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Article
Impact of iron-reducing bacteria on the corrosion rate of carbon steel under simulated geological disposal conditions Marta K. Schütz, Michel L. Schlegel, Marie Libert, and Olivier Bildstein Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00693 • Publication Date (Web): 19 May 2015 Downloaded from http://pubs.acs.org on May 19, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 35
Environmental Science & Technology
Impact of iron-reducing bacteria on the corrosion rate of carbon steel under simulated geological disposal conditions Marta K. Schütz§,ǁ,¥,†,*, Michel L. Schlegel #,ǂ,¥, Marie Libert §,¥, Olivier Bildstein §,¥ §
CEA, DEN, DTN/SMTA/LMTE, 13108 Saint Paul lez Durance, France
#
CEA, DEN, DANS/DPC/SEARS/LISL, F91191 Gif-sur-Yvette, France
* Corresponding author: [email protected] KEYWORDS: biocorrosion, iron-reducing bacteria, magnetite, hydrogen, carbon steel, corrosion rate, geological disposal 1
ABSTRACT. The current projects for the disposal of high-level radioactive waste rely on
2
underground burial and confinement by metallic envelopes that are susceptible to corrosion
3
processes. The impact of microbial activity must be fully clarified in order to provide biological
4
parameters for predictive reactive transport models. This study investigates the impact of
5
hydrogenotrophic iron-reducing bacteria (Shewanella oneidensis strain MR-1) on the corrosion
6
rate of carbon steel under simulated geological disposal conditions by using a geochemical
7
approach. It was found that corrosion damage changes mostly according to the experimental
8
solution (i.e. chemical composition). Magnetite and vivianite were identified as the main
9
corrosion products. In the presence of bacteria, the corrosion rate increased by a factor 1.3
ACS Paragon Plus Environment
1
Environmental Science & Technology
Page 2 of 35
10
(according to weight loss analysis) to 1.8 (according to H2 measurements) and the detected
11
amount of magnetite diminished. The mechanism likely to enhance corrosion is the
12
destabilization and dissolution of the passivating magnetite layer by reduction of structural Fe(III)
13
coupled to H2 oxidation.
14 15
INTRODUCTION
16
The disposal of high-level radioactive waste (HLW) in deep geological formations is the
17
solution investigated for HLW management in many countries. In these repositories, long-term
18
confinement of HLW would be ensured by a multi-barrier system involving several natural (e.g.
19
clay minerals) and/or engineered barriers (e.g. carbon steel, stainless steel, glass, concrete).1 An
20
assessment of the long-term behavior of the radioactive waste and confining materials in the
21
geological environment is crucial to demonstrate the disposal safety. This assessment entails an
22
understanding of corrosion of the successive metallic barriers in contact with water coming from
23
the geological formation.
24
Corrosion can be described as a process coupling the oxidation of metals (anodic reaction) and
25
the reduction of an oxidizing agent (cathodic reaction) in the presence of an electrolyte. For
26
example, iron corrosion by water under anoxic conditions usually lead to the release of Fe2+
27
which precipitates as Fe(OH)2 according to:
28 29
Fe + 2H2O → Fe2+ + 2OH- + H2 → Fe(OH)2 + H2
(1)
30 31 32
Fe(OH)2 is metastable in solution and oxidizes to form magnetite (Fe3O4).2 The overall chemical reaction describing iron oxidation and magnetite formation can be written:
ACS Paragon Plus Environment
2
Page 3 of 35
Environmental Science & Technology
33 34
3Fe + 4H2O → Fe3O4 + 4H2
(2)
35 36
However, this simple description does not apply in the geological environment, where
37
physicochemical conditions change with time and promote the formation of several distinct solid
38
corrosion products at the metal surface. In addition, these solid products can further protect the
39
metal from corrosion, depending on their chemistry and morphology. The current consensus is
40
that the most aggressive forms of corrosion (e.g. localized, stress corrosion cracking) will occur
41
during initial transient conditions in the repository, and more predictable generalized corrosion
42
will take place later.3 Several studies have suggested that decreasing long-term corrosion rates
43
may result from the presence of a passivating layer made of magnetite in direct contact with the
44
metal surface.4-10 However, changes in the environmental conditions (e.g. pH, redox potential,
45
CO2 pressure, presence of microorganisms) can affect the stability of passivating layers and,
46
therefore, the overall susceptibility of the metal to corrosion.
47
Corrosion associated to the action of microorganisms is known as either microbiologically
48
influenced corrosion (MIC) or biocorrosion. MIC is usually related to the presence of biofilms
49
and to bacterial metabolism11,12 which can control physicochemical properties at the metal
50
surface,13-15 alter the properties and stability of passivating layers,16-18 and eventually modify
51
(increase or decrease) the kinetics of cathodic and/or anodic reactions.19,20 Of specific concern is
52
the role of iron-reducing bacteria (IRB), for which either inhibitory or enhancing effects have
53
been hypothesized.21-24 Indeed, magnetite destabilization and dissolution under anaerobic
54
conditions has been observed as a result of the activity of bacteria using hydrogen (H2) to reduce
55
structural Fe(III).17,18,25 This mechanism is therefore believed to influence the corrosion rate. In
56
fact, corrosion processes can provide all the necessary chemical compounds for IRB anaerobic
ACS Paragon Plus Environment
3
Environmental Science & Technology
Page 4 of 35
57
bioactivity, such as H2 as electron donor and Fe(III) from Fe hydr(oxides) as electron acceptor.
58
Hydrogen is a potent energetic substrate for anaerobic microbial communities, especially in
59
geological formations containing small amounts of biodegradable organic matter.26-30 In addition,
60
the extreme conditions (e.g. radiation, desiccation, high temperature and pressure) that will
61
prevail during the early lifetime of the geological repository should not prevent microbial
62
survival and subsequent growth when conditions will become more favorable: radiation dose rate
63
< 60 Sv.h-1, temperature < 70°C, and water saturation > 0.97.20,31 Therefore, the effect of
64
microbial activity on corrosion needs to be investigated and quantified in the framework of
65
safety assessment of HLW geological disposal. Our previous work demonstrated the impact of
66
hydrogenotrophic IRB on corrosion process by combining geochemical and electrochemical
67
techniques.18 Short term corrosion rates (20 days) were estimated from aqueous Fe concentration
68
(in the case of geochemical measurements) and from exchanged charge (in the case of
69
electrochemical measurements).
70
The objective of the present study is to assess the impact of hydrogenotrophic IRB on carbon
71
steel corrosion mechanisms and corrosion rate on a longer timeframe (5 months) and using
72
additional techniques. Corrosion rates were determined by weight loss analysis and H2
73
measurements in order to compare these two methods. The presence and nature of corrosion
74
products was assessed by spectroscopic and structural techniques to relate the discrepancies in
75
corrosion rate to the nature of corrosion products. Shewanella oneidensis strain MR-1 has been
76
chosen as model organism for this study because it is able not only to oxidize organic matter but
77
also hydrogen, an abundant energy source in such a disposal environment.28
78 79
MATERIALS AND METHODS
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Page 5 of 35
Environmental Science & Technology
80
Experimental solutions. Two experimental solutions with distinct chemical compositions
81
were used in this study: M1 and S1 (see chemical composition in Table 1). M1 is a minimal
82
medium used to cultivate IRB species but with a chemical composition slightly modified in order
83
to be representative of the groundwater from the argillaceous Callovo-Oxfordian formation of
84
the French Underground Research Laboratory (FURL, Bure, France).18,32 For comparison
85
purposes, a solution (S1) with simplified chemical composition was also used. Its composition
86
consists in a mixture of 10 mM Na2SO4, 2 mM NaHCO3, and vitamins (0.08 mM nicotinic acid,
87
0.01 mM thiamine-HCl, 0.40 µM biotine). The sulfate concentration is similar to that measured
88
in the FURL groundwater.32 The pH in the M1 and S1 was adjusted to ~7 with NaOH, and
89
solutions were sterilized by autoclaving (121°C, 20 min), except for the thermolabile
90
components (e.g. vitamins) which were filter-sterilized through a 0.22 µm filter and added to the
91
autoclaved solutions.
92 93
Table 1. Chemical composition of the M1 and S1 solutions. Concentration Compound M1
S1
Na2SO4
-
10 mM
(NH4)2SO4
9 mM
-
Na2SeO4
11 µM
-
HEPES
17 mM
-
NaHCO3
2 mM
2 mM
K2HPO4
0.5 mM
-
KH2PO4
0.3 mM
-
CoSO4.7H2O
5 µM
-
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Environmental Science & Technology
NiSO4.6H2O
5 µM
-
NaCl
10 µM
-
H3BO3
45 µM
-
ZnSO4.7H2O
0.8 µM
-
Na2MoO4.2H2O
3 µM
-
CuSO4.5H2O
0.2 µM
-
MnSO4.H2O
1 µM
-
MgSO4.7H2O
0.8 mM
-
CaCl2.2H2O
0.4 mM
-
FeSO4.7H2O
4 µM
-
arginine
0.11 mM
-
glutamate
0.13 mM
-
serine
0.19 mM
-
nicotinic acid
0.08 mM
0.08 mM
thiamine-HCl
0.01 mM
0.01 mM
biotine
0.40 µM
0.40 µM
Page 6 of 35
94 95
Bacterial culture. Shewanella oneidensis strain MR-1 (ATCC 700550TM) was used as model
96
of hydrogenotrophic IRB in the biotic experiments. Pre-cultures were obtained aerobically at the
97
beginning of stationary growth phase in Luria Bertani Broth (LB) medium (5 g.L-1 NaCl, 10 g.L-
98
1
99
medium by centrifugation (4000 rpm, 20 min), washed once with sterile M1 or S1 solutions and
100
then inoculated into the batch reactors. The initial bacterial concentration was determined by
101
epifluorescence method with LIVE/DEAD® BacLight™ kit33,34, and equalled 108 cells.mL-1.
tryptone, 5 g.L-1 yeast extract) after 24 h at 30°C. Bacterial cells were harvested from the LB
102
Corrosion experiments. One- and 5-months experiments were performed in 250 mL batch
103
reactors at 30oC under anaerobic atmosphere (N2:CO2 ratio of 90:10%) and abiotic (without
ACS Paragon Plus Environment
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Page 7 of 35
Environmental Science & Technology
104
bacteria) or biotic (with bacteria) conditions. The experimental conditions were detailed in our
105
previous study.18 All experiments were started under aseptic conditions by introducing 140 mL
106
of experimental solution (either M1 or S1) and one cylindrical coupon sample (A37 low carbon
107
steel polished up to 600 grit) into glass reactors without agitation. The coupons were laterally
108
embedded in a diallyl phthalate glass-fiber resin in order to expose only a basal surface of 0.8
109
cm2. Biotic conditions were achieved by adding 1 mL of bacterial inoculum (108 cells.mL-1).
110
Then, the reactors were sealed with rubber plugs and aluminum caps and flushed with anaerobic
111
gas that was sterilized by passing through a 0.22 µm filter.
112
Sample analyses. Aqueous Fe concentration ([Fe]aq) was analyzed by inductively coupled
113
plasma optical emission spectrometry (ICP-OES, Varian VISTA-MPX) after 0.02 µm filtration
114
and 2% (v/v) HNO3 acidification. Analysis of H2 in the headspace was carried out by micro gas
115
chromatography (GC, Varian CP-4900) using a thermal conductivity detector with N2 as carrier
116
gas. The total H2 concentration ([H2]) was calculated as the sum of the contributions from the
117
gaseous and aqueous phases (determined using Henry’s law), normalized to the solution volume
118
to allow direct comparison with [Fe]aq. The experiments were usually performed in triplicate,
119
with two coupons used for the weight loss analysis, and one for the characterization of solid
120
corrosion products.
121
After experiment, all coupon samples were dried in an M-Braun LABstar MB10 anaerobic
122
glovebox (O2 concentration
Article
Impact of iron-reducing bacteria on the corrosion rate of carbon steel under simulated geological disposal conditions Marta K. Schütz, Michel L. Schlegel, Marie Libert, and Olivier Bildstein Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00693 • Publication Date (Web): 19 May 2015 Downloaded from http://pubs.acs.org on May 19, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 35
Environmental Science & Technology
Impact of iron-reducing bacteria on the corrosion rate of carbon steel under simulated geological disposal conditions Marta K. Schütz§,ǁ,¥,†,*, Michel L. Schlegel #,ǂ,¥, Marie Libert §,¥, Olivier Bildstein §,¥ §
CEA, DEN, DTN/SMTA/LMTE, 13108 Saint Paul lez Durance, France
#
CEA, DEN, DANS/DPC/SEARS/LISL, F91191 Gif-sur-Yvette, France
* Corresponding author: [email protected] KEYWORDS: biocorrosion, iron-reducing bacteria, magnetite, hydrogen, carbon steel, corrosion rate, geological disposal 1
ABSTRACT. The current projects for the disposal of high-level radioactive waste rely on
2
underground burial and confinement by metallic envelopes that are susceptible to corrosion
3
processes. The impact of microbial activity must be fully clarified in order to provide biological
4
parameters for predictive reactive transport models. This study investigates the impact of
5
hydrogenotrophic iron-reducing bacteria (Shewanella oneidensis strain MR-1) on the corrosion
6
rate of carbon steel under simulated geological disposal conditions by using a geochemical
7
approach. It was found that corrosion damage changes mostly according to the experimental
8
solution (i.e. chemical composition). Magnetite and vivianite were identified as the main
9
corrosion products. In the presence of bacteria, the corrosion rate increased by a factor 1.3
ACS Paragon Plus Environment
1
Environmental Science & Technology
Page 2 of 35
10
(according to weight loss analysis) to 1.8 (according to H2 measurements) and the detected
11
amount of magnetite diminished. The mechanism likely to enhance corrosion is the
12
destabilization and dissolution of the passivating magnetite layer by reduction of structural Fe(III)
13
coupled to H2 oxidation.
14 15
INTRODUCTION
16
The disposal of high-level radioactive waste (HLW) in deep geological formations is the
17
solution investigated for HLW management in many countries. In these repositories, long-term
18
confinement of HLW would be ensured by a multi-barrier system involving several natural (e.g.
19
clay minerals) and/or engineered barriers (e.g. carbon steel, stainless steel, glass, concrete).1 An
20
assessment of the long-term behavior of the radioactive waste and confining materials in the
21
geological environment is crucial to demonstrate the disposal safety. This assessment entails an
22
understanding of corrosion of the successive metallic barriers in contact with water coming from
23
the geological formation.
24
Corrosion can be described as a process coupling the oxidation of metals (anodic reaction) and
25
the reduction of an oxidizing agent (cathodic reaction) in the presence of an electrolyte. For
26
example, iron corrosion by water under anoxic conditions usually lead to the release of Fe2+
27
which precipitates as Fe(OH)2 according to:
28 29
Fe + 2H2O → Fe2+ + 2OH- + H2 → Fe(OH)2 + H2
(1)
30 31 32
Fe(OH)2 is metastable in solution and oxidizes to form magnetite (Fe3O4).2 The overall chemical reaction describing iron oxidation and magnetite formation can be written:
ACS Paragon Plus Environment
2
Page 3 of 35
Environmental Science & Technology
33 34
3Fe + 4H2O → Fe3O4 + 4H2
(2)
35 36
However, this simple description does not apply in the geological environment, where
37
physicochemical conditions change with time and promote the formation of several distinct solid
38
corrosion products at the metal surface. In addition, these solid products can further protect the
39
metal from corrosion, depending on their chemistry and morphology. The current consensus is
40
that the most aggressive forms of corrosion (e.g. localized, stress corrosion cracking) will occur
41
during initial transient conditions in the repository, and more predictable generalized corrosion
42
will take place later.3 Several studies have suggested that decreasing long-term corrosion rates
43
may result from the presence of a passivating layer made of magnetite in direct contact with the
44
metal surface.4-10 However, changes in the environmental conditions (e.g. pH, redox potential,
45
CO2 pressure, presence of microorganisms) can affect the stability of passivating layers and,
46
therefore, the overall susceptibility of the metal to corrosion.
47
Corrosion associated to the action of microorganisms is known as either microbiologically
48
influenced corrosion (MIC) or biocorrosion. MIC is usually related to the presence of biofilms
49
and to bacterial metabolism11,12 which can control physicochemical properties at the metal
50
surface,13-15 alter the properties and stability of passivating layers,16-18 and eventually modify
51
(increase or decrease) the kinetics of cathodic and/or anodic reactions.19,20 Of specific concern is
52
the role of iron-reducing bacteria (IRB), for which either inhibitory or enhancing effects have
53
been hypothesized.21-24 Indeed, magnetite destabilization and dissolution under anaerobic
54
conditions has been observed as a result of the activity of bacteria using hydrogen (H2) to reduce
55
structural Fe(III).17,18,25 This mechanism is therefore believed to influence the corrosion rate. In
56
fact, corrosion processes can provide all the necessary chemical compounds for IRB anaerobic
ACS Paragon Plus Environment
3
Environmental Science & Technology
Page 4 of 35
57
bioactivity, such as H2 as electron donor and Fe(III) from Fe hydr(oxides) as electron acceptor.
58
Hydrogen is a potent energetic substrate for anaerobic microbial communities, especially in
59
geological formations containing small amounts of biodegradable organic matter.26-30 In addition,
60
the extreme conditions (e.g. radiation, desiccation, high temperature and pressure) that will
61
prevail during the early lifetime of the geological repository should not prevent microbial
62
survival and subsequent growth when conditions will become more favorable: radiation dose rate
63
< 60 Sv.h-1, temperature < 70°C, and water saturation > 0.97.20,31 Therefore, the effect of
64
microbial activity on corrosion needs to be investigated and quantified in the framework of
65
safety assessment of HLW geological disposal. Our previous work demonstrated the impact of
66
hydrogenotrophic IRB on corrosion process by combining geochemical and electrochemical
67
techniques.18 Short term corrosion rates (20 days) were estimated from aqueous Fe concentration
68
(in the case of geochemical measurements) and from exchanged charge (in the case of
69
electrochemical measurements).
70
The objective of the present study is to assess the impact of hydrogenotrophic IRB on carbon
71
steel corrosion mechanisms and corrosion rate on a longer timeframe (5 months) and using
72
additional techniques. Corrosion rates were determined by weight loss analysis and H2
73
measurements in order to compare these two methods. The presence and nature of corrosion
74
products was assessed by spectroscopic and structural techniques to relate the discrepancies in
75
corrosion rate to the nature of corrosion products. Shewanella oneidensis strain MR-1 has been
76
chosen as model organism for this study because it is able not only to oxidize organic matter but
77
also hydrogen, an abundant energy source in such a disposal environment.28
78 79
MATERIALS AND METHODS
ACS Paragon Plus Environment
4
Page 5 of 35
Environmental Science & Technology
80
Experimental solutions. Two experimental solutions with distinct chemical compositions
81
were used in this study: M1 and S1 (see chemical composition in Table 1). M1 is a minimal
82
medium used to cultivate IRB species but with a chemical composition slightly modified in order
83
to be representative of the groundwater from the argillaceous Callovo-Oxfordian formation of
84
the French Underground Research Laboratory (FURL, Bure, France).18,32 For comparison
85
purposes, a solution (S1) with simplified chemical composition was also used. Its composition
86
consists in a mixture of 10 mM Na2SO4, 2 mM NaHCO3, and vitamins (0.08 mM nicotinic acid,
87
0.01 mM thiamine-HCl, 0.40 µM biotine). The sulfate concentration is similar to that measured
88
in the FURL groundwater.32 The pH in the M1 and S1 was adjusted to ~7 with NaOH, and
89
solutions were sterilized by autoclaving (121°C, 20 min), except for the thermolabile
90
components (e.g. vitamins) which were filter-sterilized through a 0.22 µm filter and added to the
91
autoclaved solutions.
92 93
Table 1. Chemical composition of the M1 and S1 solutions. Concentration Compound M1
S1
Na2SO4
-
10 mM
(NH4)2SO4
9 mM
-
Na2SeO4
11 µM
-
HEPES
17 mM
-
NaHCO3
2 mM
2 mM
K2HPO4
0.5 mM
-
KH2PO4
0.3 mM
-
CoSO4.7H2O
5 µM
-
ACS Paragon Plus Environment
5
Environmental Science & Technology
NiSO4.6H2O
5 µM
-
NaCl
10 µM
-
H3BO3
45 µM
-
ZnSO4.7H2O
0.8 µM
-
Na2MoO4.2H2O
3 µM
-
CuSO4.5H2O
0.2 µM
-
MnSO4.H2O
1 µM
-
MgSO4.7H2O
0.8 mM
-
CaCl2.2H2O
0.4 mM
-
FeSO4.7H2O
4 µM
-
arginine
0.11 mM
-
glutamate
0.13 mM
-
serine
0.19 mM
-
nicotinic acid
0.08 mM
0.08 mM
thiamine-HCl
0.01 mM
0.01 mM
biotine
0.40 µM
0.40 µM
Page 6 of 35
94 95
Bacterial culture. Shewanella oneidensis strain MR-1 (ATCC 700550TM) was used as model
96
of hydrogenotrophic IRB in the biotic experiments. Pre-cultures were obtained aerobically at the
97
beginning of stationary growth phase in Luria Bertani Broth (LB) medium (5 g.L-1 NaCl, 10 g.L-
98
1
99
medium by centrifugation (4000 rpm, 20 min), washed once with sterile M1 or S1 solutions and
100
then inoculated into the batch reactors. The initial bacterial concentration was determined by
101
epifluorescence method with LIVE/DEAD® BacLight™ kit33,34, and equalled 108 cells.mL-1.
tryptone, 5 g.L-1 yeast extract) after 24 h at 30°C. Bacterial cells were harvested from the LB
102
Corrosion experiments. One- and 5-months experiments were performed in 250 mL batch
103
reactors at 30oC under anaerobic atmosphere (N2:CO2 ratio of 90:10%) and abiotic (without
ACS Paragon Plus Environment
6
Page 7 of 35
Environmental Science & Technology
104
bacteria) or biotic (with bacteria) conditions. The experimental conditions were detailed in our
105
previous study.18 All experiments were started under aseptic conditions by introducing 140 mL
106
of experimental solution (either M1 or S1) and one cylindrical coupon sample (A37 low carbon
107
steel polished up to 600 grit) into glass reactors without agitation. The coupons were laterally
108
embedded in a diallyl phthalate glass-fiber resin in order to expose only a basal surface of 0.8
109
cm2. Biotic conditions were achieved by adding 1 mL of bacterial inoculum (108 cells.mL-1).
110
Then, the reactors were sealed with rubber plugs and aluminum caps and flushed with anaerobic
111
gas that was sterilized by passing through a 0.22 µm filter.
112
Sample analyses. Aqueous Fe concentration ([Fe]aq) was analyzed by inductively coupled
113
plasma optical emission spectrometry (ICP-OES, Varian VISTA-MPX) after 0.02 µm filtration
114
and 2% (v/v) HNO3 acidification. Analysis of H2 in the headspace was carried out by micro gas
115
chromatography (GC, Varian CP-4900) using a thermal conductivity detector with N2 as carrier
116
gas. The total H2 concentration ([H2]) was calculated as the sum of the contributions from the
117
gaseous and aqueous phases (determined using Henry’s law), normalized to the solution volume
118
to allow direct comparison with [Fe]aq. The experiments were usually performed in triplicate,
119
with two coupons used for the weight loss analysis, and one for the characterization of solid
120
corrosion products.
121
After experiment, all coupon samples were dried in an M-Braun LABstar MB10 anaerobic
122
glovebox (O2 concentration
