was 2.11 g/h and 1.96 g/h respectively, 19 while the maximum instantaneous bio-oxidation rates reached with pure oxygen were 6.60 20 and 6.15 respectively. That is to say, bio-oxidation rates triple their value when air is 21 replaced with O2. 22 20 This response of the bioreactor reveals that during continuous operation, when air was 1 injected, the oxygen transfer rate was insufficient to meet demand by the cells. It can 2 therefore be stated that, during continuous operation, the oxygen concentration 3 surrounding the biofilm must be lower than in the midst of the liquid phase. 4 It is possible to calculate the rate of oxygen demand by the cells if the bio-oxidation 5 stoichiometry (equation 3) and the rate of Fe2+ bio-oxidation (figure 5) are known. In 6 steady state conditions, this must necessarily be equal to the rate of oxygen transfer from 7 the gaseous to the liquid phase. It is possible to solve (Eq. 4) for kL·a if CL and CL* are 8 known. Figure 8 shows the mean values of kL·a calculated this way from the results shown 9 in figures 5 and 6 during continuous operation. 10 It can be observed in figure 8 that the values calculated for kL·a display no trend; the mean 11 value is 0.028 s-1, with a standard deviation of 0.0026 s-1. This result is consistent with the 12 assayed flow conditions, invariable throughout continuous operation. 13 Once O2 is in the liquid phase, it must travel to the vicinity of the particles of packing 14 material, on whose surface the biofilm is adhered. Akin to the mathematical treatment 15 used for gas-liquid transfer, there can be considered to be a boundary layer surrounding 16 the packing material, across which O2 is transferred at a rate given by the integrated 17 expression of Fick’s law: 18 QO = kS · s · (CL - CLS) (10) 19 Where kS is the mass transfer coefficient for O2 transfer across the boundary layer 20 surrounding the packing material, s is the specific surface area of the packing material and 21 CLS is the concentration of O2 on the surface of the packing material. 22 Oxygen transfer rate surrounding the biofilm will be highest when CLS=0. Assuming O2 is 23 depleted, based on the limiting effect of oxygen discussed earlier, kS· s can be estimated if 24 21 CL (figure 6), the bio-oxidation rate (figure 5) and the bio-oxidation stoichiometry (equation 1 3) are known. As occurred with the results given for kL·a, values for kS · s show no trend 2 for similar reasons; the mean value is 0.023 s-1, with a standard deviation of 0.0027 s-1. 3 The real mean value must necessarily be equal or lower, depending on the value of CLS. 4 However, given the similarity between the calculated average values of kL·a and kS·s, it 5 can be postulated that resistance to O2 transfer from the gaseous to the liquid phase must 6 be of the same order of magnitude as that surrounding the biofilm. 7 Under these limiting oxygen conditions, the saturation concentration for oxygen CL* 8 critically affects the bio-oxidation rate. CL* decreases as [Cu2+] in feed rises because of the 9 salting-out effect (figure 6). The average values for the percentage drop in CL* due to Cu2+ 10 being present calculated from the results shown in figure 6, for [Cu2+] 0, 5, 10, 15 y 20 g/L 11 respectively, are the following: 0, 1.73, 3.94, 6.30, 8.18. Therefore, assuming O2 is 12 depleted on the surface of the particles of packing material, the drop in CL* due to Cu2+ 13 being present would account for a drop in the bio-oxidation rate of as much as 35% 14 regarding the bio-oxidation rate achieved when Cu2+ is absent. In other words, it must be 15 understood that, in addition to the biotic effect, part of the effect Cu2+ has on bio-oxidation 16 is thermodynamic (salting out effect). 17 18 Reversibility of the inhibitory effect associated to Cu2+. 19 Obviously, this physical effect on bio-oxidation can be reversed once [Cu2+] = 0 in the feed, 20 but it remains unknown whether the biological effect is equally reversible. So as to answer 21 this question, an experiment consisting in suddenly removing Cu2+ from the feed was 22 carried out; this was done at the end of the period when continuous bio-oxidation 23 happened with 20 g/L of [Cu2+]. Figure 9 shows the concentrations of Fe2+ measured in the 24 effluent and the bio-oxidation rate during the transition stage. 25 22 In figure 9, it can be seen that removing Cu2+ from the feed is matched by an immediate 1 and gradual drop in Fe2+ concentration that ends with an asymptote. As a result, the bio- 2 oxidation rate rose and, in under 60 hours, even surpassed the initial bio-oxidation rate, 3 when [Cu2+] = 0 g/L. This result reveals that the inhibitory effect associated with Cu2+ is 4 reversible, and that the biofilm was even capable of growth during this time. 5 6 CONCLUSIONS 7 It is possible to achieve the adaptation of ferrooxidant cells to tolerate as much as 20 g/L 8 Cu2+ in continuous operation. In this situation, the bio-oxidation rate drops almost 25% with 9 respect to when Cu2+ is absent. Close to 10% of this decrease can be attributed to lower 10 O2 solubility because of the salting-out effect. However, the bio-oxidation rate can be 11 increased three fold if the feed of air is replaced with pure oxygen. The effect of Cu2+ on 12 continuous Fe2+ bio-oxidation is reversible. 13 The fact that bio-oxidation inhibition by Cu2+ is not exclusively caused by biological factors, 14 and that the kinetic delay is partly due to thermodynamic reasons offers the prospect of 15 improving bioreactor design in the field of bioleaching applications, which contributes to a 16 more versatile and competitive view of this clean technology. 17 18 Acknowledgements 19 The authors wish to thank the FEDER INTERCONECTA program (project TC-20111083) 20 for the financial support given for this study. The research group would like to thank Mr. Charles 21 Nisbet for his work translating this document from Spanish and revising it prior to publication. 22 23 References [1] W.G. Davenport, M. King, M. Schlesinger, A.K. Biswas, fourth ed., Extractive Metallurgy of Copper, Elsevier Science Ltd. Oxford (2002). [2] Watling H.R., The bioleaching of sulphide minerals with emphasis on copper sulphides — A review. Hydrometallurgy 84 : 81–108 (2006). [3] Rosa Faria, L., Ávila, R.J., Albis, V., Gomes, M., Creating a critical snapshot of the bioleaching sector by using patent databank analysis, Hydrometallurgy 175 : 155–163 (2018) [4] London Metal exchange, https://www.lme.com/en-gb/metals/nonferrous/copper/#tabIndex=0, 2018 (accessed 30 April 2018). [5] Shiers, D.W., Collinson, D.M., Watling, H.R., Life in heaps: a review of microbial responses to variable acidity in sulphide mineral bioleaching heaps for metal extraction, Research in Microbiology 167 : 576-586 (2016). 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[16] Carranza, F., Palencia, I., Romero, R., Silver catalyzed IBES process: application to a Spanish copper-zinc sulphide concentrate. Hydrometallurgy 44 : 29-42 (1997). [17] Palencia, I., Romero, R., Carranza, F., Mazuelos, A., Treatment of secondary copper sulphides (chalcocite and covellite) by the BRISA process. Hydrometallurgy 66 : 85-93 (2002). 31 0 10 20 30 40 50 60 70 80 90 100 050 100 150 200 250 300 350 400 convertion (%) time (h) 0 g/L 5 g/L 10 g/L 15 g/L 20 g/L abiotic 2.5 g/L abiotic 10 g/L abiotic 20 g/L Figure 2: Batch Fe2+ bio-oxidation with 0, 5, 10, 15 and 20 g/L of Cu2+, and abiotic Fe2+ oxidation with 0, 2.5, 10 and 20 g/L of Cu2+. Modified 9K medium at pH 1.25. Agitation at 180 rpm. 30 ºC Temperature. 32 0.00 0.05 0.10 0.15 0.20 0.25 0.30 100 120 140 160 180 200 Q0(g/Lh) stirring rate (r.p.m) Figure 3: Oxygen transfer rate (Q0) in 250 mL Erlenmeyer flasks at different stirring rates measured using the sulphite method; temperature: 30 ºC. 33 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0 1000 2000 3000 4000 5000 6000 7000 8000 F(t) time (min) Experimental Model Figure 4: Cumulative residence time distribution function F(t). Liquid flow rate = 240 mL/h, air flow rate = 750 mL/h. Negative step input signal. Cu2+ as tracer, at a concentration of 20.2 g/L. Perfect Mix Model.36 34 1.00 1.50 2.00 2.50 3.00 0.0 5.0 10.0 15.0 20.0 25.0 Biooxidation rate (g/Lh) [Cu2+] (g/L) Figure 5: Continuous operation. Mean Fe2+ bio-oxidation rate against [Cu2+] in feed, within the range 0 to 20 g/L. Feed: [Fe2+] = 7 g/L pH = 1.5. Average liquid feed flow: 240 mL/h. Air flow: 750 mL/h. Temperature: 30ºC. Operating time: 700 h. Error bars represent the standard deviation. 35 0.0 5.0 10.0 15.0 20.0 25.0 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 0100 200 300 400 500 600 700 [Cu2+] (g/L) Oxygen concentration (mg/L) Time (h) CL CL* Copper concentration Figure 6: Continuous operation. CL y CL* against time for different [Cu2+] in feed, within the range 0 to 20 g/L. Feed: [Fe2+] = 7 g/L pH = 1.5. Average liquid feed flow: 240 mL/h. Air flow: 750 mL/h. Temperature: 30ºC. 36 0 1 2 3 4 5 6 7 8 020 40 60 80 100 120 [Fe2+] (g/L); QFe (g/Lh) time (min) QFe; [Cu(II)]=15 g/L QFe; [Cu(II)]=20 g/L [Fe(II)]; [Cu(II)]=15 g/L [Fe(II)]; [Cu(II)=20 g/L Figure 7: Results for [Fe2+] and bio-oxidation rate QFe when the current of air was replaced with a current of oxygen, [Cu2+] in feed being 15 and 20 g/L. 37 0.000 0.010 0.020 0.030 0.040 0.0 5.0 10.0 15.0 20.0 25.0 kL·a (1/s) [Cu2+] (g/L) Figure 8: kL·a versus [Cu2+] in feed within the range 0 a 20 g/L. Feed: [Fe2+] = 7 g/L pH = 1.5. Average liquid feed flow: 240 mL/h. Air flow: 750 mL/h. Temperature: 30ºC. Error bars represent the standard deviation. 38 0 1 2 3 4 5 024 48 72 96 120 144 168 192 216 240 [Fe2+] (g/L); Biooxidation rate (g/Lh) time (h) [Fe(II)] Biooxidation rate Figure 9: Results for [Fe2+] and bio-oxidation rate shown against time after removing 20 g/L of Cu2+ from the feed.