Attachment of bacteria Acidithiobacillus ferrooxidans to pyrite was investigated in two dif- ferent environment: fresh water and saline water (water with 35 g/L of NaCl), in both cases at pH 4. Adsorption isotherms were tted to the Langmuir and Freundlich models. The results showed that the bacteria adhere to pyrite to a greater extent in fresh water than in saline water. The Langmuir and Freundlich models tted well the data obtained in fresh water, showing a coecient of determination (R2) approximately equal to 0.8 for both models. On the other hand, in saline water the models did not show a good coecient of determination with a value approximately equal to 0.4 for both models. Keywords: Langmuir, Freundlich, Isotherm, Acidithiobacillus ferrooxidans , Pyrite 1. Introduction Acidithiobacillus ferrooxidans is a chemolithoautotrophic bacterium that obtains the car- bon from the CO 2of the air, to generate their cellular mass, and the energy (electrons) via the oxidation of inorganic compounds (e.g. minerals). Due to its ability to oxidize ferrous and reduced sulfur compunds, A. ferrooxidans has been widely used in leaching when processing 5 copper oxides and copper sul des (Donati et al., 2007; Rawlings, 2005). Moreover, A. ferroox- idans has been studied as pyrite depressant in the otation process (Chandraprabha et al., 2004, 2005; 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). In general, bacteria tend to adhere to solid surfaces forming bio lms rather than remain free in solution as planktonic cells because they 10 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 nd protection against hostile environments and a high concentration of nutrient (Flemming and Wingender, 2010; Schaechter, 2009; Chandraprabha and Natarajan, 2006). In Chile most of the mines are located in the north of the country where arid conditions prevail. Due to the water scarcity in this region, mining companies have started to change the source of water that they currently use, from groundwater to seawater. The use of seawater is 15 becoming increasingly important in mining, especially in the leaching and otation processes. Microorganisms, including bacterium Acidithiobacillus ferrooxidans , have been used in the otation and leaching processes (Chandraprabha et al., 2004, 2005; Hosseini et al., 2005; Mehra- bani 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). In both processes, the adherence of microorganisms 20 to minerals is an important step. For example, bacterium A. ferrooxidans must adhere to the mineral before leaching it in the direct bio-oxidation mechanism(Schaechter, 2009); in bio ota- tion it has been shown that adhesion is part of the pyrite bio-depression mechanism (Donati et al., 2007; Ohmura et al., 1993). Langmuir isotherm is an adsorption model that has been used to determine qualitatively 25 the degree of attachment of A. ferrooxidans to minerals (Xia et al., 2013). The model assumes that: (i) bacteria form a monolayer over the mineral, (ii) all sites on the surface are energetically equivalent, (iii) the attached bacteria do not interact with each other and (iv) the bacteria do not move on the surface. The Langmuir isotherm model is presented in Equation (1): Xe=XmKLCe 1 + K LCe(1) Equation (1) can be easily linearized as follows: 30 1 Xe=1 XmKLCe+1 Xm(2) where, X eis the attached bacteria per gram of mineral (bacteria/g), C eis the equilibrium concentration of bacteria in solution (bacteria/mL), K Lis the Langmuir adsorption equilibrium constant (mL/bacteria) and X mis the maximum adsorption capacity per unit mass of adsorbent (bacteria/g). X mand K Lare tted from experimental data. 2 Freundlich isotherm is an empirical model given by the Equation (3): 35 Xe= K FCe1 n (3) Equation (3) can be written in the logarithmic form as follows: logX e= logKF+1 nlogCe (4) where, X eis the attached bacteria per gram of mineral (bacteria/g), C eis the equilibrium concentration of bacteria in solution (bacteria/mL), n indicates the adsorption e ectiveness and represents the inherent properties of the adsorbent and K Fis a constant related to the adsorption capacity ((bacteria =g)(mL=bacteria)1=n). K Fand n are tted from experimental 40 data. Because the use of bacteria and seawater in mining is gaining attention, it is important to study the adsorption behavior to pyrite in a saline environment. The present work studies the kinetics of attachment of Acidithiobacillus ferrooxidans to pyrite in fresh water and in water with 35 g/L NaCl (saline water), which is the concentration of sodium chloride in seawater. In 45 addition, the attachment behavior will be modeled using Langmuir and Freundlich equations to determine which of these two models represent the real behavior. 2. Experimental 2.1. Mineral The pyrite used corresponds to hand picked mineral samples that were manually crushed. 50 The samples were dry screened between 37 m-212m (mesh # 70 and # 400) and cleaned 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 55 The bacteria used correspond to Acidithiobacillus ferrooxidans strain ATCC19859. The bacteria were grown at 30 /uni2103in sterile basal medium containing 0.4 g/L of ammonium sulfate 3 ((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- 60 rity99.5%, Merck) was used as substrate. The sterile medium was inoculated with an active 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- 65 trifuge, at 5 /uni2103. The pellet obtained was re-suspended in a sulfuric acid (H 2SO4) solution at 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. 70 2.3. Attachment kinetics and isotherms Attachment kinetics experiments of A. ferrooxidans to pyrite were carried out in fresh water and saline water at pH 4. 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. The experiments were conducted at di erent initial concentration 75 of bacteria in order to construct the isotherm curves later. These tests were performed in a 50 mL Erlenmeyer ask, where 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 with di erent cell concentrations. 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 80 ask. This experiment was carried out only with bacteria and water, no mineral was added. The concentration of cells in solution was measured at di erent 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. 85 The adsorption isotherms were obtained by plotting the data at equilibrium for each initial concentration of bacteria. Subsequently, the Langmuir and Freundlich adsorption isotherms 4 models were tted to the experimental results. 3. Results and Discussion 3.1. Attachment kinetics 90 Figure 1 shows the concentration of bacteria A. ferrooxidans remaining in fresh water over time when they were contacted with pyrite. These attachment kinetics experiments were carried out at pH 4 and at di erent levels of initial concentration of bacteria. It is observed that the initial concentration of bacteria in solution progressively decreased and reaches a relatively constant value at aproximately 60 min, which indicates that the bacteria are being adsorbed on 95 pyrite. Then, the equilibrium was reached at 60 min, time from which the adsorption no longer occurs or it occurs to a lesser extent. The trend described previously was observed independently of the initial concentration of bacteria. In the control experiment (results not shown), it was determined that A. ferrooxidans concentration does not decrease over time, showing that the bacteria do not attach to internal walls of the ask. Figure 2 shows the concentration of 100 bacteria in saline water as a function of time. The concentration of bacteria quickly decreased and reached a concentration relatively constant at approximately 15 min. When comparing Figure 1 with Figure 2, it is possible to observe that a lower concentration of bacteria remained in fresh water than in saline water, independently of their initial concentration. This indicates that a greater number of bacteria attach to pyrite surface in fresh water than in saline water. 105 5 Figure 1: Concentration of bacteria A. ferrooxidans remaining in solution (fresh water) over time when they are contacted with pyrite. The attachment kinetics experiments were conducted in fresh water at pH 4 at di erent initial concentrations of bacteria. Figure 2: Concentration of bacteria A. ferrooxidans remaining in solution (saline water) over time when they are contacted with pyrite. The attachment kinetics experiments were conducted in saline water (35 g/L NaCl) at pH 4 at di erent initial concentrations of bacteria. 6 3.2. Adsorption isotherms Figure 3 shows the linear tting of isotherms obtained in fresh water and saline water to the Langmuir model, while, Figure 4 shows the linear tting to Freundlich model. Adsorption isotherms were plotted with data of bacteria concentrations at the equilibrium from the attach- ment kinetics experiments. From the slope and the intersection with the Y axis, the constants of 110 both models were obtained. In Table 1 the values of the Langmuir and Freundlich constants are presented. It is observed that the two models showed a better coecient of determination for the experimental data obtained in fresh water than in saline water. In fresh water both models presented a R2>0.8, while, in saline water, R2were approximately 0.4. Therefore, according to the Langmuir model, bacteria could attach as a monolayer to pyrite in fresh water. In saline 115 water is not possible to assume that because the coecient of determination (R2) was too low. The Freundlich model is appropriate to predict the attachment behavior of bacteria to pyrite in fresh water but not in saline water. The value of X mfrom the Langmuir model, indicates the maximum adsorption capacity per unit mass of adsorbent (bacteria/g). This value was equal to 5109bacteria/g in fresh water and in saline water. However, due to the low determination 120 coecient in saline water, the result in this case should not be considered. Figure 5 shows the adsorption isotherms and their adjustments to the Langmuir and Freundlich models. Is possible to observe that in fresh water the adsorption of A. ferrooxidans is higher than in saline water, showing higher attachment densities (number of attached bacteria per mass of mineral). 7 Figure 3: Linear tting of the isotherms obtained in fresh water and saline water (35 g/L NaCl) to the Langmuir model. Figure 4: Linear tting of the isotherms obtained in fresh water and saline water (35 g/L NaCl) to the Freundlich model. 8 Table 1: Parameters obtained from the Freundlich and Langmuir models for the adsorption isotherms in fresh water and in saline water (35 g/L NaCl). Model Parameter Fresh water Saline water LangmuirXm(bacteria/g) 5 1095109 KL(mL/bacteria) 3 :881091:14109 R20.85 0.44 Freundlichn 2.02 1.07 KF(bacteria=g)(mL=bacteria)1=n1:681051:85101 R20.81 0.41 Figure 5: Adsorption isotherms in fresh water and in saline water (35 g/L NaCl) and its tting to the Langmuir and Freundlich models. 4. Conclusions 125 The attachment density of bacteria A. ferrooxidans to pyrite was higher in fresh water than in saline water (35 g/L NaCl), showing that not only the characteristics of the adsorbent and the absorbate in uence the adherence, but also the characteristics of the environment where it 9 is carried out the adsorption. Freundlich and Langmuir models show to predict well the adhesion behavior of A. ferroox- 130 idans to pyrite in fresh water, however, in saline water the models show a low coecient of determination indicating that they do not predict the adhesion behavior in this type of solution. The good t of the experimental data to the Langmuir model suggests that the bacteria adhere as a monolayer on the surface of the pyrite when the solution correspond to fresh water. Acknowledgements 135 The authors would like to acknowledge the Biohydrometallurgy laboratory of Department of Chemical Engineering, Biotechnology and Materials, University of Chile, for allowing the use the installations of and equipment.