Bioelectrochemistry 122 (2018) 40–50

Contents lists available at ScienceDirect

Bioelectrochemistry

journal homepage: www.elsevier.com/locate/bioelechem

Corrosion behavior of X65 steel in seawater containing sulfate reducing

bacteria under aerobic conditions
Qiushi Li, Jihui Wang ⁎, Xuteng Xing, Wenbin Hu
Tianjin Key Laboratory of Composite and Functional Materials, School of Materials Science and Engineering, Tianjin University, Tianjin 300072, China

a r t i c l e i n f o a b s t r a c t

Article history: The corrosion behavior of X65 steel was investigated in the seawater inoculated with sulfate reducing bacteria
Received 22 November 2017 (SRB) under the aerobic environment by electrochemical impedance techniques and immersion tests. The cor-
Received in revised form 4 March 2018 roded morphologies and the composition of the corrosion products were investigated. The variation of the solu-
Accepted 10 March 2018
tion parameters including the bacterium number, the pH value and the soluble iron concentration were also
Available online 11 March 2018
investigated. The results indicated that in the SRB-containing system, the impedance responses presented a de-
Keywords:
pressed semi-circle in the initial period, which then turned into the blocked electrode characteristic during the
Sulfate reducing bacteria later immersion. The biofilm, mainly composed of extracellular polymeric substances, Fe(OH)3, γ-FeOOH and
Aerobic environment α-Fe2O3, formed and degraded with the SRB growth. The soluble iron concentration initially increased, then rap-
X65 steel idly decreased and later slowly increased. In the SRB-containing seawater under the aerobic environment, the
Microbiologically influenced corrosion X65 steel was corroded in the initial immersion. The corrosion became inhibited with the forming of the biofilm
during the subsequent immersion. The inhibition efficiency rapidly increased in the logarithmic phase, remained
stable in the stationary phase and then decreased in the declination phase. In the corrosion process, the biofilm
metabolized by SRB played a key role in the corrosion inhibition of X65 steel.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction
Corrosion under the presence of microorganisms has been recog-
nized as a common phenomenon in both natural environments and in-
dustrial processes. Microorganisms can be widely found in water
environments. Microorganisms tend to attach to the surface of metals
and form particular biofilms. This phenomenon changes the physical
and chemical properties at the interface between the metal and the so-
lution. These changes of the physical and chemical properties have con-
siderable influence on the corrosion behavior of metals. The corrosion
process in the presence of microorganisms is known as microbiologi-
cally influenced corrosion (MIC).
Generally, sulfate reducing bacteria (SRB) are considered to be one
of the typical microorganisms that can cause MIC, consequently acceler-
ating the corrosion under the anaerobic environment [1–4]. Various re-
searches have been reported to study the influence of SRB on the
corrosion behavior of metals [5–7]. The studies of corrosion mecha-
nisms by SRB have been reviewed, including the initial cathodic depo-
larization theory [8], the anodic depolarization [9], the sulfide-induced
stress corrosion cracking [10–12] and the biocatalytic cathodic sulfate
⁎ Corresponding author.
E-mail addresses: qsli@tju.edu.cn (Q. Li), jhwang@tju.edu.cn (J. Wang),
wbhu@tju.edu.cn (W. Hu).
reduction (BCSR) theory [13]. Recently, further researches have re-
ported on the electron transfer process based on the BCSR theory
[14,15]. Because the iron is insoluble, the electrons released by Fe0 oxi-
dation are extracellular to SRB cells. To use the extracellular electrons
for the sulfate reduction, which happens in the cytoplasm through bio-
catalysis, a transfer process of the electrons across the cell wall is essen-
tial, this is called the extracellular electron transfer (EET) [16,17]. Two
types of EET have been reported. One is the direct electron transfer
[18–21] and the other is the mediated electron transfer [22]. The two
transfer methods explain how the SRB acquires electrons under differ-
ent conditions. The mechanisms illuminate the way by which SRB accel-
erate corrosion under anaerobic environment.
Apart from the acceleration effect, SRB can also lead to microbially
influenced corrosion inhibition (MICI). Videla et al. have investigated
that under anaerobic environment, SRB-induced corrosion can be af-
fected by the conditions of the generated inorganic sulfide derived
from the SRB environment. The physical and chemical properties of
these sulfides are influenced by environmental parameters. Thin adher-
ent films of sulfide on the surface are protective, while bulky precipi-
tates work inversely, enhancing corrosion rate [23–25]. The possible
mechanisms of MICI under anaerobic environment have been discussed
by Zuo and Videla in their overview articles [26,27] and it can be
achieved in conditions in which (1) corrosive ingredients are removed
or hindered from reaching the metal surface, (2) the growth of the cor-
rosion causing bacteria is inhibited by antimicrobials secreted by the

https://doi.org/10.1016/j.bioelechem.2018.03.003
1567-5394/© 2018 Elsevier B.V. All rights reserved.
 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50
coexisting bacteria present within the biofilm, (3) stable and protective
films are formed on the metal surface.
As SRB have always been regarded as a strict anaerobe, most re-
searches have been focused on the effects of SRB on the corrosion be-
havior of metals under anaerobic environments. Recent studies report
that SRB can exist under the aerobic condition because of the gene ex-
pression of superoxide dismutase and catalase enzymes [28–32]. Wan
et al. have reported that SRB have a slow growth under the aerobic con-
dition [33]. Few studies have reported the effects on the corrosion influ-
enced by SRB under aerobic environments. It is not clear whether the
corrosion is accelerated or inhibited by SRB in the presence of oxygen.
For a corrosion process under aerobic environment at neutral pH, the
oxygen reduction is the dominating reaction at the cathode. This reac-
tion is sensitive to electrode surface conditions and electrolyte environ-
ments, which would be affected by the SRB growth. Thus, the influence
of bacterial metabolites on the cathodic reduction of oxygen is still an
open topic.
In this paper, a corrosion condition in the presence of SRB at neutral
pH under the aerobic environment is proposed. The corrosion behavior
of X65 steel is investigated under the proposed condition. The abiotic
system is used as the contrast test. Electrochemical impedance mea-
surements are conducted to determine whether the corrosion is accel-
erated or inhibited under the proposed condition. The corroded
morphologies of the X65 steel are observed using the scanning electron
microscope (SEM). The composition and valence of the corrosion prod-
ucts are determined using the X-ray photoelectron spectroscopy. Fur-
thermore, the evolution of solutions during the experiment are
monitored by the most probable number (MPN) method and induc-
tively coupled plasma mass spectrometry. The corrosion mechanism
of the X65 steel under the proposed condition is developed based on
the comprehensive analysis of electrochemical impedance measure-
ments, surface characterizations and solution evolutions.
2. Materials and methods
2.1. Materials
The chemical composition of the PSL2 X65 steel is shown in Table 1.
Coupons with the dimension of 10 mm × 10 mm × 3 mm were cut from
a tube sample. The coupons for electrochemical measurements were
encapsulated by epoxy resin after welding with copper wires. All cou-
pons were ground by SiC paper up to 1500 grit, then cleaned with eth-
anol. The ground coupons were then packaged with aluminum foil
tightly and sterilized in autoclave at 121 °C for 20 min.
2.2. Culture and media
The SRB strain used in this work was obtained from the Institute of
Oceanology, Chinese Academy of Sciences. The SRB strain was isolated
from rust layers of carbon steels immersed in Bohai Sea (Qingdao,
China). The SRB strain was identified to be a Desulfovibrio sp. strain by
the comparison of 16S rDNA sequences with the reference strains held
in GenBank database. The 16S rRNA gene sequences of the present
SRB strain were available in the GenBank database with the accession
number of MF461625 [34].
A modified Postgate's culture medium, composed of (per liter of ul-
trapure water) NaCl 36.27 g, K2HPO4 0.65 g, NH4Cl 1.0 g, MgSO4 2.0 g,
CaCl2 0.1 g, Na2SO4 0.5 g, yeast extract 1.0 g and sodium lactate 4 mL
(electron donor), was used for bacterial culture. The pH value of the me-
dium was adjusted between 7.2 and 7.3 by adding the NaOH solution.
Table 1
Chemical compositions (wt%) of PSL2 X65.
Alloy C Si Mn Ni Cr Mo Cu V Nb
X65 0.09 0.26 1.30 0.15 0.04 0.17 0.13 0.04
41
For sterilization, the medium was autoclaved at 121 °C for 20 min and
cooled to room temperature before the experiment.
2.3. Electrochemical measurements
The corrosion behaviors of X65 steel in both the abiotic system and
the SRB-containing system were measured by an electrochemical work-
station (Autolab 302N, Metrohm, Switzerland) with a three-electrode
system. Modified glass cells with the capacity of 300 mL were used for
the electrochemical impedance spectroscopy (EIS) measurement. As
an abiotic control system, the modified cell was filled with 200 mL sterile
medium. As a SRB-containing system, 2 mL SRB seed culture (3 days cul-
tured) was used to inoculate the 198 mL sterile medium to provide an
initial bacterium concentration of approximately 1.0 × 105 cfu·mL−1.
Two X65 steel coupons were placed in the modified cell. One was served
as a working electrode (WE) and the other was for the parallel test. The
working area of the WE was 1 cm2. A platinum plate was used as counter
electrode (CE) and an Ag/AgCl electrode (saturated KCl) served as refer-
ence electrode (RE). After assembling with corresponding electrodes
and coupons, the modified cells were hermetically sealed and placed
in an incubator at 30 °C during the whole experiment. EIS measurements
were carried out at open-circuit potential (OCP), using a frequency range
from 100 kHz to 0.01 Hz and an amplitude of 10 mV. The data were fitted
by ZSimpWin software after measurements.
2.4. Surface characterizations
Immersion tests were simultaneously conducted for the surface
characterizations and solution analysis. The immersion tests are parallel
to the electrochemical measurements. Each immersion test contained
two coupons and at each time, two sets of the immersion test were pre-
pared for each condition. Coupons for surface analysis were taken out
from immersion after 8 h, 2 days, 5 days and 11 days. The coupons
were rinsed by ultrapure water and immersed in an immobile liquid
containing 2.5% glutaraldehyde for 1 h in order to immobilize the bio-
film. Then, graded dehydration was applied to the coupons with 30,
50, 70, 90 and 100 vol% ethanol (each for 15 min). The whole operation
process was conducted on a clean bench.
The surface and cross-sectional morphologies were observed by the
scanning electron microscope (SEM, S-4800, Hitachi, Japan) at 5 kV ac-
celerating voltage. The elemental composition and valence of the corro-
sion products were measured by the energy dispersive spectroscopy
(EDS, Genesis XM2, EDAX, USA) at 15 kV accelerating voltage and X-
ray photoelectron spectroscopy (XPS, PHI 5000 VersaProbe, ULVAC,
Japan). C 1s peak at 284.6 eV was set as the reference to correct the
shifts.
2.5. Solution analysis
After the coupons were removed from the immersion vessels, the
solution in the vessels were shaken and mixed evenly. Three sets of so-
lutions were taken from each vessel to measure the bacterium number,
pH and concentration of soluble iron respectively. The number of active
SRB in each SRB-containing system was estimated using the most prob-
able number (MPN) method according to ASTM D4412-15 [35]. The pH
values for the abiotic system and the SRB-containing system after differ-
ent time of immersion were measured by a pH meter (PHSJ-4A, Shang-
hai Electronics Science Instrument, China). The pH measurements were
conducted immediately after solutions were taken out of the vessels to
reduce the influence of volatilization and oxygen. The concentration of
soluble iron for abiotic and SRB-containing systems at different time
were determined by Inductively Coupled Plasma Mass Spectrometry
0.03
(ICP-MS, 7700x, Agilent, USA).
42 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50
3. Results
3.1. EIS results
At OCP, EIS measurements were conducted on abiotic and SRB-
containing systems for 11 days. Fig. 1 illustrates the impedance re-
sponses for X65 steel exposed to the abiotic system. The Nyquist plots
are shown in Fig. 1(A) and (B) and the corresponding Bode plots are
shown in Fig. 1(A′) and (B′). As shown in Fig. 1(A) and (B), in the abiotic
condition, the Nyquist plots display one depressed semi-circle at high
and low frequencies during the whole period. The diameter of the de-
pressed semi-circles fluctuates with time. The diameter reaches a max-
imum value at 7 d and decreases afterwards. The Bode plots also
confirm the characteristic of one time constant, shown in Fig. 1(A′)
and (B′).
Fig. 2 illustrates the impedance responses for X65 steel exposed to
the SRB-containing system. As shown in Fig. 2(A) and (B), in the SRB-
containing system, the Nyquist plots at 3 h, 8 h and 1 d display one de-
pressed semi-circle at high and low frequencies. A large increase of the
diameter from 3 h to 8 h is observed. The Nyquist plots turn into incom-
plete capacitive loops after 2 days of immersion. The impedance re-
sponses present blocked electrode characteristics, which may be
caused by the formation of a compact film covering the entire surface
of X65 steel [36]. The diameter of the incomplete capacitive loops
changes with time, increasing from 2 d to 3 d, remaining static from
Fig. 1. Electrochemical impedance spectra of X65 steel exposed to the abiotic system for different time. (A) Nyquist plots from 3 h to 3 d, (B) Nyquist plots from 4 d to 11 d, (A′) Bode plots
from 3 h to 3 d, (B′) Bode plots from 4 d to 11 d.
3 d to 5 d and decreasing afterwards. As shown in Fig. 2(A′), the peaks
of the phase at 8 h and 1 d are widened in the corresponding Bode
plots. The Bode plots confirm the occurrence of two time constants
after 2 days of immersion.
As demonstrated from EIS results shown in Fig. 1, the impedance re-
sponses remain one time constant during the whole experiment in the
abiotic system. In the SRB-containing system, as shown in Fig. 2, the sig-
nals perform one time constant within the first day and two time con-
stants after 2 days.
To extract the electrochemical parameters in different corrosion pro-
cesses, two equivalent circuits are used to fit the EIS results, as shown in
Fig. 3. In the equivalent circuit models, Q represents the characteristic
parameter of constant phase element (CPE). The impedance of CPE
can be calculated as [37].
Z CPE ¼ 1=ðjωÞα Q ð1Þ
where α is dispersion coefficient related to the heterogeneous condition
of the electrode surface.
Equivalent circuit for one time constant is shown in Fig. 3(A). Rs is
the solution resistance, Rct is the charge transfer resistance and Qdl is
the double layer capacitance. Equivalent circuit for two time constants
is shown in Fig. 3(B). Rs, Rct and Qdl represent the same as in Fig. 3(A).
Rs, pore represents the resistance of the solution in the pores embedded
in the biofilm. The value of Rs, pore depends on the compactness of the
 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50
Fig. 2. Electrochemical impedance spectra of X65 steel exposed to the SRB-containing system for different time. (A) Nyquist plots from 3 h to 3 d, (B) Nyquist plots from 4 d to 11 d, (A′)
Bode plots from 3 h to 3 d, (B′) Bode plots from 4 d to 11 d.
biofilm and the conductivity of the solution in the pores. Qf is the capac-
itance of the biofilm, which is determined by the thickness and the spe-
cific surface area of the biofilm. The fitted results of parameters for the
Fig. 3. Equivalent circuit models used for EIS fitting analysis. (A) The equivalent circuit for
one time constant, (B) the equivalent circuit for two time constants.
43
abiotic system and the SRB-containing system are displayed in
Tables 2 and 3 respectively.
Fig. 4 illustrates the change of Rct with time in the abiotic system and
the SRB-containing system. As shown in Fig. 4, in the abiotic system, Rct
increases in the first 7 days and then decreases with time. In the SRB-
containing system, an increase of Rct occurs at 8 h (from 2.8 to
22.3 kΩ). Rct rapidly increases between 2 d and 3 d, remains almost un-
changed from 3 d to 5 d and decreases with time afterwards. The value
of Rct during the whole experiment is larger than the initial value, which
indicates that the corrosion of X65 steel is inhibited during the immer-
sion in the seawater containing SRB. The evolution of Rct further indi-
cates the inhibition efficiency changes with time.
Fig. 5 illustrates two parameters of Rs, pore and Qf related to the bio-
film in the SRB-containing system. As shown in Fig. 5, Rs, pore increases
first and then decreases with time. Rs, pore has a maximum value and
shares a similar trend with Rct. Qf decreases between 2 d and 3 d, and re-
mains almost unchanged from 3 d to 5 d and then increases with time.
The evolution of Rs, pore and Qf indicates that the performance of the bio-
film changes with the immersion time.
3.2. Corroded morphology analysis
Fig. 6 illustrates the corroded morphologies of coupons immersed in
the abiotic system after different immersion time. As shown in Fig. 6
(A)–(D), the sizes of the corrosion products appeared on surfaces of
X65 steel exposed to the abiotic system are small. The amount of corro-
sion products increases as the immersion time increases. The surface
44 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50

Table 2
Abiotic system: the evolution of electrochemical impedance parameters with time for X65 steel immersed in the abiotic system.

Parameters Immersion time

3h 8h 1d 2d 3d 4d 5d 7d 9d 11 d

Rs (Ω·cm2) 2.8 2.6 2.5 2.4 2.3 2.3 2.3 2.3 2.4 2.7
α1 0.87 0.87 0.89 0.91 0.92 0.92 0.92 0.93 0.93 0.93
Qdl (μF·cm−2) 315.8 365.3 431.6 522.4 616.9 703.4 790.6 933.7 1052 1419
Rct (Ω·cm2) 2251 2041 2512 2411 2269 2798 2320 3424 2837 1911
Chi-square value (×10−3) 2.933 1.657 1.812 1.839 1.792 1.950 1.597 1.718 1.487 1.205

Table 3
SRB-containing system: the evolution of electrochemical impedance parameters with time for X65 steel immersed in the SRB-containing system.

Parameters Immersion time

3h 8h 1d 2d 3d 4d 5d 7d 9d 11 d

Rs (Ω·cm2) 1.7 1.1 1.1 3.2 1.1 1.5 1.8 2.9 1.4 2.1
α1 – – – 0.99 0.99 0.99 0.98 0.94 0.98 0.95
Qf (μF·cm−2) – – – 465.9 404.9 419.1 432.5 483.0 582.4 725.9
Rs, pore (Ω·cm2) – – – 29.4 43.7 23.3 20.9 6.3 16.0 8.5
α2 0.97 0.95 0.94 0.77 0.79 0.82 0.83 0.90 0.86 0.91
Qdl (μF·cm−2) 137.5 136.0 192.5 786.5 314.5 320.8 297.6 317.4 375.6 312.1
Rct (kΩ·cm2) 2.8 22.3 17.6 39.4 110.9 108.0 102.9 87.0 90.7 72.1
Chi-square value (×10−3) 2.407 7.624 4.125 4.737 0.108 0.540 0.281 0.220 0.910 0.266

becomes rougher with the changing of time. There is no integrated layer
induced by the accumulation of the corrosion products on the surface of
X65 steel.
Fig. 7 illustrates the corroded morphologies of the coupons immersed
in the SRB-containing system. As shown in Fig. 7(A), after 8 h of immer-
sion, the surface of X65 steel is covered by a large amount of corrosion
products which may be mainly composed of iron oxide compounds.
From Fig. 7(B), distinct bacterial cells can be observed on the surface at
2 d. Fig. 7(B) also shows the decrease of the corrosion products. From
the magnified images in Fig. 7(A′)-(D′), a reticular biofilm can be ob-
served. The structure of the reticular biofilm is formed and degraded
with the growth of bacterial cells. As shown in Fig. 7(A′) and (B′), the re-
ticular biofilm appears after 8 h of immersion and covers the entire surface
gradually till 2 d. The biofilm deposits quite evenly and compactly at 5 d,
shown in Fig. 7(C′). The biofilm becomes sparse after 5 days, shown in
Fig. 7(D′), potentially due to the decrease of extracellular polymeric sub-
stances (EPS) which play a crucial role in the integrity of the biofilm.
3.3. Corrosion products analysis
As observed from the corroded morphologies in Fig. 7, the biofilm
has become compactly enough at 5 d compared with other periods.
Therefore, the coupons of X65 steel immersed for 5 days were selected
for elemental analysis. Fig. 8 shows the full XPS spectra of the surface of
X65 steel immersed in the abiotic system and the SRB-containing sys-
tem for 5 days. As shown in Fig. 8, the peaks of Fe 3p, Fe 3s, C 1s, O 1s
and Fe 2p are observed in both the abiotic system and the SRB-
containing system. While the peaks of N 1s, P 2s and P 2p can be only
observed in the SRB-containing system. The variation of relative con-
tents of C, N, O and P in the SRB-containing system compared with
those in the abiotic system is due to the forming of the biofilm. The
main composition of biofilm is EPS, an organic component derived
from bacterial metabolism.
Fig. 9 shows the high-resolution spectra of O 1s and Fe 2p3/2 for the
X65 steel exposed to the abiotic system and the SRB-containing system
for 5 days. As shown in Fig. 9(A), the O 1s spectrum of the X65 steel in
the abiotic system can be divided into two peaks at 531.3 and
530.1 eV. The O 1s peak at 531.3 eV can be attributed to OH– and the
peak at 530.1 eV can be attributed to O2– [38,39]. The Fe 2p3/2 spectrum
of X65 steel in the abiotic system shows a divalent peak at 709.9 eV and
two trivalent peaks at 711.1 and 712.8 eV [39,40]. The results reveal that
the corrosion products on the surface of the X65 steel exposed to the
abiotic system may be primarily composed of Fe(OH)3, γ-FeOOH,
Fe3O4 and α-Fe2O3.

Fig. 4. Time dependence of Rct in the abiotic system and the SRB-containing system. Fig. 5. The variation of Rs, pore and Qf with time in the SRB-containing system.
 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50
Fig. 6. Corroded morphologies of X65 steel exposed to the abiotic system for (A) 8 h, (B) 2 days, (C) 5 days and (D) 11 days.
For the coupons of X65 steel in the SRB-containing system, as shown
in Fig. 9(B), three peaks of O 1s at 532.0, 531.2 and 530.1 eV are calcu-
lated. The peak at 532.0 eV can be attributed to \\(C_O)\\O\\ and
HO\\C groups, which widely exist in the organic components of EPS.
The dominant peak at 531.2 eV can be attributed to OH– and the oxygen
of\\(C_O)\\O\\group. The third peak at 530.1 eV can be attributed to
O2– [38,41]. The Fe 2p3/2 spectrum of the X65 steel in the SRB-
containing system shows two trivalent peaks at 711.1 and 712.8 eV.
The peaks for Fe2+ are not included in the spectrum. The results indicate
that the surface of the X65 steel exposed to the SRB-containing system is
covered by the corrosion products composed of Fe(OH)3, γ-FeOOH and
α-Fe2O3 along with the organic products mainly composed of EPS.
Fig. 10 shows the cross-sectional SEM/EDS analysis of biofilm formed
on X65 steel after 5 days of immersion in the SRB-containing system.
The distinctions between the epoxy resin, the biofilm and the substrate
(X65 steel) can be clearly observed. Fig. 10 shows a biofilm layer of ap-
proximately 3 μm tightly adhered to the substrate. The biofilm layer is
rich in C, O and Fe. The result also provides evidence that the biofilm
consists of EPS and iron oxide compounds. Furthermore, the content of
C is higher in the outer part than that in the inner part, which means
that EPS are the dominant ingredients in the outer part of the biofilm.
3.4. Solution analysis
The MPN method is used to evaluate the bacterial growth in the me-
dium. Fig. 11 shows the time dependence of the bacterium number after
the inoculation of SRB. As shown in Fig. 11, the number of active SRB re-
mains low before 8 h of inoculation. The number rapidly increases and
remains stationary from 2 d to 5 d, and then decreases after 5 d. Conse-
quently, four stages are identified based on the variate number of SRB,
consisting of the lag phase, the logarithmic phase, the stationary phase
and the declination phase.
The pH values of the test systems are measured at 8 h, 2 d, 5 d, 8 d
and 11 d. Fig. 12 shows the variation of pH with time in the abiotic sys-
tem and the SRB-containing system. As shown in Fig. 12, the initial pH of
the two systems is controlled at about 7.23 in both the abiotic and SRB-
containing systems. In the abiotic system, the pH remains almost
45
unchanged during the 11 days of immersion. In the SRB-containing sys-
tem, a large decrease of pH occurs at 8 h (about 6.27). Then, the values
begin to increase gradually with time to be almost neutral (about 6.99).
The observed trend of pH in the SRB-containing system is similar to the
previous reports which investigate the corrosion in the presence of SRB
in a solution at neutral pH [42,43].
Fig. 13 shows the variation of the soluble iron concentration in the
abiotic system and the SRB-containing system. As shown in Fig. 13, in
the abiotic system, the concentration of iron in the solution keeps in-
creasing during the entire experiment (up to 2141.4 μg·L−1), due to
the continuous dissolution of iron from the anode. In the SRB-
containing system, the concentration of the soluble iron remains
lower than that in the abiotic system throughout the experiment. As
shown in the magnified figure in Fig. 13, an increase of the concentra-
tion of the soluble iron is observed for the first two days of immersion
(up to 220.1 μg·L−1). A large decrease of the concentration of the solu-
ble iron occurs from 3 d to 5 d (down to 15.2 μg·L−1). Afterwards, the
concentration of the soluble iron begins to slowly increase again. The
trend of the concentration of the soluble iron indicates that although
the iron is continuously dissolved in the solution, a large amount of sol-
uble iron react with other substance to form a precipitate.
4. Discussion
4.1. Corrosion behaviors of X65 steel in different systems
In the abiotic system, the increase of R ct in the first 7 days is
caused by the accumulation of corrosion products on the surface
and the subsequent decrease is due to the porous structure of the
corrosion product layer. Furthermore, due to the porous structure,
the adherent corrosion products composed of iron oxide compounds
are not efficient enough to protect the steel against further corrosion.
By combining the results of morphology and elemental analysis, the
X65 steel is continuously corroded in the abiotic system without an
efficient layer to protect it.
In the SRB-containing system, the SRB growth and the corrosion of
iron are occurring simultaneously during the early stage of immersion.
46 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50

Fig. 7. Corroded morphologies of X65 steel exposed to the SRB-containing system for (A) 8 h, (B) 2 days, (C) 5 days and (D) 11 days. (A′), (B′), (C′) and (D′) The magnified images for 8 h,
2 days, 5 days and 11 days respectively.

The bacterial cells of SRB attach to the surface of X65 steel and produce the productivity of EPS, which is directly related to the bacterial activity.
EPS through the metabolism. As these EPS accumulate, a biofilm is The biofilm is rapidly thickening and densifying in logarithmic phase
formed, gradually covering the entire surface and with time, becoming because of the fast reproduction and metabolism of bacteria. The biofilm
increasingly thick and compact. The biofilm development depends on development slows down and remains stable when bacterial growth
 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50
Fig. 8. Full XPS spectra of the surface of X65 steel immersed in the abiotic system and the
SRB-containing system for 5 days.
enters stationary phase. The biofilm degrades subsequently with the
death of bacteria in declination phase.
As shown in Fig. 4, an increase of Rct occurs at 8 h, indicating the cor-
rosion is inhibited. Meanwhile, the bacterial cells and the biofilm are
discovered on the surface, as shown in Fig. 7(A) and (A′). Consequently,
this increase of Rct reveals that this inhibition of the corrosion is influ-
enced by both the initial SRB attachment and the forming of the biofilm.
The impedance responses start to appear two time constants at 2 d, as
shown in Fig. 2(A). At this moment, the biofilm has covered the surface
of the X65 steel completely, shown in Fig. 7(B) and (B′). The variation of
Rct in Fig. 4 as well as the evolution of morphologies in Fig. 7 follow the
Fig. 9. O 1s and Fe 2p3/2 spectra of the surface of X65 steel immersed in (A) abiotic and (B) SRB-containing systems for 5 days.
47
same trend with the bacterial growth in Fig. 11. Rct rapidly increases in
the logarithmic phase, remains almost unchanged in the stationary
phase, and decreases in the declination phase. It is indicated that the
biofilm compactness and the coverage integrity have decisive effects
on the corrosion inhibition efficiency. The X65 steel is corroded in the
initial immersion. The corrosion of the X65 steel becomes inhibited
with the forming of the biofilm during the subsequent immersion. The
inhibition efficiency is influenced by the growth of SRB.
The corrosion inhibition effect of biofilm is also illustrated by the re-
sults of the soluble iron concentration, shown in Fig. 13. In the SRB-
containing system, the variation of soluble iron concentration is affected
by two competitive factors: the anodic dissolution of iron and the
formation of iron sulfide precipitates. The anodic dissolution is a contin-
uous process and its rate depends on the corrosion rate. The precipita-
tion of iron sulfide is also a continuous process and its rate depends
on the generating ability of S2−. The generating ability of S2− corre-
sponds to the growth of SRB. In the first two days, which is the early
stage of the SRB growth, SRB have a weak ability to generate S2−. This
weak ability results in a low precipitation rate. In addition, during this
period, the biofilm has not yet spanned over the entire surface of X65
steel. Therefore the corrosion is relatively unhindered, making the an-
odic dissolution rate still high. Hence, the increase of the soluble iron
concentration for the first two days can be attributed to the fact that
the anodic dissolution rate is faster than the precipitation rate. From
2 d to 5 d, the decrease of the soluble iron concentration can be attrib-
uted to the decrease of the corrosion rate and the increase of the amount
of S2− generated by SRB. After 5 days of immersion, the increase of the
soluble iron concentration can be attributed to the degraded biofilm and
the weakened S2− generating ability.
The trend of Rs, pore can be also illustrated by the results of the SRB
growth in Fig. 11. The biofilm has formed on the metal surface
completely at 2 d, shown in Fig. 7(B) and (B′). A certain number of mi-
cropores reserving the electrolyte solution are embedded in the biofilm.
The period from 2 d to 3 d is the logarithmic phase of the SRB growth, as
48 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50
Fig. 10. Cross-sectional SEM and EDS elemental line scan analysis of the biofilm formed on
X65 steel immersed in the SRB-containing system for 5 days.
shown in Fig. 11, which is the fastest period of SRB reproduction and
metabolism. The metabolic products are generated with a high rate dur-
ing this period. The biofilm is rapidly thickening and densifying, and
consequently, the porosity of the biofilm is reduced. Because of the re-
duced porosity, Rs, pore increases during the period from 2 d to 3 d.
From 3 d to 5 d, the structure of the biofilm remains stable during the
stationary phase of the SRB growth. This should results in an unchanged
Fig. 11. SRB growth in the medium.
Fig. 12. Time dependence of pH measured in the abiotic system and the SRB-containing
system.
Rs, pore. However, the ions in the external solution can get into the micro-
pores of the biofilm by diffusion, which leads to the increase of the solu-
tion conductivity in the biofilm pores. Consequently, Rs, pore decreases
with time from 3 d to 5 d. After 5 d, the growth of SRB enters the decli-
nation phase. The biofilm starts to degrade with the increasing death
rate of SRB, shown in Fig. 7(D) and (D′). Due to the degradation of the
biofilm, Rs, pore decreases with time after 5 d.
Because Qf is determined by the thickness and the specific surface
area of the biofilm, the variation of Qf can clearly illustrate the
formation-degradation process of the biofilm on the surface of X65
steel. The capacitance of a layer can be described as [44,45]:
C ¼ εε0 S=D ð2Þ
where ε0 is the permittivity of vacuum with the value of 8.85 × 10−
12
F·m−1. ε is the permittivity of the layer, which is considered as a con-
stant in this experiment. S is the specific surface area of the layer. D is the
thickness of the layer. According to Eq. (2), either the decrease of the
specific surface area or the increase of the thickness can lead to a de-
crease of the capacitance. From 2 d to 3 d, the porosity of the biofilm
Fig. 13. Time dependence of the concentration of the soluble iron measured in the abiotic
system and the SRB-containing system.
 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50
reduces, which results in the decrease of the specific surface area of the
biofilm. The decrease of Qf is due to the increasing compactness and
thickness of the biofilm. From 3 d to 5 d, remains nearly unchanged dur-
ing the stationary phase. After 5 d, Qf increases during the declination
phase in consequence of the decreasing thickness and the increasing po-
rosity of the biofilm.
4.2. Inhibition mechanisms of SRB under the aerobic environment
Oxygen is a preferred oxidant in the corrosion system under the aer-
obic environment. The formation of an even and compact biofilm on the
surface of X65 steel can be proposed as a potential inhibition mecha-
nism of SRB for X65 steel under the aerobic environment. The even
and compact biofilm formed by the SRB metabolism, shown in Figs. 7
and 10, acts as a barrier that can hinder the transfer process of oxygen
and prevent corrosive Cl− and H2S from reaching the surface of X65
steel. The biofilm has covered the surface of X65 steel completely at
2 d. A blocked characteristic of the impedance response occurs, shown
in Fig. 2(A) and (B). Because of the barrier effect of the biofilm, the cor-
rosion rate of X65 steel is slowed down.
In addition to acting as a barrier, the biofilm also contains EPS com-
ponent which also has effects on the corrosion inhibition. Stadler et al.
have demonstrated that the EPS generated by different SRB have differ-
ent influences on the corrosion behavior of pure iron and carbon steel.
In certain cases, the corrosion is inhibited due to the specific complex-
binding ability of the EPS to chelate metal ions [46]. During the experi-
ment, EPS are continuously generated with the SRB growth and accu-
mulated on the surface of X65 steel. Because of the inherent corrosion
inhibition of EPS, EPS component is another possibility to cause the cor-
rosion inhibition of X65 steel in the SRB-containing system.
The EPS can also exhibit the protective property to X65 steel by
preventing the bacterial cells of SRB from adhering to the surface of
X65 steel. Observed from the surface morphologies and the cross-
sectional morphology in Figs. 7 and 10 respectively, the bacterial cells
of SRB mainly exist on the surface of the biofilm, instead of the interface
between the biofilm and the substrate. SRB can accelerate the corrosion
of X65 steel by obtaining electrons from the substrate. The process of
obtaining electrons by SRB requires the direct adhesion of the bacterial
cells of SRB to the substrate. A mechanism of EPS called ‘microbial foot-
prints’ prevents the adhesion of the bacterial cells, which has been re-
ported by Marshall et al. and Neu [47–50]. The footprint-EPS excreted
by microbes label the surface as ‘already exploited’. These labels can
protect the surface against the future adhesion of the bacteria. The pro-
cess of obtaining electrons by SRB is hindered by the excretion of
footprint-EPS. Hence, the corrosion is inhibited by EPS.
Based on the above discussion, because of the barrier effect of the in-
tegrated biofilm, the corrosion of X65 steel is inhibited after the lag
phase of the SRB growth. Due to the inherent corrosion inhibition and
the ‘microbial footprints’ of EPS, the corrosion can be even inhibited in
the lag phase of the SRB growth, although there is not an integrated bio-
film formed on the surface of X65 steel.
This paper is addressed in the effect of SRB on the corrosion
behavior of X65 steel under an aerobic condition of the overall envi-
ronment. Although the overall environment is aerobic, the biofilm
may consume the oxygen in the solution. The consumption of oxy-
gen by the biofilm may result in a reduced concentration of oxygen
at the interface between the biofilm and the substrate. The specific
concentration of oxygen underneath the biofilm deserves further
study.
5. Conclusions
In the abiotic system, the X65 steel is continuously corroded without
an efficient layer to protect it. Rct increases in the first 7 days of immer-
sion and then decreases with time. The surface of X65 steel is covered by
49
the corrosion products primarily composed of Fe(OH)3, γ-FeOOH, Fe3O4
and α-Fe2O3.
In the SRB-containing system, the X65 steel is corroded in the initial
immersion. The corrosion of the X65 steel becomes inhibited with the
forming of the biofilm during the subsequent immersion. Rct rapidly in-
creases between 2 d and 3 d, remains almost unchanged from 3 d to 5 d
and decreases with time afterwards. Rs, pore first increases and then de-
creases with time. Qf decreases between 2 d and 3 d, remains almost un-
changed from 3 d to 5 d and then increases with time. The surface of X65
steel is covered by the corrosion products composed of Fe(OH)3, γ-
FeOOH and α-Fe2O3 along with the organic products mainly composed
of EPS.
Due to the formation of the biofilm, the SRB inhibit the corrosion of
X65 steel to a certain extent. The inhibition efficiency is influenced by
the growth of SRB. The efficiency rapidly increases in the logarithmic
phase, remains stable in the stationary phase and decreases in the dec-
lination phase.
Acknowledgements
We are indebted to Dun Zhang, Jiajia Wu, Yan Sun and Juna Chen
from Key Laboratory of Marine Environmental Corrosion and Bio-
fouling, Institute of Oceanology, Chinese Academy of Sciences for their
support in microbiological methodology. This work was supported by
National Basic Research Program of China (2014CB046801); National
Natural Science Foundation of China (51471117); and Key Project of
Tianjin Natural Science Foundation (13JCZDJC29500).
References
[1] W.A. Hamilton, Sulphate-reducing bacteria and anaerobic corrosion, Annu. Rev.
Microbiol. 39 (1985) 195–217.
[2] B.W.A. Sherar, I.M. Power, P.G. Keech, S. Mitlin, G. Southam, D.W. Shoesmith, Char-
acterizing the effect of carbon steel exposure in sulfide containing solutions to
microbially induced corrosion, Corros. Sci. 53 (2011) 955–960.
[3] P. Beese, H. Venzlaff, J. Srinivasan, J. Garrelfs, M. Stratmann, K.J.J. Mayrhofer, Moni-
toring of anaerobic microbially influenced corrosion via electrochemical frequency
modulation, Electrochim. Acta 105 (2013) 239–247.
[4] W. Dec, M. Mosiałek, R.P. Socha, M. Jaworska-Kik, W. Simka, J. Michalska, The effect
of sulphate-reducing bacteria biofilm on passivity and development of pitting on
2205 duplex stainless steel, Electrochim. Acta 212 (2016) 225–236.
[5] M. Stipaničev, F. Turcu, L. Esnault, E.W. Schweitzer, R. Kilian, R. Basseguy, Corrosion
behavior of carbon steel in presence of sulfate-reducing bacteria in seawater envi-
ronment, Electrochim. Acta 113 (2013) 390–406.
[6] E. Huttunen-Saarivirta, P. Rajala, L. Carpén, Corrosion behaviour of copper under bi-
otic and abiotic conditions in anoxic ground water: electrochemical study,
Electrochim. Acta 203 (2016) 350–365.
[7] F. Guan, X. Zhai, J. Duan, J. Zhang, K. Li, B. Hou, Influence of sulfate-reducing bacteria
on the corrosion behavior of 5052 aluminum alloy, Surf. Coat. Technol. 316 (2017)
171–179.
[8] C.A.H. von Wolzogen Kuehr, L.S. Van der Vlugt, Graphitization of cast iron as an
electro-biochemical process in anaerobic soils, Hague 8 (1934) 147–165.
[9] R.A. King, J.D.A. Miller, J.S. Smith, Corrosion of mild steel by iron sulphides, Br.
Corros. J. 8 (1973) 137–141.
[10] T.S. Rao, K.V.K. Nair, Microbiologically influenced stress corrosion cracking failure of
admiralty brass condenser tubes in a nuclear power plant cooled by freshwater,
Corros. Sci. 40 (1998) 1821–1836.
[11] V.P. Kholodenko, S.K. Jigletsova, V.A. Chugunov, V.B. Rodin, V.S. Kobelev, S.V. Karpov,
Chemicomicrobiological diagnostics of stress corrosion cracking of trunk pipelines,
Appl. Biochem. Microbiol. 36 (2000) 594–601.
[12] T. Wu, J. Xu, C. Sun, M. Yan, C. Yu, W. Ke, Microbiological corrosion of pipeline steel
under yield stress in soil environment, Corros. Sci. 88 (2014) 291–305.
[13] T. Gu, K. Zhao, S. Nesic, A new mechanistic model for MIC based on a biocatalytic ca-
thodic sulfate reduction (BCSR) theory, in: Corrosion 2009, Atlanta, Georgia, Paper
No. 09390, NACE International.
[14] D. Enning, J. Garrelfs, Corrosion of Iron by sulfate-reducing bacteria: new views of an
old problem, Appl. Environ. Microbiol. 80 (2014) 1226–1236.
[15] D. Xu, Y. Li, F. Song, T. Gu, Laboratory investigation of microbiologically influenced
corrosion of C1018 carbon steel by nitrate reducing bacterium Bacillus licheniformis,
Corros. Sci. 77 (2013) 385–390.
[16] D. Xu, Y. Li, T. Gu, Mechanistic modeling of biocorrosion caused by biofilms of sulfate
reducing bacteria and acid producing bacteria, Bioelectrochemistry 110 (2016)
52–58.
[17] R. Jia, J.L. Tan, P. Jin, D.J. Blackwood, D. Xu, T. Gu, Effects of biogenic H2S on the mi-
crobiologically influenced corrosion of C1018 carbon steel by sulfate reducing
Desulfovibrio vulgaris biofilm, Corros. Sci. 130 (2018) 1–11.
50 Q. Li et al. / Bioelectrochemistry 122 (2018) 40–50
[18] H.T. Dinh, J. Kuever, M. Mussmann, A.W. Hassel, M. Stratmann, F. Widdel, Iron cor-
rosion by novel anaerobic microorganisms, Nature 427 (2004) 829–832.
[19] H. Venzlaff, D. Enning, J. Srinivasan, K.J.J. Mayrhofer, A.W. Hassel, F. Widdel, M.
Stratmann, Accelerated cathodic reaction in microbial corrosion of iron due to direct
electron uptake by sulfate-reducing bacteria, Corros. Sci. 66 (2013) 88–96.
[20] D. Xu, T. Gu, Carbon source starvation triggered more aggressive corrosion against
carbon steel by the Desulfovibrio vulgaris biofilm, Int. Biodeterior. Biodegrad. 91
(2014) 74–81.
[21] P.F. Beese-Vasbender, S. Nayak, A. Erbe, M. Stratmann, K.J.J. Mayrhofer, Electrochem-
ical characterization of direct electron uptake in electrical microbially influenced
corrosion of iron by the lithoautotrophic SRB Desulfopila corrodens strain IS4,
Electrochim. Acta 167 (2015) 321–329.
[22] P. Zhang, D. Xu, Y. Li, K. Yang, T. Gu, Electron mediators accelerate the microbiolog-
ically influenced corrosion of 304 stainless steel by the Desulfovibrio vulgaris biofilm,
Bioelectrochemistry 101 (2015) 14–21.
[23] H.A. Videla, Corrosion of mild steel induced by sulfate-reducing bacteria. A study of
passivity breakdown by biogenic sulfides, NACE-8 International Corrosion Confer-
ence Series, NACE International, Houston, Texas 1986, pp. 162–171.
[24] H.A. Videla, C.L. Swords, R.G.J. Edyvean, Corrosion products and biofilm interactions
in the SRB influenced corrosion of steel, CORROSION, NACE International, 2002 Den-
ver, Colorado, Paper No. 557.
[25] H.A. Videla, L.K. Herrera, R.G.J. Edyvean, An updated overview of SRB induced corro-
sion and protection of carbon steel, CORROSION, NACE International, 2005 Houston,
Texas, Paper No.488.
[26] R. Zuo, Biofilms: strategies for metal corrosion inhibition employing microorgan-
isms, Appl. Microbiol. Biotechnol. 76 (2007) 1245–1253.
[27] H.A. Videla, L.K. Herrera, Understanding microbial inhibition of corrosion. A compre-
hensive overview, Int. Biodeterior. Biodegrad. 63 (2009) 896–900.
[28] E.C. Hatchikian, Y.A. Henry, An iron-containing superoxide dismutase from the strict
anaerobe Desulfovibrio desulfuricans (Norway 4), Biochimie 59 (1977) 153–161.
[29] A.J. Pierik, R.B.G. Wolbert, G.L. Portier, M.F.J.M. Verhagen, W.R. Hagen, Nigerythrin
and rubrerythrin from Desulfovibrio vulgaris each contain two mononuclear iron
centers and two dinuclear iron clusters, Eur. J. Biochem. 212 (1993) 237–245.
[30] E.D. Coulter, D.M. Kurtz Jr, A role for rubredoxin in oxidative stress protection in
Desulfovibrio vulgaris: catalytic electron transfer to rubrerythrin and two-iron super-
oxide reductase, Arch. Biochem. Biophys. 394 (2001) 76–86.
[31] K.U. Kjeldsen, C. Joulian, K. Ingvorsen, Oxygen tolerance of sulfate-reducing bacteria
in activated sludge, Environ. Sci. Technol. 38 (2004) 2038–2043.
[32] A. Dolla, M. Fournier, Z. Dermoun, Oxygen defense in sulfate-reducing bacteria, J.
Biotechnol. 126 (2006) 87–100.
[33] Y. Wan, D. Zhang, H. Liu, Y. Li, B. Hou, Influence of sulphate-reducing bacteria on en-
vironmental parameters and marine corrosion behavior of Q235 steel in aerobic
conditions, Electrochim. Acta 55 (2010) 1528–1534.
[34] J. Wu, H. Liu, P. Wang, D. Zhang, Y. Sun, E. Li, Oxygen reduction reaction affected by
sulfate-reducing bacteria: different roles of bacterial cells and metabolites, J.
Microbiol. 57 (2017) 344–350.
[35] ASTM D4412-15, Standard Test Methods for Sulfate-reducing Bacteria in Water and
Water-formed Deposits, ASTM International, West Conshohocken, PA, 2015.
[36] H. Castaneda, X.D. Benetton, SRB-biofilm influence in active corrosion sites formed
at the steel-electrolyte interface when exposed to artificial seawater conditions,
Corros. Sci. 50 (2008) 1169–1183.
[37] M.E. Orazem, B. Tribollet, Electrochemical Impedance Spectroscopy, First ed. John
Wiley & Sons Inc., Hoboken, 2008 233–236.
[38] S. Chen, P. Wang, D. Zhang, Corrosion behavior of copper under biofilm of sulfate-
reducing bacteria, Corros. Sci. 87 (2014) 407–415.
[39] H. Yan, J. Wang, Y. Zhang, W. Hu, Preparation and inhibition properties of molybdate
intercalated ZnAlCe layered double hydroxide, J. Alloys Compd. 678 (2016)
171–178.
[40] K. Wang, J. Wang, W. Hu, Evaluation of temperature effect on the corrosion process
of 304 stainless steel in high temperature water with electrochemical noise, Mater.
Des. 82 (2015) 155–163.
[41] X. Fu, M.J. Jenkins, G. Sun, I. Bertoti, H. Dong, Characterization of active screen
plasma modified polyurethane surfaces, Surf. Coat. Technol. 206 (2012) 4799–4807.
[42] M.A. Javed, P.R. Stoddart, S.A. Wade, Corrosion of carbon steel by sulphate reducing
bacteria: initial attachment and the role of ferrous ions, Corros. Sci. 93 (2015) 48–57.
[43] Y. Chen, Q. Tang, J.M. Senko, G. Cheng, B.Z. Newby, H. Castaneda, L.K. Ju, Long-term
survival of Desulfovibrio vulgaris, on carbon steel and associated pitting corrosion,
Corros. Sci. 90 (2015) 89–100.
[44] C. Cote, O. Rosas, R. Basseguy, Geobacter sulfurreducens: an iron reducing bacterium
that can protect carbon steel against corrosion? Corros. Sci. 94 (2015) 104–113.
[45] Z. Li, G.L. Song, S. Song, Effect of bicarbonate on biodegradation behaviour of pure
magnesium in a simulated body fluid, Electrochim. Acta 115 (3) (2014) 56–65.
[46] R. Stadler, W. Fuerbeth, K. Harneit, M. Grooters, M. Woellbrink, W. Sand, First eval-
uation of the applicability of microbial extracellular polymeric substances for corro-
sion protection of metal substrates, Electrochim. Acta 54 (2008) 91–99.
[47] R. Stadler, L. Wei, W. Fürbeth, M. Grooters, A. Kuklinski, Influence of bacterial
exopolymers on cell adhesion of Desulfovibrio vulgaris on high alloyed steel: corro-
sion inhibition by extracellular polymeric substances (EPS), Mater. Corros. 61
(2010) 1008–1016.
[48] K.C. Marshall, R. Stout, R. Mitchell, Mechanism of the initial events in the sorption of
marine bacteria to surfaces, J. Gen. Microbiol. 68 (1971) 337–348.
[49] T.R. Neu, Microbial “footprints” and the general ability of microorganisms to label
interfaces, Can. J. Microbiol. 38 (1992) 1005–1008.
[50] T.R. Neu, Significance of bacterial surface-active compounds in interaction of bacte-
ria with interfaces, Microbiol. Rev. 60 (1996) 151–166.