In the present work electrostatic forces are evaluated as the cause of attachment of Acidithiobacil- lus ferrooxidans to pyrite. Streaming potential of A. ferrooxidans and pyrite was measured in two environment, fresh water and saline water (water with 35 g/L of NaCl) at di erent pH's values. At the same time, attachment kinetics of A. ferrooxidans to pyrite were carried out in fresh and saline water at pH 4. The results showed that A. ferrooxidans and pyrite present lower streaming potential (comparing absolute values) in saline water than in fresh water, indicating the compression in the electric double layer caused by Cl{and Na+ions. Simultaneously, it was determined that the bacteria present a higher attachment to pyrite in fresh water than in saline water. This suggests that electrostatics forces do not play a leading role in the adhesion mechanism. Keywords: Streaming potential, Acidithiobacillus ferrooxidans , Pyrite 1. Introduction When particles are immersed in a uid, they develop a surface electrical charge which is compensated by an equivalent charge distribution (of opposite sign) in the aqueous phase (Stern layer). In the transient between the Stern layer and the bulk, a di use layer of counterions is formed. Both layers together form the so-called double electric layer. The potential between 5 the Stern layer and the di use layer is known as zeta potential. At high ionic strength, the double electric layer is compressed, which means that the zeta potential approaches zero at a smaller distance from the particle surface compared to when there is a low ionic strenght (Wills Corresponding author at: Department of Metallurgical Engineering and Materials, Universidad T ecnica Federico Santa Mar a, Chile. Email address: francisca.san@usm.cl (F. San Martin) Preprint submitted to ... March 28, 2019 and Finch, 2015; Hunter, 2013). Zeta potential is a parameter widely used to characterize the charge of mineral and mi- 10 crobial surfaces (Chen et al., 2008; Devasia et al., 1993; Liu et al., 2006; Solari et al., 1992). Zeta potential can be determined by the following techniques: microelectrophoresis, streaming potential measurements and electroosmosis. Microelectrophoresis is the most common way to measure zeta potential, however it has the disadvantage that only can be used for particles smaller than 10 m (Hunter, 2013). Streaming potential could be measured for bigger particles 15 (>10m) and it is related to the zeta potential ( ) by the following equation (Fuerstenau, 1956): =4 E P(1) where,is the viscosity, is the dielectric constant, is the speci c conductivity, P is the applied pressure di erence and E is the streaming potential. The mineral charge depends on the adsorption density of the ions that determine the poten- 20 tial on its surface. In the case of many minerals, including pyrite, the ions that determine the potential are H+and OH{, therefore, the charge varies with the pH. For example, at low pH, pyrite acquires a positive charge due to the formation of FeOH 2+and FeSH 2+on its surface, as the pH rises, the FeO{and FeS{species become predominant and the mineral becomes negatively charged. 25 Bacterium A. ferrooxidans is surrounded by substances called exopolysaccharides (EPS) which give them their surface properties. EPS have a di erent composition depending on the growth substrate of the bacteria. For example, it has been determined that EPS from A. ferrooxidans grown in iron (II) sulfate contain mainly sugars (52.2%), while, if the growth sub- strate is sulfur, the major component of EPS corresponds to lipids (53.8%) (Gehrke et al., 1998). 30 EPS have an in uence on the adherence of bacteria to minerals. It has been demonstrated that A. ferrooxidans without EPS presents less attachment to minerals than the bacteria with EPS (Yu et al., 2011; Harneit et al., 2006; Chandraprabha and Natarajan, 2013). Bacteria present charge due to functional groups such as amino (NH 2), carboxyl (COOH), and hydroxyl (OH), which are constituents of lipids, sugars and proteins on their EPS (Sharma et al., 2003; Chen 35 et al., 2008; Devasia et al., 1993). 2 Nowadays, with the incorporation of seawater in the otation and leaching processes, it is important to study the properties of minerals in this type of water. On the other hand, the use of bacteria in metallurgical processes is becoming more relevant because it represents a sustainable alternative to the reagents currently used (Chandraprabha et al., 2004, 2005; 40 Hosseini et al., 2005; Mehrabani et al., 2011; Misra et al., 1996; Nagaoka et al., 1999; Ohmura et al., 1993; San Mart n et al., 2018; Donati et al., 2007; Rawlings, 2005). The present study aims to determine the behavior of surface charges of A. ferrooxidans and pyrite in fresh water and saline water (water with NaCl at the concentration of seawater, 35 g/L) by streaming potential measurements, in order to know if electrostatic forces are the responsibles of the attachment of 45 the bacteria to pyrite. 2. Experimental 2.1. Mineral The pyrite used corresponds to hand picked mineral samples that were manually crushed. The samples were dry screened between 37 m-212m (mesh # 70 and # 400) and cleaned 50 with 6N hydrochloric acid solution to remove the oxidized species from their surfaces. The purity of the pyrite was ascertained by X-ray di raction and it was determined to be higher than 99%. 2.2. Microorganisms The bacteria used correspond to Acidithiobacillus ferrooxidans strain ATCC19859. The 55 bacteria were grown at 30 /uni2103in sterile basal medium containing 0.4 g/L of ammonium sulfate ((NH 4)2SO4, purity 99.5%, Merck), 0.056 g/L of di-potassium hydrogen phosphate trihy- drate (K 2HPO 43H2O, purity 99%, Merck) and 0.4 g/L of magnesium sulfate heptahydrate (MgSO 47H2O, purity 99.5%, Merck) at pH 1.6. Iron sulfate heptahydrate (FeSO 47H2O, pu- rity99.5%, Merck) was used as substrate. The sterile medium was inoculated with an active 60 inoculum of A. ferrooxidans and a 33% (wt/v) solution of iron sulfate heptahydrate obtaining a concentration of 0.05 M of FeSO 47H2O. At the end of the incubation, the solution containing the cells was ltered using Whatman 42 lter paper to remove precipitated solids. The ltrate was then centrifuged at 12,000 rpm for 20 min in a Sorvall RC-5B refrigerated Superspeed Cen- trifuge, at 5 /uni2103. The pellet obtained was re-suspended in a sulfuric acid (H 2SO4) solution at 65 3 pH 2. The re-suspended cells were ltered using a 0.22 m Millipore membrane in order to obtain metabolite free centrifuged iron-free cells. Finally, cells retained in the membrane were re-suspended in pH 2 H 2SO4solution again. Bacterial concentration was monitored by direct counting in an Axio. Lab. A1 Zeiss microscope using a Neubauer counter. 2.3. Streaming potential 70 In order to evaluate the role of surface charges on the attachment of A. ferrooxidans to pyrite, the streaming potential was measured. Electrophoresis is the most common way to determine the charge of particle surfaces, however, it only can be used to particles smaller than 10m. Since, the mineral sample used in this work was between 37 and 212 m, streaming potential is a more appropriate method. The streaming potential was measured using the 75 Particle Charge Detector Mutek PCD 03. Streaming potential of A. ferrooxidans and pyrite were measured in presence of fresh water and saline water at di erent pH's (4, 6, 8, 10 and 12). Fresh water corresponds to distilled water, while, destilled water with NaCl at a concentration of 35 g/L (concentration of sodium chloride in seawater), was used as saline water. Pyrite (0.5 g) was mixed with 10 mL of water and conditioned with it for 5 min at the desired pH. 80 The resulting slurry was added to the measuring cell of the Particle Charge Detector and the streaming potential was measured. The same procedure was executed with A. ferrooxidans by mixing 150 L of a solution with metabolite free centrifuged iron-free cells with 9.85 mL of water. The concentration of bacteria was 2.39 108bacteria/mL (0.2805 g of biomass). In all experiments the pH was adjusted by adding either a solution of potassium hydroxide (KOH) 85 or sulfuric acid (H 2SO4). All experiments were conducted in duplicate. 2.4. Attachment kinetics Attachment kinetics experiments of A. ferrooxidans to pyrite were carried out in fresh water and saline water at pH 4. The experiments were conducted at an initial concentration of bacteria equal to 2.52 108bacteria/mL. These tests were performed in a 50 mL Erlenmeyer ask, where 90 1 g of pyrite was contacted with 19.5 mL of water and 0.5 mL of a solution with metabolite free centrifuged iron-free cells of A. ferrooxidans . The resulting slurry was agitated on a rotary shaker. A control experiment was carried out in fresh and in saline water to evaluate if bacteria attach to the internal walls of the ask. This experiment was carried out only with bacteria and 4 water, no mineral was added. The concentration of cells in solution was measured at di erent 95 times by direct counting in a Neubauer camera using a microscope Axio. Lab. A1 Zeiss. The number of cells attached to the mineral was calculated as the di erence between the cells in the liquid at a certain time and the initial cell concentration. The attachment kinetics experiments were carried out in duplicate. 3. Results and Discussion 100 The streaming potential of A.ferrooxidans and pyrite in fresh water at di erent pH's is presented in Figure 1. At all the pH range studied (4-12), both bacteria and pyrite were negatively charged. Other authors (Chandraprabha et al., 2004; Devasia and Natarajan, 2010) have reported isoelectric points (IEP) of pyrite equals to 3.25 and 2.9. Therefore, at the pH range studied it is not possible to nd pyrite positively charged. Alike, IEP values equals to 105 2.3 and 2.0 have been found for A. ferrooxidans , far from the lower pH analyzed in this study (Chandraprabha et al., 2004; Devasia and Natarajan, 2010; Chen et al., 2008; Misra et al., 1996; Sharma et al., 2003). Figure 2 shows the streaming potential of A.ferrooxidans and pyrite in saline water at di erent pH's. Pyrite presented two IEP (pH 5 and 10.2), this implies that it was positively 110 charged at pH <5 and at pH >10.2. From pH 4 to 6, the streaming potential of pyrite become increasingly negative but, from pH 6 up, it becomes progressively positive until it reaches pH 12. The bacteria showed an IEP equal to 4.6, then, it was positively charge at pH <4.6. The positive charge of pyrite at low pH is due to the formation of FeOH 2+and FeSH 2+ species. By raising the pH, pyrite streaming potential become progressively negative due to the 115 formation of FeOand FeSon the mineral surface. In the case of bacteria, the negative charge in most of the pH range studied, may be due to the presence of sugars. It has been determined that 52.2% of the EPS of A. ferrooxidans cultured in iron (II) sulfate correspond to sugars (monosaccharides) (Gehrke et al., 1998). The monosaccharides have carboxyl groups (COOH), which can lose its proton, leaving a negative charge (COO{). The anomalous behavior of the 120 pyrite streaming potential in saline water (Figure 2), can be explained with the theory of the electric triple layer (Liu et al., 2006). The magnitude of streaming potential of pyrite and A. ferrooxidans was lower in saline water than in fresh water. This is explained by the presence of sodium (Na+) and chloride 5 (Cl{) ions which increase the ionic strength and compress the electric double layer. 125 Figure 1: Streaming potential of A. ferrooxidans and pyrite in fresh water. 6 Figure 2: Streaming potential of A. ferrooxidans and pyrite in saline water (35 g/L NaCl or 0.6 M ). Figure 3 shows the attachment kinetics of A. ferrooxidans to pyrite in fresh water and in saline water. The dotted lines correspond to the controls. The controls are experiments that were carried out without mineral to determine if the bacteria adhere to the internal walls of the ask. It can be observed that in the controls the density of attached bacteria (bacteria attached per gram of pyrite) did not increase over the time, indicating that they did not adhere to the 130 internal walls of the ask. When pyrite is added, the density of attached bacteria increases. This occurs both for fresh water and saline water. It can also be observed that the adhesion of the bacteria to pyrite is greater in fresh water than in saline water. Due to the compression of the electrical double layer in saline water, it would be expected that the attachment of A. ferrooxidans to pyrite would be greater in this kind of water than in 135 fresh water. However, this does not happen but the opposite, in fresh water attachment density of the bacteria to the mineral is higher than in saline water. This suggests that the surface charges are not responsible for adhesion. These results are in agreement with the results of Tan and Chen (2012) who also determined that the adhesion mechanism is not related to surface 7 charges. 140 Figure 3: Attachment kinetics of A. ferrooxidans to pyrite in fresh and saline water (35 g/L NaCl or 0.6 M). The controls are experiments carried out without mineral (only bacteria and water). 4. Conclusions In saline water (water with 35 g/L of NaCl), A. ferrooxidans and pyrite showed streaming potentials very low compared to that obtained in fresh water. This indicates the compression in double electrical layer due to the Na+and Cl{ions. At the same time, a higher attachment density of A. ferrooxidans to pyrite was shown in fresh water than in saline water. This suggests 145 that electrostatic forces are not the responsibles of the attachment of A. ferrooxidans to pyrite. Acknowledgements In saline water (water with 35 g/L of NaCl), A. ferrooxidans and pyrite showed streaming potentials very low compared to that obtained in fresh water. This indicates the compression in double electrical layer due to the Na+and Cl{ions. At the same time, a higher attachment 150 density of A. ferrooxidans to pyrite was shown in fresh water than in saline water. This suggests that electrostatic forces are not the responsibles of the attachment of A. ferrooxidans to pyrite. 8