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Title: Oxidation of elemental sulfur, tetrathionate and ferrous iron by thepsychrotolerant Acidithiobacillus strain SS3
Authors: Daniel Kupka, Maria Liljeqvist, Pauliina Nurmi, Jaakko A. Puhakka, Olli H.Tuovinen, Mark Dopson
PII: S0923-2508(09)00159-4
DOI: 10.1016/j.resmic.2009.08.022
Reference: RESMIC 2788
To appear in: Research in Microbiologoy
Received Date: 20 May 2009
Revised Date: 19 August 2009
Accepted Date: 25 August 2009
Please cite this article as: D. Kupka, M. Liljeqvist, P. Nurmi, J.A. Puhakka, O.H. Tuovinen, M. Dopson.Oxidation of elemental sulfur, tetrathionate and ferrous iron by the psychrotolerant Acidithiobacillusstrain SS3, Research in Microbiologoy (2009), doi: 10.1016/j.resmic.2009.08.022
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For publication 1
Oxidation of elemental sulfur, tetrathionate and ferrous iron by the 2
psychrotolerant Acidithiobacillus strain SS3 3
4
Daniel Kupka,a Maria Liljeqvist,b Pauliina Nurmi,c,1 Jaakko A. Puhakka,d Olli H. Tuovinenc,d 5
Mark Dopsonb,* 6
7
aInstitute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, SK-043 53 Košice, Slovakia 8
bDepartment of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden cDepartment of 9
Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FI-33101 Tampere, 10
Finland 11
dDepartment of Microbiology, Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, 12
USA 13
Received 20 May 2009; accepted 25 August 2009 14
Email addresses: Daniel Kupka ([email protected]), Maria Liljeqvist ([email protected]), Pauliina Nurmi 15
([email protected]), Jaakko A. Puhakka ([email protected]), Olli H. Tuovinen ([email protected]), Mark 16
Dopson ([email protected])*Correspondence and reprints 17
18 1Present address: MTT Agrifood Research Finland, Biotechnology and Food Research, FI-31600 Jokioinen, Finland. 19
20
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Abstract 21
22
Mesophilic iron and sulfur-oxidizing acidophiles are readily found in acid mine drainage 23
sites and bioleaching operations, but relatively little is known about their activities at 24
suboptimal temperatures and in cold environments. The purpose of this work was to 25
characterize the oxidation of elemental sulfur (S0), tetrathionate (S4O62-) and ferrous iron 26
(Fe2+) by the psychrotolerant Acidithiobacillus strain SS3. The rates of elemental sulfur and 27
tetrathionate oxidation had temperature optima of 20° and 25°C, respectively, determined 28
using a temperature gradient incubator that involved narrow (1.1°C) incremental increases 29
from 5° to 36°C. Activation energies calculated from the Arrhenius plots were 61 and 89 kJ 30
mol-1 for tetrathionate and 110 kJ mol-1 for S0 oxidation. The oxidation of elemental sulfur 31
produced sulfuric acid at 5°C and decreased the pH to approximately 1. The low pH inhibited 32
further oxidation of the substrate. In media with both S0 and Fe2+, oxidation of elemental 33
sulfur did not commence until all available ferrous iron was oxidized. These data on 34
sequential oxidation of the two substrates are in keeping with upregulation and 35
downregulation of several proteins previously noted in the literature. Ferric iron was reduced 36
to Fe2+ in parallel with elemental sulfur oxidation, indicating the presence of a sulfur:ferric 37
iron reductase system in this bacterium. 38
39
Keywords Acidithiobacillus strain SS3; Iron oxidation; Psychrotolerant; Redox coupling; 40
Qulfur oxidation; Temperature dependency 41
42
43
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1. Introduction 44
45
Bioleaching processes utilize acidophilic Fe2+ and elemental sulfur (S0) oxidizing 46
microorganisms to aid in the solubilization of metals from sulfide minerals [27, 31]. In 47
bioleaching, the sulfur entity in sulfide minerals is oxidized chemically by Fe3+ or directly by 48
bacteria with oxygen as the electron acceptor. Typical intermediates from these reactions are 49
secondary sulfide minerals (e.g. CuS from CuFeS2), thiosulfate (S2O32-), polythionates 50
(SnO62-), polysulfides (Sn
2-) and elemental sulfur (S0) [34]. These sulfur compounds are 51
further oxidized to sulfate in acid-producing reactions. S0 is biologically oxidized to sulfate in 52
the presence of oxygen according to the net equation [1] (the major sulfate species in acidic 53
solutions is bisulfate (pKa2 1.92)). 54
2S0 + 3O2 + 2H2O → 2HSO4- + 2H+ [1] 55
The use of Fe3+ as an external electron acceptor for Acidithiobacillus ferrooxidans 56
mediated oxidation of elemental sulfur [eqn. 2] under oxic and anoxic conditions has been 57
reported [6, 28, 35]. 58
S0 + 6Fe3+ + 4H2O → HSO4- + 6Fe2+ + 7H+ [2] 59
In bioleaching processes, S0 can precipitate on sulfide mineral surfaces and slow down 60
the diffusion of reactants and products to and from the mineral surface, thereby passivating 61
the surface [22, 33, 42]. The passivating S0 layer may be removed or partially alleviated by 62
sulfur-oxidizing acidophilic bacteria and archaea [9, 11], but oxidation of such sulfur rims is 63
relatively slow. Bioleaching and associated iron oxidation involves acidic sulfate-containing 64
solutions which promote the precipitation of schwertmannite (eqn. 3) and jarosites such as K-65
jarosite (eqn. 4). 66
8Fe3+ + SO42- + 14H2O Fe8O8(OH)6SO4 + 22H+ [3] 67
3Fe3+ + K+ + 2SO42- + 6H2O KFe3(SO4)2(OH)6 + 6H+ [4] 68
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The chemical and biological oxidation of the S-entity in sulfide minerals is central to 69
metal dissolution, acid consumption or formation, and passivation in biological leaching 70
processes. Understanding of the biological oxidation of inorganic sulfur compounds at low 71
temperatures is therefore requisitely important for management of bioleaching processes and 72
environmental problems with mine waste rocks in boreal environments. Bacterial oxidation 73
of sulfide minerals at low temperatures has been reported [1, 12, 13, 25] and a number of 74
acidophilic iron-oxidizing strains have been isolated that are capable of growth at low 75
temperatures [5, 14]. In our previous work, we characterized growth and iron oxidation of an 76
Acidithiobacillus ferrooxidans strain SS3 at low temperatures [24]. Strain SS3 is 77
psychrotolerant (optimum growth at 20° to 40°C, but also grows at 4°C). The 16S rRNA 78
gene sequence of strain SS3 [24] aligned in a clade with other isolates from boreal 79
environments, including clone T7 from a pilot scale bioheap [29] and strain NO-37 from an 80
acid mine drainage site [19]. In this study, we have investigated the oxidation of S0 and 81
tetrathionate (S4O62-) by Acidithiobacillus strain SS3 and determined the corresponding 82
activation energy values for the oxidation of these substrates. In addition, redox coupling 83
between Fe3+ and S0 was demonstrated in this work. 84
85
2. Materials and methods 86
87
2.1 Microorganisms and growth conditions 88
89
Acidithiobacillus strain SS3 [24] was used throughout this study. Based on 16S rRNA 90
gene sequence similarity, strain SS3 aligns in a clade with the newly proposed 91
‘Acidithiobacillus ferrivorans’ sp. nov. [16]. Cultures were incubated in mineral salts 92
medium (pH 2.5) that contained (per liter) 3 g (NH4)2SO4, 0.1 g KCl, 0.5 g K2HPO4, 0.5 g 93
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MgSO4·7H2O, and 0.01 g Ca(NO3)2. For routine subcultures, the medium was amended with 94
5 g S0 L-1 (156 mM) and/or 80 mM Fe2+ as energy source. S0 (precipitated) was from 95
Lachema (Brno, Czech Republic; mean diameter of 30 µm and specific surface area of 0.175 96
m2 g-1). 97
98
2.2 Oxidation of tetrathionate and S0 in temperature gradient incubator 99
100
Oxidation rates of tetrathionate and S0 were measured over a temperature range of 5° to 101
30° ± 0.5°C in a temperature gradient incubator (Test Tube Incubator, Terratec®, Australia). 102
The temperature interval was approximately 1.1°C and the oscillation at 35 min-1. The 103
mineral salts medium was adjusted to pH 2.5 with H2SO4 and supplemented with trace 104
element solution [9] and filter-sterilized 5 mM K2S4O6 or 5 g L-1 (156 mM) S0 (autoclaved at 105
105°C for 30 min). The media were inoculated with approximately 1 × 108 cells from a batch 106
culture of Acidithiobacillus strain SS3 previously grown with the respective substrate. Cell 107
numbers were determined by counting 4’-6-diamidino-2-phenylindole stained cells under 108
epifluorescence microscopy. Changes in the tetrathionate concentration [21] and the pH were 109
measured at intervals. The oxidation data were fitted to the Ratkowsky equation according to 110
eqn. 5 [30]. 111
)1()(1 ))((min
maxTTceTTbt
−⋅−⋅−⋅= [5] 112
where T = temperature (°C); Tmin and Tmax = the minimum and the maximum temperature, 113
respectively; b and c = fitting parameters. The parameter time t is derived from the time it 114
takes for half the tetrathionate concentration to be consumed or for the pH to be decreased by 115
half. The greatest value for the square root of 1/t is derived from the optimum temperature 116
(Topt) shown by the inflection in the fitted curve. Chi-square (distribution when the null 117
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hypothesis is true) and R-square (index of correlation) values were calculated with a 95% 118
confidence interval together with the errors for parameters b and c. Microcal Origin 6.0 119
software was used for construction of the Ratkowsky equation with a non-linear regression 120
model. 121
Linearization of the Arrhenius equation (eqn. 6) based on the rates of tetrathionate 122
oxidation and pH decrease between 6° and 18°C was used to calculate the activation energy 123
(Ea). 124
AlnRT
ln +−= aEk [6] 125
where k = rate constant; A = frequency coefficient; Ea = activation energy (J mol-1); R = gas 126
constant; and T = absolute temperature (K). The rate constants for tetrathionate oxidation 127
were calculated from half-life data, the time it takes for half the tetrathionate concentration to 128
be oxidized (T½=0.693/k). The slope of the linear portion of the Arrhenius plot gives the Ea. 129
Similarly, the rate constants for sulfur oxidation were calculated from the corresponding 130
increases in sulfate concentration over time. Since tetrathionate and elemental sulfur 131
oxidation produces sulfuric acid, rate constants were also estimated from time courses of 132
decreases in pH. 133
134
2.3 Oxidation of elemental sulfur and iron 135
136
Elemental S0 (5 g L-1) was added to the mineral salts medium and autoclaved for 30 min 137
at 105ºC. Filter-sterilized ferrous sulfate and ferric sulfate solutions were added as required 138
and the pH was adjusted to 2.5 with either H2SO4 or KOH. The inocula for all experiments 139
were taken from an iron-grown culture of Acidithiobacillus strain SS3 in the late exponential 140
phase at 4°C (specific growth rate ≈ 0.012 h-1). A fraction of Fe2+ and Fe3+ was introduced 141
into each flask with inoculum. Cultures (100 mL) in 250 mL shake flasks were incubated 142
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aerobically at 4°C and at 180 rpm. Samples were removed at intervals for pH, redox, iron 143
and sulfate analysis. Suspended solids were separated by filtration using 0.22 µm Millipore 144
syringe filters. The Fe2+ concentration was determined spectrophotometrically with the o-145
phenanthroline method [17], Fe3+ with UV/Vis spectroscopy at 300 nm [4] and total soluble 146
iron by atomic absorption spectrometry. The sulfate concentration was determined by the 147
BaCl2 nephelometric method [41]. Sulfur oxidation was also monitored by following changes 148
in pH due to acid formation over time using a combined red-rod type pH electrode and PHM 149
210 pH meter (Radiometer Analytical). Subcultures were routinely inoculated into shake 150
flasks containing S0 to ensure that the bacterial culture was active during the long incubation 151
period. Results are presented as averages (number of replicates (n) = 3) ± SD. 152
153
3. Results 154
155
3.1. Temperature dependency of tetrathionate and S0 oxidation 156
157
The temperature dependency of tetrathionate and S0 oxidation by Acidithiobacillus strain 158
SS3 was determined over a range of 5° to 30°C. The lag phases before tetrathionate oxidation 159
was initiated were <12 h and 360 h for 30° and 5°C, respectively (Fig. 1a). The rates of 160
substrate oxidation were dependent on the temperature. From the data fitted to the Ratkowsky 161
equation (eqn. 5), a temperature optimum of 25°C was calculated for tetrathionate oxidation 162
(Fig. 1b). This was consistent with a temperature optimum of 26°C (Fig. 1c) calculated from 163
the corresponding pH decreases. The Ea values for tetrathionate oxidation were calculated on 164
the basis of decreases in the substrate concentration and pH over time. Linearity in the 165
corresponding Arrhenius plots (Fig. 2a,b) was apparent between 6° and 18°C, whereas rate 166
constants below and above these temperatures deviated from the linearity. The Arrhenius 167
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plots yielded Ea values of 61 (R2 = 0.68) and 89 (R2 = 0.90) kJ mol-1 for tetrathionate 168
oxidation and pH decrease, respectively. 169
For S0 oxidation, a pH decrease was first observed after 280 h (~12 days) of incubation 170
at 5°C, whereas it started almost immediately at 25°C. In contrast to tetrathionate oxidation, a 171
lower temperature optimum of 20°C was calculated for S0 oxidation when the data were 172
fitted to the Ratkowsky equation (Fig. 1d). The temperature range for S0 oxidation was larger 173
than for tetrathionate and lacked a distinct temperature optimum peak. The S0 oxidation rate 174
constants between 6° and 18°C also gave a linear plot for lnk versus the reciprocal 175
temperature (Fig. 2c) with an Ea of 110 kJ mol-1 (R2 = 0.89). However, the Arrhenius plot for 176
S0 oxidation based on the decrease in pH did not give a straight line with high probability 177
over the same temperature range (Fig. 2d), casting doubt on the calculated Ea value of 48 kJ 178
mol-1, although the data unequivocally demonstrated a strong association between the 179
oxidation rate and the temperature. The Arrhenius plots in Figs. 2 b-c may also display 180
biphasic linearity, but this possibility was not investigated further. 181
182
3.2. Oxidation of Fe2+ and S0 at 4°C 183
184
Oxidation of 5 g S0 L-1 (156 mM) by Acidithiobacillus strain SS3 at 4°C started after a 185
30-day lag phase upon the first subculture with iron-grown cells (Fig. 3). The medium 186
initially contained 9 mM Fe2+ carried over with the inoculum. During the lag period, the 187
initial 9 mM Fe2+ was completely oxidized, while the sulfate concentration and pH only 188
slightly changed. The oxidation of S0 yielded a linear increase in sulfate formation over time 189
in a semi-log plot (Fig. 3a) and a linear pH decrease due to H2SO4 production (Fig. 3b). Upon 190
approximately 60% S0 oxidation, the linear decrease in the pH reached a prohibitively low 191
value of 1.1 ± 0.1 that inhibited further S0 oxidation. Determination of the sulfur oxidation 192
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rate using the H+ formation rate has been demonstrated [7, 18]. The diprotic H2SO4 193
dissociates in two steps and thus the relationship between the pH and the rate of S0 oxidation 194
changes when the pH approaches the second dissociation constant of H2SO4 (pKa2 = 1.92). 195
The rate constants from these linear regression lines were 0.045 ± 0.0013 d-1 for net sulfate 196
production and 0.043 ± 0.0013 d-1 for pH change. In the absence of Acidithiobacillus strain 197
SS3, the sulfate concentration did not increase (Fig. 3a) and the pH was more or less constant 198
(Fig. 3b). 199
In the next experiment at 4°C, the concentration of Fe2+ was increased from 9 to 67 mM 200
Fe2+ while the initial S0 concentration was 156 mM (5 g L-1). The concentration of Fe3+ 201
increased to a maximum of 67 mM upon the initial Fe2+ oxidation (Fig. 4a), also indicated by 202
the change in color to reddish-orange with time. The first order rate constant estimated from 203
exponential Fe2+ oxidation was 0.012 h-1. This value corresponds to the generation time 58 h 204
(g = ln2/k) and is in agreement with previously observed estimates with Fe2+ alone [24]. 205
Concurrently, the concentration of soluble sulfate decreased, indicating the precipitation of 206
Fe(III) hydrosulfates, and subsequent changes in sulfate concentration fluctuated while the 207
Fe3+ continued to precipitate (Fig. 4c). A net increase in the concentration of soluble sulfate 208
was distinct only after 4 months (Fig. 4c). The initial increase in the pH was due to proton 209
consumption coupled with Fe2+ oxidation, whereas the decrease in the pH after 5 days was 210
attributed to Fe(III)-precipitation in the form of schwertmannite (eqn. [4]) or jarosite (eqn. 211
[5]) (Fig. 4b). The pH did not decrease to a prohibitively low pH value (≈ pH <1.2) in this 212
experiment. The long time course reflects the slow precipitation of Fe(III), which also 213
removes sulfate from the solution phase and thereby masks any net increase in sulfate 214
concentration until Fe(III) precipitation approaches equilibrium conditions. 215
216
3.3. Redox coupling between S0 and Fe3+ at 4°C 217
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Previous experiments in this study showed that the subambient incubation temperature 218
slowed down bacterial metabolism to the extent that it should be possible to discern major 219
phases in redox reactions due to substrate conversions. Thus, the possibility was explored that 220
Fe3+ was an external electron acceptor for S0 oxidation in growing cultures of strain SS3. 221
When the SS3 culture was grown at 4ºC with S0 and amended with Fe3+, the apparent 222
formation of soluble sulfate was delayed until the low concentration of Fe2+ (10 mM) carried 223
over with the inoculum was completely oxidized (Fig. 5a,c). The pH decreased in two phases 224
(Fig. 5a,b): (a) the initial pH decrease was attributed to ferric iron hydrolysis and 225
precipitation (eqns. 3 and 4) and (b) the latter phase was attributed to sulfuric acid formation 226
from S0 oxidation (eqns. 1 and 2). The first phase, Fe2+ oxidation and Fe3+ precipitation, 227
resulted in partial removal of soluble sulfate via precipitation as Fe(III) hydroxysulfates, 228
which masked fractional increases in sulfate formation upon initial S0 oxidation. As sulfur 229
oxidation ensued, the pH gradually decreased to low levels, which were increasingly 230
prohibitive to Fe2+ oxidation. The decrease in pH to <1.2 was found to be inhibitory to 231
Acidithiobacillus strain SS3 in this and other Fe2+ oxidation experiments such that some Fe2+ 232
accumulated in the medium toward the end of the incubation. This suggested that S0 233
oxidation was coupled with reduction of Fe3+ to Fe2+ due to parallel sulfur oxidation (Fig. 5c). 234
Thus, the low pH brought about by S0 oxidation slowed down Fe2+ re-oxidation (i.e. the 235
recycling of Fe2+ and Fe3+), while the S0-dependent Fe3+ reduction continued. In the 236
corresponding sterile control, the concentration of Fe2+ remained below the detection limit of 237
36 µM (2 mg Fe2+ L-1) (based on parameters of linear regression of the calibration line) [8]. 238
Parallel changes in Fe3+ concentration (Fig. 5d) showed an initial decrease followed by a 239
steep increase when S0 oxidation ensued. Fe(III)-hydroxysulfates that were precipitated in the 240
preceding stage of Fe2+ oxidation were re-dissolved when the pH decreased to <1.4 for 241
several months, causing the Fe3+ concentration to increase (Fig. 5d). The final concentration 242
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of Fe3+ was close to the sum of the initial concentrations of Fe2+ + Fe3+. In the chemical 243
control, ferric iron precipitated and did not re-dissolve because the pH remained at ~2.3 244
through the time course. 245
246
4. Discussion 247
248
This study addresses the oxidation of tetrathionate and S0 by Acidithiobacillus strain SS3 249
at low temperatures, an important subject matter to characterize because of bioleaching and 250
acid rock drainage in boreal mine sites. Temperature gradient experiments showed that 251
Acidithiobacillus strain SS3 could oxidize tetrathionate and S0 even at 5°C. The temperature 252
optima for tetrathionate and S0 oxidation were between 20º and 25ºC and were consistent 253
with the previous finding that Acidithiobacillus strain SS3 is psychrotolerant [24]. The broad 254
temperature optimum for S0 oxidation suggested that the oxidation rate was not solely 255
dependent on the temperature. It was possible that the oxidation rate may have also been 256
limited by the available surface area for the microorganisms to attach to the solid S0 particles 257
required for oxidation to occur. The upper permissive temperature for a number of pure and 258
mixed cultures of A. ferrooxidans has been demonstrated to range between 36.1 and 43.6ºC 259
[26]. The activation energies calculated for tetrathionate metabolism of 89 and 61 kJ mol-1 260
were lower than that calculated for abiotic tetrathionate oxidation of 105 kJ mol-1 at pH 1.5 261
[10]. The Ea values for tetrathionate and sulfur metabolism were both greater than the 38 kJ 262
mol-1 calculated for Fe2+ oxidation by washed cell suspensions of Acidithiobacillus strain SS3 263
[24]. Cultures growing with Fe2+ have typically yielded Ea values in the 80 kJ mol-1 range, 264
clearly suggesting a strong temperature dependency of iron oxidation by A. ferrooxidans [3]. 265
In previous studies of S0 oxidation, Ea values for several chemolithotrophic acidophiles 266
including A. ferrooxidans, Acidithiobacillus caldus, and Acidithiobacillus thiooxidans ranged 267
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from 26 to 72 kJ mol-1 [2, 15]. In addition to possible surface area limitation, the underlying 268
basis of temperature dependence of S0 may be a complex subject to interpret because solid 269
substrate oxidation additionally involves cell attachment, which may also respond to the 270
temperature. In the case of acidophilic iron and sulfur oxidizers, the thermodynamic 271
properties and temperature sensitivity of cellular attachment have yet to be elucidated. 272
Our experimental results indicated that substantial S0 oxidation was delayed until the 273
available Fe2+ had been oxidized. There are planktonic and sessile cells in the S0 culture, and 274
it is unclear whether all cells are equally capable of oxidizing Fe2+. A. ferrooxidansT rus 275
genes involved in Fe2+ oxidation are more highly expressed in Fe2+ media but are also present 276
in S0 grown cells [43], but similar information on the genes involved in S0 oxidation is not 277
available. Proteomic studies have shown that several proteins of A. ferrooxidans are 278
upregulated or downregulated depending on whether growth is dependent on Fe2+ or S0 [40]. 279
The differences in the Fe2+ oxidation rates of 67 mM Fe2+ in Fig. 4 (a rate in line with 280
previous data [24]) and 10 mM Fe2+ in Fig. 5 may have an explanation in differential 281
expression of genes responsible for Fe2+ and S0 oxidation. This may occur with available 282
substrate concentrations and the 10 mM Fe2+ situation may conceivably involve low level of 283
expression of the corresponding Fe2+ oxidation gene(s). Regardless of rate differences, both 284
experiments also indicated preferential oxidation of Fe2+ over S0, which may have 285
consequences for the bioleaching of sulfide minerals as the initial intermediates include 286
polysulfides and S0 as well as polythionates [23, 34]. While these differences in substrate 287
oxidation rates cannot be interpreted with certainty with the data available, they warrant 288
further research on multiple substrate conditions, an inevitable situation in bioleaching 289
processes. Solid-phase sulfur intermediates may passivate mineral surfaces [42] and thus 290
reduce the bioleaching rate because these sulfur species may not be efficiently removed until 291
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the available Fe2+ has been oxidized. The dynamics of iron and sulfur oxidation and reduction 292
in bioleaching situations is complex as it involves solid and solution phase reactions. 293
In this study, Acidithiobacillus strain SS3 utilized three different electron donor/acceptor 294
pairs at suboptimal temperatures, namely Fe2+/O2, S0/O2, and S0/Fe3+. The cultures were 295
grown under fully aerobic conditions as previous measurements of Fe2+ oxidation at higher 296
rates with a similar experimental setup demonstrated 95% O2 saturation [24]. The S0/Fe3+ 297
couple represents a mechanism of S0 oxidation whereby the electrons from S0 proceed via 298
carriers to Fe3+ reduction. The ability to reduce Fe3+ with S0 was already reported for A. 299
ferrooxidans by Brock and Gustafson [6]. As a result of the low pH and high Fe3+/Fe2+ ratio 300
after approximately 100 days, the medium redox potential measured by Pt-Ag/AgCl system 301
reached 650 mV (equivalent to 840 mV with the SHE), suggesting that the Fe3+/Fe2+ redox 302
pair could be thermodynamically competitive with the O2/H2O couple as an electron acceptor 303
during bacterial sulfur oxidation. 304
Different strains of A. ferrooxidans and ‘A. ferrivorans’ can have highly variable rates of 305
Fe2+ and S0 oxidation under aerobic and anaerobic conditions depending on their growth 306
history [38]. Sugio and co-workers [35-37, 39] reported the presence of a ferric ion reducing 307
system that catalyzes the reduction of Fe3+ with S0 in A. ferrooxidans. The authors proposed a 308
sulfur oxidation mechanism involving Fe3+ reduction via the sulfide:ferric ion 309
oxidoreductase, sulfur:ferric ion oxidoreductase, and sulfite:ferric ion oxidoreductase systems 310
in A. ferrooxidans. The accumulation of Fe2+ in media was observed under anaerobic 311
conditions and under aerobic conditions in the presence of inhibitors of the bacterial iron 312
oxidation enzyme system [35-37]. In the present study, the accumulation of Fe2+ from Fe3+ 313
reduction may have resulted from the decrease in pH to low values prohibitive to iron 314
oxidation [32] and relatively slow S0 oxidation and sulfate removal due to Fe(III)-315
precipitation. Sand [32] reported substantial accumulation of Fe2+ at pH <1.3 in aerobic A. 316
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ferrooxidans cultures which were supplemented with both S0 and Fe3+, again suggesting that 317
sulfur oxidation by A. ferrooxidans under oxic conditions is coupled with Fe3+ reduction. Fe2+ 318
oxidation may have been inhibited under these particular conditions because of the 319
prohibitively low pH value. 320
As shown in this work, subambient incubation temperatures allow temporal isolation of 321
redox reactions and pH changes associated with single and dual substrate oxidation, and 322
precipitation and chemical reactions are also relatively slow. Under these low temperature 323
and low pH conditions, it was possible to demonstrate the coupling between S0 oxidation and 324
Fe3+ reduction. The temperature dependence of the oxidation of solid-phase and soluble 325
substrates by Acidithiobacillus strain SS3 can be characterized by activation energy values 326
within a similar range, although the underlying biochemical oxidation mechanisms are very 327
different. The low temperature oxidation of the staple substrates of A. ferrooxidans and ‘A. 328
ferrivorans’, inorganic iron and sulfur compounds has relevance in management of 329
biogeochemical reactions in acid mine drainage in the arctic climate where the prevailing 330
temperatures are dominantly suboptimal for typical mesophilic microorganisms. How these 331
suboptimal temperatures impact the biological leaching reactions in bioleach mines remains 332
unclear. The oxidative acidic leaching reactions are highly exergonic and consequently heat 333
evolution in the interior zones of sulfide ore heaps as well as gob piles is inevitable even in 334
the winter time [20]. Strains such as the psychrotrophic Acidithiobacillus strain SS3 offer a 335
biological tool to simulate biogeochemical reactions and control iron and sulfur oxidation 336
rates at low temperatures, a mission that is impossible to accomplish with strictly chemical 337
reaction sequences. 338
339
Acknowledgements 340
341
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We thank S. Sääf for expert technical assistance. D. Kupka acknowledges research 342
support from the Science and Technology Assistance Agency (contract number APVV-51-343
027705) and the VEGA Agency projects No-2/0159/08. Work carried out at Umeå University 344
and the Tampere University of Technology was within the European Commission project 345
‘BioMinE’ under the Sixth Framework Program for Research and Development (European 346
project contract NMP1-CT-500329-1). O.H. Tuovinen was supported in part by the Finnish 347
Funding Agency for Technology and Innovation (Finland Distinguished Professor Program, 348
402/06). 349
350
References 351
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2. Ahonen, L., Tuovinen, O.H. (1990) Kinetics of sulfur oxidation at suboptimal 356
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475
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FIGURE LEGENDS 476 477 Fig. 1 Temperature gradient results for (a) tetrathionate oxidation rates by Acidithiobacillus 478
strain SS3 at 5° and 30°C; Ratkowsky plots (time in hours) of the relationship between 479
temperature and tetrathionate calculated for (b) tetrathionate metabolism and (c) pH decrease; 480
and (d) Ratkowsky plots for S0 oxidation as measured by the decrease in pH. The solid lines 481
in panels (b), (c) and (d) represent the best fit of the linear portion of the tetrathionate and S0 482
oxidation rates. 483
484
Fig. 2 Arrhenius plots for the metabolism of tetrathionate and sulfur. Tetrathionate 485
metabolism was measured by a decrease in the tetrathionate concentration by cyanolysis (a) 486
and a decrease in pH (b), whereas S0 oxidation was calculated using an increase in sulfate 487
concentration (c) and a decrease in pH (d). 488
489
Fig. 3 Time course of (a) 5 g S0 L-1 (156 mM) oxidation by Acidithiobacillus strain SS3 at 490
4ºC inoculated from a ferrous-iron-grown culture at 4°C plotted as the log sulfate 491
concentration ( ) and the Fe2+ concentration ( ); (b) corresponding changes in pH (b). 492
Open symbols indicate abiotic control. Data points are averages ± SD (n = 3). 493
494
Fig. 4 Fe2+ oxidation by Acidithiobacillus strain SS3 in the presence of S0 at 4°C inoculated 495
from a ferrous-iron-grown culture at 4°C. Initially, the medium contained 156 mM S0 and 67 496
mM Fe2+. Symbols are: a: Fe2+ ( ), Fe3+ ( ), and total iron ( ); b: pH ( ) and redox ( ); 497
and c: sulfate plotted as the semilog ( ). Data points are averages ± SD (n = 3). 498
499
Fig. 5 Time course of S0 oxidation coupled with Fe3+ reduction by Acidithiobacillus strain 500
SS3 at 4ºC inoculated from a ferrous-iron-grown culture at 4°C: (a) sulfate; (b) pH; (c) Fe2+; 501
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(d) Fe3+. Open symbols indicate sterile controls. Data points are averages ± SE (n = 2). 502
Initially, the medium contained 156 mM S0 and 35 mM Fe3+. About 10 mM Fe2+ was carried 503
over with the inoculum to the culture medium. No Fe2+ was added to the uninoculated control 504
and the Fe2+ concentration in the control was below the detection limit for the entire 505
experiment. 506
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