Department Keyword: A. ferrooxidans, pyrite, pyrrhotite ... · PDF fileDraft 1 1 Kinetics of Pyrite, Pyrrhotite and Chalcopyrite Dissolution by 2 Acidithiobacillus ferrooxidans 3 4

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Kinetics of Pyrite, Pyrrhotite and Chalcopyrite Dissolution

by Acidithiobacillus ferrooxidans

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2016-0085.R1

Manuscript Type: Article

Date Submitted by the Author: 19-Mar-2016

Complete List of Authors: Kocaman, Ayse Tuba; Yildiz Technical University , Bioengineering Department Cemek, Mustafa; Yildiz Technical University , Bioengineering Department Edwards, Katrina Jane; University of Southern California, Biological Sciences Department

Keyword: A. ferrooxidans, pyrite, pyrrhotite, chalcopyrite, dissolution kinetics

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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Kinetics of Pyrite, Pyrrhotite and Chalcopyrite Dissolution by 1

Acidithiobacillus ferrooxidans 2

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Ayse Tuba Kocaman a, b, *

, Mustafa Cemek b, Katrina Jane Edwards

a, 1 5

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a Department of Biological Sciences, University of Southern California, Los Angeles, CA 8

90089, USA 9

b Faculty of Chemical and Metallurgical Engineering, Department of Bioengineering, Yildiz 10

Technical University, 34210 Esenler, Istanbul, TURKEY 11

1 Deceased 12

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*Corresponding author 15

Ayse Tuba Kocaman

16

[email protected] 17

+90(505) 697 7135 18

19

Mustafa Cemek 20

[email protected] 21

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Kinetics of Pyrite, Pyrrhotite and Chalcopyrite Dissolution by 26

Acidithiobacillus ferrooxidans 27

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Abstract 29

The main objective of this study was to investigate the dissolution kinetics of pyrite, 30

pyrrhotite, and chalcopyrite. Crushed minerals were reacted with A. ferrooxidans (25°C). The 31

kinetics of dissolution was investigated by monitoring pH and Fe2+

, Fe3+

ion concentrations in 32

the leaching solutions. Pyrite, pyrrhotite and chalcopyrite dissolution by A. ferrooxidans were 33

found to be chemically controlled processes. The dissolution rates of the minerals with 34

bacteria increased in the order of pyrrhotite, pyrite, and chalcopyrite. The attached cell 35

numbers to mineral surfaces increased in the same order. A. ferrooxidans was found to 36

enhance the dissolution rates of the minerals. The acid-insoluble trait of pyrite and acid-37

soluble trait of the other two minerals affected the pH changes in the leaching solutions. 38

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Keywords: A. ferrooxidans, pyrite, pyrrhotite, chalcopyrite, dissolution kinetics 40

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1. Introduction 51

Over the last few decades, the mining industry has had the growing interest in 52

bioleaching of sulphide minerals as a means of metal recovery. “Bioleaching” is described as 53

biological oxidation of metal sulphides (Rawlings and Silver 1995), and the mobilization of 54

metal cations involving biological oxidation from otherwise insoluble ores (Vera et al. 2013). 55

Bioleaching is a vital alternative to traditional extraction methods because of its low cost, 56

environmental benefits, and broad efficiency for low-grade and recalcitrant ores (Rawlings 57

and Silver 1995). Pyrite (FeS2) and pyrrhotite (Fe1-xS) are the examples of sulphide minerals 58

composed entirely of Fe and S, whereas other sulphide minerals such as chalcopyrite 59

(CuFeS2) contain economically valuable metals as well (Bulaev et al. 2012). Although pyrite 60

is not an important economic mineral by itself, many studies on biologically mediated pyrite 61

oxidation have been conducted (Bellenberg et al. 2015; Konhauser et al. 2011; Edwards et al. 62

2003; Singer and Stumm 1970) because it is commonly associated with valuable minerals 63

such as sphalerite, chalcopyrite, and galena (Chandra and Gerson 2010). Pyrrhotite (Fe1-xS) is 64

the second common iron sulphide in nature. Although extracting copper by bioleaching is 65

difficult due to the refractoriness of chalcopyrite, it has been studied intensively because of its 66

high crustal abundance (Brierly and Brierly 2013). 67

Iron-oxidizing bacteria obtain metabolic energy through the oxidation of iron- and 68

sulfur-bearing minerals. In doing so, they dissolve economically valuable metals (Sand et al. 69

1995; Ehrlich and Fox 1967). Acidithiobacillus ferrooxidans (Acidithiobacillus was formerly 70

known as “Thiobacillus”, Kelly and Wood 2000) was the first to be isolated and is the best-71

studied iron and/or sulfur-oxidizing bacterium (Vera et al. 2013). 72

Most studies on bioleaching of sulphide minerals have focused mainly on the effect of 73

mixed bacterial cultures (Romo et al. 2013), mineral morphology (Dong et al. 2013), bacterial 74

attachment (Yang et al. 2014), mineral particle size (Olubambi et al. 2009), pulp density 75

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(Pradhan et al. 2010), the presence of various metallic ions in solution (Wong et al. 2015), 76

and temperature on the dissolution kinetics of sulphide minerals (Vilcaez et al. 2008). A 77

significant number of individual studies focused on the bioleaching of pyrite (Edwards et al. 78

1998; Florian et al. 2011), pyrrhotite (Korehi et al. 2014) and chalcopyrite (Zhu et al. 2015).79

To date, more studies on chemical leaching kinetics of sulphide minerals were carried 80

out (Perez and Dutrizac 1991; Dreisinger and Abed 2002; Aydogan et al. 2006; Adebayo et 81

al. 2003; Havlik and Kammel 1995) compared to bioleaching kinetics (Pradhan et al. 2010; 82

Abhilash et al. 2013). On the other hand, very few studies compared the bioleaching rates of 83

pyrite, pyrrhotite, and chalcopyrite (Lei et al. 2007). The objective of this study was to 84

investigate the dissolution kinetic mechanisms of pyrite, pyrrhotite, chalcopyrite, and 85

compare the bioleaching rates in the presence of A. ferrooxidans. 86

2. Materials and Methods 87

2.1. Bacterial Cultivation 88

A. ferrooxidans strain ATCC 23270 was purchased from the American Type Culture 89

Collection (ATCC). Cells were cultured in a medium containing solution A, and solution B. 90

Solution A contained (in g/L): (NH4)2SO4 0.8; MgSO4.7H2O 2.0; K2HPO4 0.4; and 0.5% 91

Wolfe’s mineral solution (V/V) (Wolin et al. 1963). Solution B contained 20 g FeSO4.7H2O 92

per liter of solution. The ratio in which solutions A and B were mixed for preparing the final 93

medium was 4:1. Solution A was prepared as described above, and the pH was adjusted to 2.3 94

with H2SO4, then sterilized by autoclaving at 121°C for 15 minutes. Solution B was prepared 95

separately and filtered through a 0.2 µm membrane filter (Acrodisc). Solution A and solution 96

B were then aseptically combined. Batch cultures of A. ferrooxidans were grown at 120 rpm 97

on a rotary shaker at 25°C for seven days before harvesting for bioleaching experiments. 98

2.2. Minerals 99

The following sulphide minerals were used in the bioleaching experiments: Pyrite 100

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(FeS2; Ward’s, Scientific, Huanzala, Peru), pyrrhotite (Fe1-xS, x=0 to 0.2; Ward’s, Scientific, 101

Galax, Virginia, USA), and chalcopyrite (CuFeS2; Ward’s, Scientific, Durango, Mexico). All 102

minerals were crushed using a mortar and pestle and sieved to a grain size of ≤ 355µm. 103

Crushed minerals were cleaned by following three steps: First, they were repeatedly rinsed 104

with distilled water and sonicated in an ultrasonic cleaner for 1-2 minutes to remove fine 105

particles. Second, they were treated with 10% HCl for 2 hours to remove any preexisting 106

oxide layers, then rinsed with 100% ethanol (Koptec, 200 proof) and dried in a shallow layer 107

(Edwards et al. 2001). Finally, 2 grams of the cleaned, crushed minerals were added to each 108

of sterile 250-ml flasks and autoclaved for 8 min. 109

2.3. Experimental Procedures 110

Cells of the A. ferrooxidans grown in the ferrous sulfate medium for one week in 111

shake culture were harvested by centrifugation at 10,000 rpm for 20 minutes and then washed 112

once by centrifugation at 10,000 rpm for 15 min in filter-sterilized distilled water adjusted to 113

pH 1.5 with H2SO4. Cells were re-suspended in salts medium (solution A) at pH 2.3. Two mL 114

of cell suspension (initial cell number ≈ 6.5x106 cells mL

1-) was inoculated into 250 mL 115

flasks containing 2 grams of sterile crushed minerals and 35 mL of salts medium (solution A) 116

(Edwards et al. 2001). Duplicate sets for each mineral were prepared. Bioleaching flasks (A. 117

ferrooxidans + medium + crushed minerals) and control flasks (medium + crushed minerals) 118

were incubated without shaking at 25°C for 30 days. 119

2.4. Analytical Techniques 120

The leaching solutions were analyzed for the concentration of bacteria unattached to 121

mineral particles, total Fe, Fe2+

, Cu2+

ions, and pH. For cell counting, 2µL of culture samples 122

were spread evenly on a 1% agarose-coated fluoroslide printed with 4 mm diameter circles 123

(Emerson and Moyer 2002). After air-drying, the samples were stained with DAPI (200 124

µg/mL) and fifty fields/circle were counted at 100X magnification using an epifluorescent 125

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Axiostar Plus microscope. From each flask, a 1-mL sample was removed daily, filtered 126

through a 0.2 µm membrane, and pH values were determined using a pH-meter (Thermo 127

Scientific). For dissolved iron measurements, a 1-mL sample was collected from each flask, 128

filtered through a 0.2µm membrane and diluted ten times with 1 mM HCl when their 129

absorbance measurements with UV- spectrophotometer were higher than 1.0. 1-mL of 130

samples were also collected from the chalcopyrite flasks and filtered through a 0.2µm 131

membrane and diluted 100 times with distilled water to measure Cu2+

ion concentrations. All 132

ion concentrations were measured using the UV-spectrophotometer (Shimadzu UV-1601). 133

Any decrease in volume due to sampling was adjusted to the original volume by addition of 134

an equivalent volume of sterile solution A. 135

The original ferrozine method was modified to measure total Fe and Fe2+

ion 136

concentrations (Stookey 1970). Ferrozine reagent (2 mM Ferrozine in 50 mM Hepes) was 137

used to determine both total Fe and Fe2+

ion concentrations whereas a reducing reagent (6.25 138

mM hydroxylamine hydrochloride) was used to determine total Fe ion concentrations. The 139

absorbance values at 562 nm were used to calculate iron ion concentrations. Standards 140

containing different known concentrations of FeSO4 in 1mM HCl were prepared. Fe3+

141

concentrations were calculated as the difference between total Fe and Fe2+

. 142

To measure Cu2+

ion concentrations, 0.1 mM Bathocuprione disulfonic acid (Aldrich) 143

and reducing reagent (0.3 mM hydroxylamine hydrochloride) were used. The method was 144

modified from that of Moffett et al. (1985). Standards containing different known 145

concentrations of CuSO4 in distilled water were prepared. For each sample, a full spectrum 146

was run between 400-800 nm. A peak for Cu2+

concentrations can be detected at 484 nm. The 147

area of the peak was measured using the Shimadzu UV Probe Version 2.20 software. Cu2+

148

concentrations in the leaching solutions were calculated by using the peak area. 149

The XRD patterns and chemical compositions of original pyrite, pyrrhotite and 150

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chalcopyrite, were characterized by X-ray diffraction (XRD, Philips Panalytical X’Pert-Pro, 151

CuKα radiation), and X-ray fluorescence spectrometry (XRF, Philips Panalytical Minipal 4), 152

respectively. 153

3. Results and Discussion 154

Leaching experiments in the presence and absence of A. ferrooxidans were carried out 155

to investigate the dissolution kinetic mechanisms of pyrite, pyrrhotite and chalcopyrite. In the 156

following, we record our findings on pH, Fe2+

, Fe3+

, Cu2+

concentration variations in the 157

leaching solutions, XRD and XRF analysis of untreated minerals. 158

The mineral structure is an important factor, which determines the mechanism and 159

chemistry of dissolution. Sand et al. (2001) reported that metal sulphides are degraded 160

through a chemical attack of Fe3+

ions and/or protons. Most metal sulphides, including pyrite, 161

pyrrhotite, and chalcopyrite, are semiconductors. In semiconductors, the valence band has the 162

highest energy level and is filled with electrons. In pyrite, the valance bands are derived only 163

from orbitals of metal atoms, whereas the valance bands in pyrrhotite and chalcopyrite are 164

derived from both metal and sulfur orbitals. Thus, the bonding between the metal and sulfur 165

atoms in the pyrite crystal lattice can be broken only by Fe3+

ions. In contrast, both Fe3+

ions 166

and protons can break metal-sulfur bonds in many other metal sulphides including pyrrhotite 167

and chalcopyrite. Consequently, pyrite is insoluble in acid while the others are acid-soluble 168

(Sand et al. 2001). Schippers and Sand (1999) proposed a thiosulfate mechanism for the 169

oxidation of acid-insoluble metal sulphides and a polysulfide mechanism for the oxidation of 170

acid-soluble metal sulphides. Thus for acid-insoluble pyrite, the reactions of the thiosulfate 171

mechanism are as follows (Eq. 1-2): 172

FeS2 + 6Fe3+

+ 3H2O → S2O32-

+ 7Fe2+

+ 6H+ (1) 173

S2O32-

+ 8Fe3+

+ 5H2O → 2SO42-

+ 8Fe2+

+ 10H+ (2) 174

For acid-soluble metal sulphides (MS) including pyrrhotite and chalcopyrite, the polysulfide 175

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mechanism reaction is as follows (Eq. 3): 176

MS + Fe3+

+ 2H+ → M

2+ + H2S

*+ + Fe

2+ (3) 177

Fe3+

ions are the most reactive oxidants in the oxidation of sulphide minerals. The 178

dissolution reactions of pyrite, pyrrhotite and chalcopyrite with Fe3+

are shown in equations 4, 179

5 and 6 (Singer and Stumm 1970; Nicholson and Scharer 1994; Dutrizac and MacDonald 180

1974). 181

FeS2 + 14 Fe3+

+ 8H2O → 15Fe2+

+ 2SO42-

+ 16H+

(4) 182

Fe1-xS + (8-2x) Fe3+

+ 4 H2O → (9-3x) Fe2+

+ SO42-

+ 8H+ (5) 183

CuFeS2(s) + 16Fe3+

+ 8H2O → Cu2+

+ 17Fe2+

+ 2SO42-

+ 16H+ (6) 184

4Fe2+

+ 4 H+ + O2 → 4 Fe

3+ + 2H2O (Fe-oxidizers) (7) 185

S0 + 3/2 O2 (aq) + H2O → H2SO4 (S-oxidiziers) (8) 186

As the most abundant metal sulphides on Earth, pyrite, pyrrhotite and chalcopyrite are 187

well-studied sulphide minerals. However, the oxidation processes of these minerals are 188

complicated and not yet fully understood (Evangelou 1995; Chirita and Rimstidt 2014; 189

Ahmadi et al. 2011). Because the dissolution mechanisms of chalcopyrite in acidic solutions 190

are still under debate, a general theory could not be proposed. Many researchers have studied 191

the efficient factors on dissolution rates and the possible reaction mechanisms (Li et al. 2010). 192

Stott et al. (2003) studied the bioleaching of chalcopyrite by 11 species of acidophilic bacteria 193

and archaea and investigated the effects of microorganisms, temperature and redox potential 194

on the dissolution rates. The effects of acid and ferric iron concentrations, solid/liquid ratio, 195

and synergistic effect of cupric/ferrous ions on chalcopyrite dissolution behavior were also 196

investigated (Antonijevic and Bogdanovic 2003; Hiroyoshi et al. 2004). In another study, 197

neither an increase in ferric iron nor an increase of bacterial concentration could improve the 198

dissolution rates. A limited bacterial activity and a controlled ferric iron concentration were 199

suggested for the improved bioleaching rates (Third et al. 2000). Sasaki et al. (1995) studied 200

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the chemical dissolution of pyrite with Fe3+

ions around pH 2. The results showed that pyrite 201

dissolved nonstoichiometrically in the initial stage, and later a sulfur-rich layer was formed on 202

the mineral surface. Yahya and Johnson (2002) examined the bioleaching of pyrite by two 203

strains of acidophilic bacteria. They discussed the advantages of bioleaching of sulfidic 204

minerals under low pH (<1) and low redox potential. Bhatti et al. (1993) studied the oxidation 205

of pyrrhotite by A. ferrooxidans. They observed an acid-consuming step with the formation of 206

elemental sulphur, following by an acid-producing step, which is a result of the formation of 207

solid products. Janzen at al. (2000) studied on the oxidation of 12 well-characterized 208

pyrrhotite samples by ferric iron and oxygen and reported the incomplete oxidation of 209

pyrrhotite with both oxidants. Harries et al. (2013) investigated the dissolution behavior of 210

pyrrhotite samples, which have different crystal structures, and found that the crystal structure 211

strongly influences the mineral dissolution. Plenty of researches were carried out to 212

investigate the effects of crystal structure, oxygen and ferric iron concentrations, temperature 213

and pH on the dissolution of pyrrhotite (Belzile et al. 2004). Although chalcopyrite is 214

refractory to hydrometallurgical methods, it is studied extensively due to its high crustal 215

abundance. The main factors affecting the dissolution rates of chalcopyrite are the 216

concentration of oxidants, the ferric/ferrous ion ratio in the leaching solutions, and the 217

formation of the passivation layer on the mineral surface (Klauber 2008; Li et al. 2010, 2013). 218

Olvera et al. (2014) studied the dissolution of chalcopyrite electrode in a solution composed 219

of H2SO4, FeSO4.7H2O and Fe2(SO4)3.5H2O at 25°C. According to the results, dissolution 220

of mineral increased with increasing Fe3+

concentrations and with the addition of FeS2 221

particles. Zhao et al (2015a) reported that additional Cu2+

and Fe2+

ions accelerated the 222

dissolution of chalcopyrite initially, but the formation of jarosite inhibited further dissolution. 223

In another study, Zhao et al. (2015b) studied the bioleaching of chalcopyrite with A. 224

ferrooxidans. The results showed that bacteria accelerate the copper extraction rate initially. 225

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In the later stage of bioleaching, copper extraction rate decreased due to the formation of 226

jarosite and polysulfide. 227

Fe3+

/Fe2+

ratio is another important parameter affecting the dissolution of sulphide 228

minerals (Cordoba et al. 2008; Hiroyoshi et al. 2000, 2001, 2004, 2008; Kametani and aoki 229

1985; Sandstrom et al. 2004). Microorganisms maintain high ratios of ferric to ferrous ion in 230

bioleaching solutions, thus enhance electron transfer between oxidizing agents and the 231

minerals (Wu et al. 2014). To understand the role of bacteria, Khoshkhoo et al. (2014) 232

studied on the chemical dissolution of chalcopyrite by mimicking the redox conditions of 233

thermophilic bioleaching of the mineral. Surprisingly, copper recoveries were very similar in 234

the presence and the absence of bacteria. Therefore, they concluded that the role of bacteria 235

was only to provide the oxidizing agent for chalcopyrite dissolution, by oxidizing Fe2+

to 236

Fe3+

. Similarly, Third et al. (2000) carried out a series of leaching experiments with and 237

without bacteria and found that ferric/ferrous ratio is more relevant for determining 238

chalcopyrite dissolution rates than the concentration of bacteria in the leaching solution. 239

Petersen and Dixon (2006) studied on bioleaching of a copper-gold concentrate by using 240

mesophilic, moderately thermophilic and extremely thermophilic bacteria and concluded that 241

high temperature and low redox potential supported the dissolution of chalcopyrite. In 242

contrast, the opposite conditions were sufficient for obtaining high dissolution rates of pyrite. 243

Gericke et al. (2010) found that redox control is not necessary for achieving high copper 244

extraction rates at high temperatures around 70°C, but it increases the extraction rates around 245

45°C. 246

Attacking agents for dissolution of pyrite are only Fe3+

ions (Eq. 1-2), whereas 247

attacking agents for pyrrhotite and chalcopyrite are both Fe3+

and H+

(Eq. 3). In our study, the 248

pH values continuously decreased throughout the experiments in pyrite leaching solutions due 249

to its acid-insoluble nature. The pH values in pyrrhotite and chalcopyrite leaching solutions 250

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increased at the initial stage of bioleaching due to their acid-soluble nature and then 251

decreased. Lei et al. (2007) observed significant amounts of jarosite and elemental sulfur 252

during the bio-oxidation of pyrrhotite and chalcopyrite with A. ferrooxidans while no such 253

deposition was observed for pyrite. Similarly, in our study, no deposition was observed for 254

pyrite during the experiments. For pyrrhotite and chalcopyrite, some deposits were seen after 255

a week, corresponding to the decrease in pH (Fig. 1). 256

Because bacteria both accelerate the oxidation of Fe2+

and sulphur, more Fe3+

ions 257

were involved in dissolution reactions, and more sulfate was produced in bioleaching 258

solutions compared to control solutions (Eq. 4-5). Thus, the pH values in the pyrite and 259

pyrrhotite bioleaching solutions were lower than the corresponding controls (Fig. 1). By 260

contrast, the pH values in chalcopyrite bioleaching solutions were higher than in control 261

solutions during measurements (Fig. 1). Chalcopyrite (bio)leaching processes include 262

complex oxidation-reduction reactions such as the oxidation of mineral, iron, sulphur, and 263

hydrolysis of ferric iron. The pH behavior in chalcopyrite leaching solutions may be a result 264

of combined effect of these reactions. Although chalcopyrite has the lowest %Fe content 265

compared to other minerals (Table II), it is apparent from Fig. 5c that the highest bacterial 266

oxidation of Fe was determined in chalcopyrite bioleaching solutions. Therefore, bacterial 267

oxidation of Fe, an acid-consuming reaction, is very likely to be the dominant reaction in 268

chalcopyrite bioleaching processes and resulted in high pH values of biotic solutions 269

compared to controls. On the other hand, our result is in agreement with that of Zhao et al. 270

(2013). They studied the bioleaching of chalcopyrite with A. ferrooxidans, and the pH values 271

in bioleaching system were found to be higher than the controls during the experiments. 272

Fe2+

released from minerals (Eq. 1-3) is cycled between the oxidized and reduced 273

states by bacterial oxidation of Fe2+

(Eq. 7) and consumption of Fe3+

during mineral 274

dissolution (Eq. 4-6). Because of the bacterial oxidation of Fe2+

, we observed low 275

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concentrations of Fe2+

in the bioleaching solutions (Fig. 2), and low levels of Fe3+

in control 276

(abiotic) solutions for each mineral tested (Fig. 3). 277

The state of iron depends on environmental factors, such as pH. Above pH 3, iron is 278

insoluble, and it precipitates (Bird et al. 2011). Because of that, the concentration of free Fe3+

279

ions, which plays an active role in the dissolution of minerals, decrease in the leaching 280

solutions (Eq. 4-6). Figure 4 shows the Fe3+

concentration changes by pH in the bioleaching 281

solutions of the three minerals. Fe3+

concentrations decrease with increasing pH in pyrite 282

bioleaching solution, whereas they increase up to pH values 3.0 and 3.4 for pyrrhotite and 283

chalcopyrite respectively, and then decrease. Eq. (7) explains the Fe3+

behavior in pyrite and 284

pyrrhotite bioleaching solutions. However, the oxidation of ferrous ion might be affected both 285

by bacteria (Eq. 7) and cupric ions (Eq. 9) in chalcopyrite bioleaching solutions (Cher and 286

Davidson 1955). 287

Fe2+

+ Cu2+

↔ Fe3+

+ Cu1+

(9) 288

Figure 5 compares the pH, Fe2+

and Fe3+

concentration changes by time in the 289

bioleaching and control solutions of the three minerals. 290

Mathematical modeling of fluid-solid systems such as leaching of metals from 291

minerals is crucial to gain insight into the reaction mechanisms and to interpret experimental 292

results. During the leaching processes, many dissolution products were found on the mineral 293

surfaces. Researchers suggested the formation of various leaching products on the 294

chalcopyrite surface including jarosite, chalcocite, covellite, and sulfur (Pradhan et al. 2008; 295

Watling 2006; Karimi et al. 2010; Gericke et al. 2010). When pyrrhotite is exposed to moist 296

air, an over-layer of iron (III) oxyhydroxides form on the mineral surface (Buckley and 297

Woods 1985; Pratt et al. 1994; Mycroft et al. 1995). As the oxidation proceeds, a sub-layer of 298

polysulfides forms on the mineral surface. Bhatti et al. (1993) identified the mineral 299

weathering products (elemental sulfur, K-jarosite, goethite, schwertmannite) during the 300

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bioleaching of pyrrhotite by A. ferrooxidans. Sasaki et al. (1995) studied the dissolution of 301

pyrite by Fe3+

ions around pH 2. They observed a S-rich layer on pyrite surface. According to 302

Schippers et al. (1996), more elemental sulfur forms on the mineral surfaces, which are 303

oxidized by polysulfide mechanism (such as pyrrhotite) compared to the minerals oxidized by 304

thiosulfate mechanism. 305

If the product layers forming on the mineral surfaces are porous and permeable to 306

attacking agents, mineral dissolution process is controlled by chemical reactions. The kinetic 307

equation can be written as follows: 308

kc t= 1- (1-x)1/3

(10) 309

If the product layers are impermeable to attacking agents, diffusion through the product layer 310

controls the dissolution rate of mineral. The kinetic equation can be written as follows: 311

kd t = 1- (2x/3)-(1-x)2/3

(11) 312

In these equations, kc and kd are the leaching rate constants, and x is the fraction of iron 313

oxidized (Pradhan et al. 2010). 314

Abhilash et al. (2013) studied on the bioleaching of a low-grade chalcopyrite ore by A. 315

ferrooxidans. Their kinetic data was compatible with diffusion-controlled. Pradhan et al. 316

(2010) studied on the bioleaching of complex sulphide minerals including chalcopyrite, 317

sphalerite and galena by A. ferrooxidans. The dissolution of minerals showed a good fit with 318

product diffusion model. Natural pyrite samples were leached under various conditions, and 319

the dissolution found to be controlled by surface chemical reactions (Wiersma and Rimstidt 320

1984; Antonijevic et al. 1997; Dimitrijevic et al. 1999; Dimitrijevic et al. 1996). 321

Of the two kinetic equations, equation (10) has been found to give a straight line. 322

Figure 6 indicates the plot of 1-(1-x)1/3

versus bioleaching time of pyrite, pyrrhotite, and 323

chalcopyrite. The rate constants and the R2

values for both equations (Eq. 10-11) are shown in 324

Table I. Based on the R2 values of the plots, the dissolution of pyrite, pyrrhotite and 325

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chalcopyrite by A. ferrooxidans were found to be chemically controlled processes. It is also 326

evident that the bioleaching rate of minerals increases in the order of pyrrhotite, pyrite and 327

chalcopyrite (Table I). On the other hand, we observed very different ferric/ferrous iron ratios 328

for the bioleaching solutions of the three minerals. This might be one of the reasons for 329

various dissolution behaviors. 330

Chemical analysis of sulphide minerals is shown in Table II. Iron percentage of pyrite, 331

pyrrhotite and chalcopyrite are 53.5, 66.4 and 33.7 (wt %), respectively. 332

Three main bioleaching mechanisms are generally accepted: indirect, contact, and 333

cooperative mechanisms. For the latter two, bacterial attachment onto the mineral surface is 334

essential. In the indirect mechanism, planktonic cells make Fe3+

ions, which are present in the 335

bioleaching solutions, available for mineral dissolution (Li et al. 2013). According to 336

Rodriguez et al. (2003), bioleaching process includes three stages. In the first stage, bacterial 337

cells attach to the mineral surface. In the second phase, bacterial attachment decreases due to 338

surface saturation. Therefore, the concentration of planktonic cells in the leaching solution 339

increases. In the last stage, a balance between planktonic and attached cells is reached. 340

Yang et al. (2015) studied the early stage attachment of A. ferrooxidans at pyrite and 341

chalcopyrite surfaces. They did not observe a significant difference in selectivity of 342

attachment between the minerals. Africa et al. (2013) obtained the higher levels of attachment 343

of A. ferrooxidans to pyrite surface compared to chalcopyrite surface. Zhu et al. (2015) 344

studied the relation between bacterial attachment and bioleaching rate of chalcopyrite by A. 345

ferrooxidans. They concluded that as the number of attached cells increased, the dissolution 346

rate of mineral increased. Shrihari et al. (1995) studied on bioleaching of pyrite by T. 347

ferrooxidans in shake flasks. They concluded that bacteria dissolved the mineral primarily 348

through the direct mechanism. 349

In the present study, the planktonic cell concentrations in the bioleaching solutions of 350

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each mineral reached stationary phase around the 13th

day (Fig. 7). It seems that A. 351

ferrooxidans cells were effectively oxidizing the minerals during the first two weeks of 352

incubation (Eq. 7-8). The planktonic cell concentrations in the bioleaching solutions including 353

all minerals dropped on the second day and then increased. This decrease was likely due to 354

the bacterial attachment to the mineral surfaces within two days. It is known that most of the 355

leaching bacteria grow attached on the sulphide mineral surfaces. Schippers et al. (2014) 356

reported that more than 80% of inoculated cells were lost from the bioleaching solution 357

within one day. The second day of our experiments, 86%, 84%, and 80% of inoculated cells 358

were lost from the bioleaching solutions including chalcopyrite, pyrite, and pyrrhotite, 359

respectively. Attached cell numbers were determined by the difference between the initial 360

concentration of cells and the concentration of cells remaining in the solution after two days. 361

The results for chalcopyrite, pyrite, and pyrrhotite were 5.64x106, 5.51x10

6, and 5.20x10

6 362

(cells/mL), respectively. These findings are also consistent with the rate constants of the 363

minerals (Table I). 364

The oxidation rate of Cu (0.31 mM Cu2+

day-1

) was almost five times higher than the 365

oxidation rate of Fe (0.054 mM Fe3+

day-1

) in chalcopyrite bioleaching solution (Fig. 8a). It is 366

likely that some Fe3+

precipitated and some underwent further reaction with chalcopyrite. Fe3+

367

and Cu2+

ion concentrations in chalcopyrite bioleaching solution increased as the pH 368

increased up to pH 3.4, after which both ion concentrations decreased as the pH increased 369

(Fig. 8b). 370

The X-ray diffraction (XRD) analysis was conducted for the untreated minerals (Fig. 371

9). FeS2 and FeS were the major and the minor phases of pyrite, respectively. The main peaks 372

for chalcopyrite were djurleite (Cu1.96S), anhydrite (CaSO4), and Cu (FeO2). Pyrrhotite 373

showed an unidentified peak, which might be an evidence of its amorphous structure. 374

Although investigating the effect of some other parameters on the dissolution 375

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behaviors of sulphide minerals is out of scope in this paper, they are stated in the following 376

paragraph to gain a broad perspective. 377

In a relatively early study, pyrite, pyrrhotite and chalcopyrite were subjected to 68% 378

humidity in the air at 52°C to ascertain the nature of the oxidation of these minerals. The 379

results showed that pyrite and chalcopyrite were initially oxidized to ferrous/cuprous 380

thiosulphates, and then further oxidized to ferric/cupric sulphates. Pyrrhotite was oxidized to 381

goethite and elemental sulphur (Steger and Desjardins, 1978). Iron sulphides show different 382

dissolution behaviors due to their structures. Thomas et al. (2000, 2003) studied the acidic 383

dissolution of troilite (FeS), pyrite and pyrrhotite. Pyrite was found to dissolve oxidatively, 384

troilite was found to dissolve nonoxidatively, and pyrrhotite was found to dissolve in both 385

ways. They also reported that the temperature and the supply of oxidizing agents have an 386

impact on the dissolution mechanism of pyrrhotite. For further analyzing the effect of 387

different crystal and electronic structures on dissolution behaviors, Schippers and Sand (1999) 388

selected six different metal sulphides for dissolution experiments. The oxidizing agent was Fe 389

(III) chloride, and the pH was 1.9. As a result, they observed the formation of different 390

sulphur compounds for each of the mineral. Yevenes et al. (2010) studied the dissolution of 391

various chalcopyrite concentrates in chloride solutions with additional cupric ions at 35°C. 392

They reported that the cupric ion concentrations below 0.1 g/L increased the chalcopyrite 393

dissolution rate. Another possible factor affecting the dissolution of minerals is the 394

composition of the exopolymeric layer, a special reaction compartment, where the dissolution 395

reactions take place. It can differ concerning the substrate, thus affect the dissolution rates 396

(Sand et al. 2001). 397

In summary, our results showed that bioleaching rates of minerals by A. ferrooxidans 398

increase in the order of pyrrhotite, pyrite and chalcopyrite. Considering the individual pH 399

values of each of the bioleaching solutions (Fig. 5a), different dissolution behaviors of these 400

Page 16 of 39



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minerals might be explained. Besides, the difference in crystal structures of minerals (Fig. 9), 401

the mineral compositions (Table II), the number of attached cells, the concentration of 402

oxidizing agents (Fig. 5c), ferric to ferrous ion ratio in bioleaching solutions, the (in)solubility 403

of minerals in acid, and the presence of cupric ions in chalcopyrite bioleaching solutions 404

might be responsible for observing different dissolution rates. 405

4. Conclusion 406

Aspects of dissolution kinetics of pyrite, pyrrhotite, and chalcopyrite by A. 407

ferrooxidans were studied in 30-day experiments using crushed minerals. Plots of 1-(1-x)1/3

408

versus bioleaching time has been found to give a straight line for pyrite, pyrrhotite and 409

chalcopyrite (Fig. 6). These results indicate that the dissolution of pyrite, pyrrhotite and 410

chalcopyrite by A. ferrooxidans were chemically controlled processes. From the results in 411

Table I, it can be observed that the dissolution rates of minerals increase in the order of 412

pyrrhotite, pyrite, and chalcopyrite. The attached cell numbers to mineral surfaces increase in 413

the same order. A. ferrooxidans was found to accelerate the dissolution rates of the three 414

minerals. The pH behavior of the leaching solutions, containing the three minerals, was 415

affected by the acid-insoluble nature of pyrite, and the acid-soluble natures of pyrrhotite and 416

chalcopyrite. 417

418

419

420

Acknowledgements 421

The authors would like to thank Dr. Roman Barco, Dr. Jason B. Sylvan, and Professor Dr. 422

Hanifi Sarac for discussions. The salary was provided to A.T. Kocaman by the Center for 423

Dark Energy Biosphere Investigation Science and Technology Center (C-DEBI), and this 424

research was supported by Yildiz Technical University under grant [2014-07-04-DOP03]. 425

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695

696

697

698

699

700

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Table I. The kc and kd values and correlation coefficients for each mineral 701

Mineral kc R2 kd R

2

(10-3

day-1

) (10-5

day-1

)

Chalcopyrite 1.6 0.914 9.0 0.787

Pyrite 0.6 0.904 1.0 0.674

Pyrrhotite 0.2 0.828 0.2 0.656

702

Table II. Chemical analysis of sulphide minerals (wt %) 703

Mineral Fe S Cu Zn Si Ca

Chalcopyrite 33.7 21.0 29.3 - 4.9 11.1

Pyrite 53.5 45.1 0.69 0.67 - -

Pyrrhotite 66.4 24.7 1.90 3.3 3.7 -

704

705

706

707

708

709

710

711

712

713

714

715

716

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Figure 1. The changes of pH in the biotic flasks inoculated with A. ferrooxidans and abiotic 717

controls during the leaching of pyrite, pyrrhotite, and chalcopyrite. 718

Figure 2. The changes of Fe2+

ion concentrations in the biotic flasks inoculated with A. 719

ferrooxidans and abiotic controls during the leaching of pyrite, pyrrhotite, and chalcopyrite. 720

Figure 3. The changes of Fe3+

ion concentrations in the biotic flasks inoculated with A. 721

ferrooxidans and abiotic controls during the leaching of pyrite, pyrrhotite, and chalcopyrite. 722

Figure 4. Fe3+

ion concentration changes by pH in the biotic solutions of pyrite, pyrrhotite, 723

and chalcopyrite. 724

Figure 5. The comparison of (a) pH, (b) Fe2+

, and (c) Fe3+

ion concentrations in biotic (data 725

are avarage of duplicates) and control flasks. 726

Figure 6. Plot of (1-(1-x)1/3

) versus leaching time for pyrite, pyrrhotite and chalcopyrite. 727

Figure 7. Free bacterial cell concentrations in pyrite, pyrrhotite and chalcopyrite bioleaching 728

solutions. 729

Figure 8. Cu2+

and Fe3+

ion concentration changes by time, and by pH in the chalcopyrite 730

biotic and control flasks. 731

Figure 9. XRD patterns of untreated pyrite, pyrrhotite and chalcopyrite. An unidentified 732

phase is present for pyrrhotite. 733

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Figure 1. The changes of pH in the biotic flasks inoculated with A. ferrooxidans and abiotic controls during the leaching of pyrite, pyrrhotite, and chalcopyrite.

180x53mm (300 x 300 DPI)

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Figure 2. The changes of Fe2+ ion concentrations in the biotic flasks inoculated with A. ferrooxidans and abiotic controls during the leaching of pyrite, pyrrhotite, and chalcopyrite.

180x59mm (300 x 300 DPI)

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Figure 3. The changes of Fe3+ ion concentrations in the biotic flasks inoculated with A. ferrooxidans and abiotic controls during the leaching of pyrite, pyrrhotite, and chalcopyrite.

180x56mm (300 x 300 DPI)

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Figure 4. Fe3+ ion concentration changes by pH in the biotic solutions of pyrite, pyrrhotite, and chalcopyrite. 99x59mm (300 x 300 DPI)

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Figure 5. The comparison of (a) pH, (b) Fe2+, and (c) Fe3+ ion concentrations in biotic (data are avarage of duplicates) and control flasks. 184x154mm (300 x 300 DPI)

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Figure 6. Plot of 1-(1-x)1/3 versus leaching time for pyrite, pyrrhotite and chalcopyrite. 99x60mm (300 x 300 DPI)

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Figure 7. Free bacterial cell concentrations in pyrite, pyrrhotite and chalcopyrite bioleaching solutions.

99x72mm (300 x 300 DPI)

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Figure 8. Cu2+ and Fe3+ ion concentration changes by time, and by pH in the chalcopyrite biotic and control flasks.

160x64mm (300 x 300 DPI)

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Figure 9. XRD patterns of untreated pyrite, pyrrhotite and chalcopyrite. An unidentified phase is present for pyrrhotite.

113x150mm (300 x 300 DPI)

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