Accepted Manuscript

Corrosion behavior of copper under biofilm of sulfate-reducing bacteria

Shiqiang Chen, Peng Wang, Dun Zhang

PII: S0010-938X(14)00317-5
DOI: http://dx.doi.org/10.1016/j.corsci.2014.07.001
Reference: CS 5922

To appear in: Corrosion Science

Received Date: 29 April 2014

Accepted Date: 2 July 2014

Please cite this article as: S. Chen, P. Wang, D. Zhang, Corrosion behavior of copper under biofilm of sulfate-
reducing bacteria, Corrosion Science (2014), doi: http://dx.doi.org/10.1016/j.corsci.2014.07.001

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
 Corrosion behavior of copper under biofilm of sulfate-reducing

bacteria

Shiqiang Chen a, b, Peng Wang a, Dun Zhang a, ∗

a
Key Laboratory of Marine Environmental Corrosion and Bio-fouling, Institute of
Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China
b
University of Chinese Academy of Sciences, 19 (Jia) Yuquan Road, Beijing 100049,
China

Abstract
The effect of sulfate-reducing bacteria (SRB) on corrosion behavior of copper was
investigated using surface analysis and electrochemical measurements in seawater.
Results demonstrated that SRB adhere onto copper surface to form biofilm and that
the resulting corrosion product is mainly composed of cuprous sulfide. Cuprous
sulfide and EPS are helpful for SRB adhesion on copper by providing a barrier against
copper toxicity. In SRB growth cycle, corrosion rate is related to metabolic activity.
Especially during exponential growth and stationary phases, SRB metabolism
decreases the anodic zone area and promotes localized corrosion of copper.

Keywords: A. copper; B. XPS; C. MIC

1. Introduction
Microorganisms tend to attach to surfaces and then grow, replicate, and produce
extracellular polymeric substances (EPS), thereby forming a cohesive structure known
as biofilm. This process corrodes metal substrate through a route known as
microbiologically induced corrosion (MIC). According to estimates, MIC accounts for

∗
Corresponding author. Tel./Fax: +86 532 82898960; E-mail: zhangdun@qdio.ac.cn

1
about 20 percent of all corrosion damage of metals and building materials, and the
direct cost of MIC is estimated to total $30–50 billion per year [1]. SRB are anaerobic
microorganisms that are ubiquitous in environment [2]. SRB are considered the main
microorganisms that cause MIC, and SRB-induced corrosion constitutes half of all
MIC cases [3]. Many studies have been conducted to investigate steel corrosion
induced by SRB, and several mechanisms have been reported [4-7]. For instance,
metal corrosion can be accelerated through consumption of cathodic hydrogen via
hydrogenase catalysis during SRB metabolism (cathodic depolarization theory) [4].
The metabolic product (H2S) of SRB also accelerates metal corrosion [5]. EPS, which
is the main component of SRB biofilm, affects corrosion processes by strongly
complexing action with metal ions [6]. Some SRB promote corrosion by direct
electron exchange between metal surface and microbial cells [7].
Copper and its alloys are commonly used in structures and components exposed
to seawater and other marine environments due to their corrosion resistance,
machinability, thermal and electrical conductivities. In general, copper and its alloys
are impervious to the effects of MIC because elemental copper and its compounds
have a broad spectrum of antimicrobial activity against Gram-negative and positive
bacteria, fungi, and viruses by disrupting plasma membrane integrity or damaging
DNA and proteins [8]. Microbes are rapidly killed on metallic copper surfaces at a
rate of at least 1×107–1×108 colony forming units per hour, and live microorganisms
rarely recover from copper surfaces after prolonged incubation [9]. Thus, limited
attention has been given to MIC of copper. In recent years, several investigations have
suggested that a number of microbes, such as Pseudomonas fluorescens, have
numerous survival mechanisms for tolerating copper toxicity; these mechanisms
include export of copper ions outside the cell [10], energy-dependent efflux of copper
ions [10, 11], and enzymatic detoxification/reduction [12]. Microbes are believed to
be able to adhere to copper surface and influence corrosion process [13]. To the best
of our knowledge, limited information on SRB-induced MIC in copper is available.
Thus, studies on SRB-induced corrosion of copper are highly valuable.
For engineering metal, localized corrosion is a significant factor that affects its
2
service life. It is a typical form of MIC for the characteristic of heterogeneous
electrochemistry on biofilm/metal interfaces [14-16]. In investigations of localized
metal corrosion mechanisms, it is essential to determine electrochemical parameters at
local areas of metal surface. However, conventional electrochemical methods utilized
in MIC studies are hard to verify the mechanisms of microorganism-induced localized
corrosion, because they can only obtain average data/information about an
electrochemically active surface. In recent years, localized electrochemical methods,
such as scanning reference electrode technique and scanning vibrating electrode
technique, were utilized to investigate localized corrosion processes on metals [17,
18]. These techniques can obtain localized electrochemical parameters by detecting
ionic current flows in electrolyte phase. However, in MIC investigation, the
distribution of ionic currents in electrolyte is complex and difficult to detect
accurately for existence of heterogeneous biofilm covering the metal. As a technique
for obtaining localized electrochemical information, wire beam electrode (WBE)
method has been successfully used to investigate characteristic of heterogeneous
electrochemistry in localized corrosion [19, 20]. Actually, WBE setup consists of
numerous metallic wires, which are individual electrochemical sensors. These wires
enable WBE system to measure electrochemical parameters, such as electronic
current and corrosion potential distribution, at localized areas of electrode surface [21].
Thus, WBE is speculated to be effective for investigations of localized corrosion
mechanisms under biofilms.
In present study, surface analysis techniques were used to investigate SRB
adhesion on copper surface as well as the resulting corrosion products.
Electrochemical measurement techniques and WBE method were utilized to monitor
the overall and local electrochemical processes of copper during growth cycle of SRB.
Based on these results, corrosion mechanism of copper induced by SRB was clarified.

2. Experimental

2.1. Materials
Disk coupons with a diameter of 10 mm and thickness of 4 mm were cut from

3
copper (> 99.9%, mass%) plates and used for electrochemical measurements, and
surface and component analyses. For electrochemical measurements, coupons were
embedded in a mold of non-conducting epoxy resin with their circular cross-sections
left exposed. Electrical connection was realized via a copper wire soldered to sample.
WBE used in this study was manufactured from 121 identical pure copper wires (99.9%,
mass%; 1.34 mm diameter). Total working area of electrode array (Fig. 1A) was
approximately 1.71 cm2. All wires were regularly arranged in an 11 × 11 matrix and
embedded in epoxy resin at intervals of 1 mm from each other.
Prior to experiments, the exposed surfaces of samples, including disk coupons
and WBE, were sequentially ground with a series of mesh silicon carbide emery papers
(400, 800, 1200, and 2000) to smoothen them. The samples were then rinsed with
deionized water, degreased with absolute ethyl alcohol, dried with pure nitrogen, and
subsequently sterilized by exposure to ultraviolet radiation for 30 min before use.
2.2. Microorganism cultivation
Bacterial sample was isolated from marine sludge collected from the Bohai Sea
of China. The modified Postgate’s culture solution used in this work contained 0.5 g
of KH2PO4 (Sinopharm Chemical Reagent Co., Ltd.), 1 g of NH4Cl (Sinopharm
Chemical Reagent Co., Ltd.), 0.1 g of CaCl2 (Sinopharm Chemical Reagent Co., Ltd.),
2 g of MgSO4 (Sinopharm Chemical Reagent Co., Ltd.), 0.5 g of Na2SO4 (Sinopharm
Chemical Reagent Co., Ltd.), 4 mL of sodium lactate (Sinopharm Chemical Reagent
Co., Ltd.), and 1 g of yeast extract (Thermo Fisher Biochemical) per liter of natural
seawater, which was collected from Huiquan Bay in Qingdao, China. The pH of this
solution was adjusted to 7.2 ± 0.1 using 1 M NaOH solution. In the culture medium,
sodium lactate served as electron donor and sulfate served as electron acceptor for
SRB growth.
Culture medium was poured into a 1 L beaker (as an electrolytic cell, Fig. 1B),
deoxygenated by N2 sparging for 1 h, and then autoclaved at 121 ◦C for 30 min. After
cooling, sterile WBE, rubber stopper, and beaker with culture medium were rapidly
assembled, as shown in Fig. 1B. This setup was inoculated with the 4-day-old bacteria
sample at room temperature (25 ± 2 ◦C) and subsequently sealed and stored in a
4
temperature incubator at 30 ◦C.
2.3. Surface and component analysis
Scanning electron microscopy (SEM) was utilized to observe morphologies of
biofilm over coupon surface and substrate. Biofilm was visualized after preparation
using following procedure: Samples were exposed to 2.5% glutaraldehyde for 1–2 h
and serially dehydrated with an ethanol gradient (at 30%, 50%, 70%, 90%, and 100%
for 15 min). Coupons were then dried at critical point and sputter-coated with gold
prior to observation. Biofilm and corrosion products were removed from coupon
surfaces by following procedure: Samples were treated by ultrasonic cleaning in
absolute ethanol for 15 min to remove biofilm and then subsequently treated with
10% dilute sulfuric acid for 1 min to remove corrosion products. A scanning electron
microscope (KYKY-2008B) was used to visualize biofilm and substrate
morphologies.
Chemical composition information of copper surface immersed in sterile and
SRB media for 14 d was obtained by X-ray photoelectron spectroscopy (XPS)
(Thermo ESCALAB 250, Al Kα radiation).
2.4. Electrochemical experiments
Open-circuit potential (Eoc) and electrochemical impedance spectroscopy (EIS)
experiments were conducted in a cell with three electrodes using a CHI760C (CH
Instruments, Inc.) control system in sterile and SRB media. In three-electrode system,
copper electrode, Pt wire, and silver/silver chloride (Ag/AgCl, 3M KCl) (CH
Instruments, Inc.) were used as working, counter, and reference electrodes,
respectively. Each impedance spectrum was obtained at Eoc under excitation of a
sinusoidal wave with an amplitude of 5 mV and within frequency range of 100 kHz to
10 mHz. EIS results were analyzed by fitting data using ZSimpWin software. All
electrochemical experiments were performed at 25 ± 2 ◦C.
The current distribution of WBE was measured using a test device (NI
PXI-1042Q) consisting of NI PXI-8108 embedded controller and modular instruments:
NI PXI-2535, PXI-4022, and PXI-4071, similar to those described in literature [22].
PXI-8108 is a 5-slot PXI chassis with an integrated MXI-Express controller.
5
PXI-4071 is a 7.5-digit digital multimeter. PXI-4022 is a high-speed, high-precision
guard and current amplifier that can detect picoampere current levels with
femtoampere noise with PXI-4071. PXI-2535 is a high-density field-effect transistor
switch matrix module featuring 544 crosspoints, a 4 × 136 one-wire matrix
configuration (136 channels), switching speeds reaching 50,000 crosspoints/s, and
unlimited simultaneous connections. This PXI system was directly controlled by a
computer.
After WBE was immersed in culture medium, all wire sensors were individually
connected in sequence to permit electrons to move freely between wires, similar to a
one-piece electrode. Galvanic current distribution was monitored by PXI-4071 and
PXI-4022. In the current test, each individual wire electrode and all other wires
shorted together were connected by the PXI-4071 and PXI-4022, and their galvanic
currents were recorded. All measurements were controlled by computer via
self-designed software in LabVIEW environment. In the current measurements, the
interval between two channels was 1 s. The current distribution maps were drawn
using the Surfer 10.0 software.
3. Results and discussion
3.1. Surface morphological and elemental analysis
Fig. 2 presents morphologies of film and copper substrate after immersion in
sterile medium for 2 h to 13 d. Corrosion products of copper accumulate with
exposure time in sterile medium. After 2 h of exposure, a small amount of corrosion
products randomly attaches to the copper surface (Fig. 2A). After 1 d of exposure, no
obvious morphological changes of corrosion products is observed because of short
time interval between determinations (Fig. 2C); particle size of corrosion products,
however, increases slightly. Corrosion products accumulate gradually and form a film
after 3 d of exposure (Fig. 2E). On the 13th day of immersion, a thick film with cracks
and pores forms on copper surface (Fig. 2G). Based on morphologies of substrate
after removal of corrosion products (Figs. 2B, 2D, 2F, and 2H), no obvious pitting is
observed, which indicates that uniform corrosion is the dominant corrosion form of
copper in sterile medium throughout exposure period.
6
 Fig. 3 shows evolution of copper surface morphology over different exposure
periods in SRB medium. Unlike sample in sterile medium, bacteria and EPS gradually
accumulate on copper surface with increasing exposure time in SRB medium. Several
bacterial cells appear on copper surface after 2 h of exposure (Fig. 3A). After 1 d of
exposure, bacterial cells, several of which appear to reproduce by splitting in half, (Fig.
3C) are evidently present on copper surface; this result indicates that SRB thrives on
copper surface and resists copper toxicity. Bacterial cells are generally known to
adhere onto copper surfaces with great difficulty on account of toxicity of metal. The
number of cells absorbed onto copper surface unexpectedly increases, and a
heterogeneous, porous, and three-dimensional biofilm coated with EPS is observed
after 3 d (Fig. 3E). The space structure of biofilm becomes more complicated after
exposure for 13 d (Fig. 3G).
In exponential growth phase (1st–3rd day), SRB vigorously metabolize and
produce an abundance of EPS, which rapidly adheres onto metal surface and forms a
densely packed film [23]. Increased accumulation of EPS provides an essential
condition for clustering of bacterial cells [24]. EPS are organized in well-defined
capsules around cells or excreted into culture medium. Oxygen atom of carboxyl
groups in EPS molecules forms a complex with copper ions and consequently protects
SRB against copper toxicity [23, 25, 26]. Furthermore, Sulfide from metabolism of
SRB reduces copper ion concentration by combining action. This process also reduces
copper toxicity to SRB. In this case, number of active bacteria that adhere onto copper
surface increases, and a heterogeneous, porous, and three-dimensional biofilm coated
with EPS is observed.
SEM images of substrate after removal of biofilm and corrosion products show
that copper substrate features slight damage after 2 h of exposure (Fig. 3B). After 1 d
of exposure, several small pits appear on copper substrate (Fig. 3D). As exposure time
increases, pit diameter also increases (Figs. 3F and 3H). Obvious pitting is observed
especially on sample surface after immersion for 13 d (Fig. 3H), which proves that
presence of SRB induces localized corrosion of copper.
Fig. 4 presents wide XPS spectra of copper exposed to sterile and SRB media for
7
14 d. Peaks for Cu LMM Auger peak, Cu 3p, Cu 3s, S 2p, S 2s, N 1s, C 1s, O 1s, and
Cu 2p are observed in spectra of both two copper samples. Relative proportions of C,
O and N increase after SRB injection compared with those in sterile medium. This
result is caused by adsorption of biofilm on copper surface. Cu LMM Auger peak is
known to be more highly reliable than binding energies (Eb) of photoelectrons for
investigating chemical states. Use of X-ray-induced Auger peaks increases
effectiveness of identifying chemical states of copper [27]. The definition of Auger
parameter (α ) is [28]:
α = Ek ( Auger ) − Ek ( photoelectron ) (1)

Ek ( photoelectron ) = hν − Eb ( photoelectron ) (2)

α ′ = α + hν = Ek ( Auger ) + Eb ( photoelectron ) (3)

where Ek (Auger) is kinetic energy of Auger electron, Ek (photoelectron) and Eb

(photoelectron) are kinetic energy and binding energy of photoelectron all referred to
Fermi level, respectively. Ek (Auger) is 917.6 eV either in sterile or SRB medium, and
Eb (photoelectron) is 932.2 eV in both sterile and SRB media. The α obtained in
both sterile and SRB media is 1849.6 eV according to Eqs. (1)–(3), which indicates
that Cu (І) compounds are main components of copper corrosion products in both
media [29, 30].
Fig. 5 shows high-resolution images of Cu 2p, O 1s, and S 2p spectra of copper
immersed in sterile and SRB media. In the Cu 2p spectrum (Fig. 5A), two sharp peaks
at Eb of 932.2 (Cu 2p3/2) and 951.9 (Cu 2p1/2) eV, which correspond to Cu (I)
compounds or metallic Cu, were observed [31, 32]. No characteristic peak of Cu (II)
is observed within the range of 933–945 eV. These results support the conclusion that
corrosion products of copper are mainly composed of Cu (І) compounds in both
sterile and SRB media. For copper in sterile medium, the core-level O 1s spectrum is
deconvoluted into three peaks at Eb of 531.2, 532.0, and 533.2 eV (Fig. 5B). Peak at
Eb of 531.2 eV is attributed to Cu2O in corrosion products layer [33], and peaks

detected at Eb of 532.0 and 533.2 eV are respectively attributed to C-O and C=O

groups of organic compounds in condition layer, respectively [34]. For copper in SRB

8
medium, the core-level O 1s spectrum is deconvoluted into three peaks at Eb of 532.0,
533.2, and 535 eV. Similar to results obtained in sterile medium, peaks at Eb of 532
and 533.2 eV are respectively attributed to C−O and C=O groups of organic
compounds in biofilm. Peak at Eb of 535 eV is attributed to H2O in biofilm [35], and
the relative content of H2O is calculated to be 79%. This result agrees with the fact
that biofilms are made up of up to 94% H2O [36]. Fig. 5C shows high-resolution XPS
spectrum of S 2p. S 2p peak is fitted with a doublet representing spin orbit splitting of
S 2p3/2 and S 2p1/2 peaks. Peaks at Eb of 162 and 163.2 eV in sterile medium are
ascribed to thiolate species from culture solution that are adsorbed onto copper
surface [37]. The curve-fitted S 2p spectrum is dominated by Cu2S with S 2p3/2 at
161.7 eV and S 2p1/2 at 162.9 eV [38], which indicates that Cu2S is the main corrosion
product of copper in SRB medium. Another doublet is fitted to account for
high-energy tail of spectrum. 2p3/2 components at 162.5 eV may correspond to
disulfides (S22-) [39]. Thus, it can be inferred that a series of subsequent reactions of
HS- and Cl- with Cu can occur in SRB medium and generate Cu2S as follows [40]:

Cu + HS- → Cu ( HS)ads + e- (4)

Cu + Cu ( HS)ads + HS- → Cu 2S + H 2S + e- (5)

Cu ( HS)ads + 2Cl- → CuCl-2 + HS- (6)

2CuCl-2 + HS- → Cu 2S + 4Cl- + H + (7)

The following hydrogen evolution reactions are represent reduction half-reactions in

anaerobically isolated system:

2H 2S + 2e − → 2HS− + H 2 (8)

2H + + 2e − → H 2 (9)

Ultimately, the copper surface film in SRB medium is composed of a heterogeneous

and porous biofilm inlaid with Cu2S (Fig. 3G).
3.2. Evolution of Eoc
Monitoring of Eoc provides some information on evolution of corrosion process.
Fig.6 shows evolution of Eoc as a function of exposure time for copper in sterile and
9
SRB media, respectively. Copper is relatively stable in sterile solution because of
protective Cu2O film. Compared with that in sterile solution, Eoc of copper is more
negative in SRB medium because S2- accelerates anodic dissolution of copper [41]. To
verify this point, evolution of S2- concentration with time was monitored (Fig. 6c).
From the 1st day to the 4th day, S2- concentration increases with time as Eoc rapidly
decreases. Then, S2- concentration remains stable at 3 mmol/L. Eoc value is also stable
at –0.76 V versus Ag/AgCl. This result indicates that change in Eoc value is relevant to
S2- concentration in solution. S2- produced through metabolism of SRB induces
negative shift of Eoc by accelerating anodic reaction.
3.3. EIS results
EIS is a powerful and non-destructive technique for characterizing
electrochemical reactions at metal/biofilm interfaces and studying formation of
corrosion products and biofilms during MIC [42]. Fig. 7 presents Nyquist diagrams of
copper in sterile and SRB media. In sterile medium, impedances measured within the
first 3 d of exposure exhibits a tail corresponding to Warburg impedance in
low-frequency regions, which suggests that corrosion is controlled by a diffusion
process (Fig. 7A). After 3 d of exposure, Warburg impedance disappears and
low-frequency limits of impedance progressively increase with immersion time. The
evolution of impedance spectra in terms of size and shape at different times
demonstrates that corrosion product layer is thickened and that protection afforded by
corrosion product layer is improved [43]. In SRB medium, diameters of impedance
loops decrease in exponential growth and stationary phases and increase in death
phase (Fig. 7B). These trends imply that corrosion of copper in SRB medium is
accelerated during exponential growth and stationary phases and inhibited in death
phase.
The impedance spectra were further analyzed by fitting with proper equivalent
circuits (Fig. 8). It was proven that the film on copper surface immersed in sterile
medium is composed of corrosion products and condition film (organic compounds
attached from culture solution to copper surface). Warburg impedance appears on the
first 3 d of exposure and disappears thereafter. Considering contribution of each
10
phenomenon, such as adsorption of the condition film, formation of corrosion product
film, electrical double layer, and diffusion process, a three-time constant model
containing Warburg impedance is used to fit EIS data obtained from the first 3 d of
exposure (Fig. 8A), and a three-time constant model without Warburg impedance is
used to fit EIS data obtained after 3 d of exposure in sterile medium (Fig. 8B). In SRB
medium, the film on copper surface is composed of a heterogeneous and porous
biofilm inlaid with Cu2S (Figs. 3C, 3E, and 3G). Therefore, based on contribution of
each phenomenon, such as adsorption of biofilm, formation of corrosion product film,
and electrical double layer, a three-time constant model is used to fit EIS data
obtained from SRB medium (Fig. 8C).
In the three time constant model (Fig. 8), Rs indicates resistance of electrolyte. Qdl
is constant phase element (CPE) of electrical double layer and given by:
1
Z CPE = (8)
Y 0( jω)
n

where Y0 is a parameter related to capacitance, ω is angular frequency, j is imaginary

number, and n is exponential term related to roughness of electrode surface [44]. ZW is
Warburg impedance; Rct is charge transfer resistance of electrical double layer; Qcf, Qbf,
and Qcpf are CPEs of condition film, biofilm, and corrosion product film, respectively;
and Rcf, Rbf, and Rcpf are resistances of condition film, biofilm, and corrosion product
film, respectively. Rct values, which are obtained from fitting results of copper in
sterile and SRB media, are summarized in Fig. 9. Rct evidently increases with
increasing exposure time in sterile medium (Fig. 9a). This behavior is attributed to
formation of protective corrosion product film that reduces corrosion rate of copper.
The value of Rct in SRB medium is larger than that in sterile medium on the 1st day of
exposure (Fig. 9b) because inhibitive components (corrosion products and EPS
isolated sample from environment) can form protective film on metal [45]. By
contrast, Rct decreases rapidly after 1 d of exposure and then slightly increases
thereafter in SRB media. From the resulting environmental parameters of growth
cycle of SRB [46], Rct may be inferred to decrease with increasing concentration of
corrosive metabolites (sulfides and organic acids) after 1 d of exposure (Fig. 6c).
11
However, Rct increases after 7 d of exposure because of low metabolic activity of
SRB during death phase, and increasing of biofilm and corrosion product layer
thicknesses (Fig. 3) [47]. According to analysis above, the rate of copper corrosion is
related to metabolic activity of SRB.
3.4. Current distribution of WBE
Fig. 3 shows that localized corrosion of copper is induced in SRB medium. To
study process of localized corrosion of copper in SRB medium, WBE technique was
adopted. This technique enables measurement of electrochemical parameters from
local areas on a working electrode surface and can be used to study heterogeneous
electrochemical processes on electrode surfaces [48]. Figs. 10 and 11 respectively
show current distribution maps of WBE in sterile and SRB media as a function of
immersion time. Maximum anodic current density (imax) reflects anodic reaction rate
at local sites. In general, a higher imax value suggests more serious corrosion. Fig. 10
shows that imax value decreases with immersion time, which implies that reaction at
local area is inhibited because of formation of Cu2O film. Furthermore, no significant
anodic current peaks appear on current distribution maps, implying that uniform
corrosion of copper occurs in sterile medium, and this result is consistent with that
obtained with SEM (Fig. 2). In SRB medium (Fig. 11), imax value initially increases
from 0.21 μA·cm-2 to 0.87 μA·cm-2 with immersion time in exponential growth phase
(1st–3rd day), stabilizes at 0.70 μA·cm-2 in stationary phase (4th–7th day), and then
decreases to 0.17 μA·cm-2 in death phase (9th–13th day). These results imply that imax
is related to metabolic process of SRB. Moreover, the anode location varies with
immersion time in SRB growth process. For example, anodic current peak located at
position (10, 3) vanishes after 3 day of exposure, and new anodic current peaks
located at position (2, 0) and (8, 0) are observed on the 5th day. According to Eqs.
(4)–(7), Cu2S forms at anodic area during metabolic process of SRB. The immense
accumulation of Cu2S inhibits occurrence of anodic reaction, and then new anodic
current peaks will appear in other areas [49].
In contrast to current distribution of copper in sterile medium, a distribution
characterized with large cathodic area can be found on current distribution map of
12
copper in SRB medium. This distribution might be an important factor that resulting
in localized corrosion of copper in SRB media. To clarify relationship between
localized corrosion and metabolic activity of SRB, the variation of cathodic and
anodic area ratios (r) as a function of immersion time in sterile and SRB media is
shown in Fig. 12. r in SRB media is evidently related to SRB growth as it increases in
exponential growth phase, maintains stable value in stationary phase, and decreases in
death phase (Fig. 12b). r is higher in SRB medium than in sterile medium from the 3rd
day to the 11th day of immersion. In this case, relative areas of large cathode coupled
with small anode have significant effects on acceleration of localized corrosion of
copper in SRB medium. Furthermore, stable growth of localized corrosion sites in
open circuit requires sufficient cathodic reaction to meet anodic current demands [50].
In isolated anaerobic system, hydrogen evolution, as the major cathodic reaction, is
promoted through capture and utilization of atomic hydrogen generated at cathode by
SRB [51]. In exponential growth and stationary phases, SRB with vigorous growth
and metabolism rapidly consume cathodic hydrogen, thereby promoting cathodic
reaction and resulting in appearance of a large cathodic area in current distribution
map (Figs. 11B–11D). In death phrase, consumption rate of cathodic hydrogen
decreases with decreasing metabolic activity of SRB, consequently weakening
cathodic depolarization (Figs. 11E–11G). Strength of cathodic depolarization is
undoubtedly related to SRB activity, and localized corrosion is supported by
SRB-induced cathodic depolarization.
4. Conclusion
SRB can adhere onto copper surface to form biofilm in seawater under anaerobic
conditions. Cu2S is proven to be main corrosion product of copper immersed in SRB
medium. Formation of Cu2S and EPS films on copper reduces copper toxicity to SRB,
and may facilitate SRB adhesion on copper surface. Compared with sample in sterile
solution, SRB accelerate corrosion of copper for formation of sulfide through
metabolic process. In the SRB growth cycle, corrosion process is related to metabolic
activity of SRB. Corrosion rate of copper increases in exponential growth and
stationary phases and decreases in death phase. Especially in exponential growth and
13
stationary phases, SRB metabolism decreases anodic zone area and promotes
localized copper corrosion.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant
No. 51131008) and National Key Basic Research Program of China (2014CB643304).
References
[1] R. Javaherdashti, A review of some characteristics of MIC caused by
sulfate-reducing bacteria: past, present and future, Anti-corros. Method. M, 46
(1999) 173-180.
[2] S.J. Yuan, B. Liang, Y. Zhao, S.O. Pehkonen, Surface chemistry and corrosion
behaviour of 304 stainless steel in simulated seawater containing inorganic
sulphide and sulphate-reducing bacteria, Corros. Sci., 74 (2013) 353-366.
[3] G. Muyzer, A.J.M. Stams, The ecology and biotechnology of sulphate-reducing
bacteria, Nature Reviews Microbiology, 6 (2008) 441-454.
[4] P. Angell, J.S. Luo, D.C. White, Microbially sustained pitting corrosion of 304
stainless steel in anaerobic seawater, Corros. Sci., 37 (1995) 1085-1096.
[5] W. Lee, W.G. Characklis, Corrosion of mild steel under anaerobic biofilm,
Corrosion, 49 (1993) 186-199.
[6] Z.H. Dong, T. Liu, H.F. Liu, Influence of EPS isolated from thermophilic
sulphate-reducing bacteria on carbon steel corrosion, Biofouling, 27 (2011)
487-495.
[7] H.T. Dinh, J. Kuever, M. Mussmann, A.W. Hassel, M. Stratmann, F. Widdel, Iron
corrosion by novel anaerobic microorganisms, Nature, 427 (2004) 829-832.
[8] T. Sato, Y. Fujimori, T. Nakayama, Y. Gotoh, Y. Sunaga, M. Nemoto, T. Matsunaga,
T. Tanaka, Assessment of the anti-biofouling potentials of a copper iodide-doped
nylon mesh, Appl. Microbiol. Biot., 95 (2012) 1043-1050.
[9] G. Grass, C. Rensing, M. Solioz, Metallic copper as an antimicrobial surface, Appl.
Environ. Microb., 77 (2011) 1541-1547.
[10] C.D. Miller, B. Pettee, C. Zhang, M. Pabst, J.E. McLean, A.J. Anderson, Copper
and cadmium: responses in Pseudomonas putida KT2440, Lett. Appl. Microbiol.,
49 (2009) 775-783.
[11] N. Hu, B. Zhao, Key genes involved in heavy-metal resistance in Pseudomonas
putida CD2, Fems Microbiol. Lett., 267 (2007) 17-22.
[12] R. Andreazza, S. Pieniz, L. Wolf, M.K. Lee, F.A.O. Camargo, B.C. Okeke,
Characterization of copper bioreduction and biosorption by a highly copper
resistant bacterium isolated from copper-contaminated vineyard soil, Sci. Total.
Environ., 408 (2010) 1501-1507.
[13] D. Nercessian, F.B. Duville, M. Desimone, S. Simison, J.P. Busalmen, Metabolic
turnover and catalase activity of biofilms of Pseudomonas fluorescens (ATCC
17552) as related to copper corrosion, Water Res., 44 (2010) 2592-2600.
[14] S.E. Werner, C.A. Johnson, N.J. Laycock, P.T. Wilson, B.J. Webster, Pitting of type

14
 304 stainless steel in the presence of a biofilm containing sulphate reducing
bacteria, Corros. Sci., 40 (1998) 465-480.
[15] D. Starosvetsky, O. Khaselev, J. Starosvetsky, R. Armon, J. Yahalom, Effect of
iron exposure in SRB media on pitting initiation, Corros. Sci., 42 (2000) 345-359.
[16] X. Shi, R. Avci, M. Geiser, Z. Lewandowski, Comparative study in chemistry of
microbially and electrochemically induced pitting of 316L stainless steel, Corros.
Sci., 45 (2003) 2577-2595.
[17] G. Williams, H.A. Dafydd, R. Grace, The localised corrosion of Mg alloy AZ31 in
chloride containing electrolyte studied by a scanning vibrating electrode technique,
Electrochim. Acta, 109 (2013) 489-501.
[18] H. Xu, Y. Liu, W. Chen, R.G. Du, C.J. Lin, Corrosion behavior of reinforcing steel
in simulated concrete pore solutions: A scanning micro-reference electrode study,
Electrochim. Acta, 54 (2009) 4067-4072.
[19] T. Liu, Y.J. Tan, B.Z.M. Lin, N.N. Aung, Novel corrosion experiments using the
wire beam electrode. (IV) Studying localised anodic dissolution of aluminium,
Corros. Sci., 48 (2006) 67-78.
[20] N.N. Aung, Y.J. Tan, Monitoring pitting-crevice corrosion using the WBE-noise
signatures method, Mater. Corros., 57 (2006) 555-561.
[21] Y.J. Tan, Understanding the effects of electrode inhomogeneity and
electrochemical heterogeneity on pitting corrosion initiation on bare electrode
surfaces, Corros. Sci., 53 (2011) 1845-1864.
[22] W. Wang, X. Zhang, J. Wang, The influence of local glucose oxidase activity on
the potential/current distribution on stainless steel: A study by the wire beam
electrode method, Electrochim. Acta, 54 (2009) 5598-5604.
[23] Q. Bao, D. Zhang, D. Lv, P. Wang, Effects of two main metabolites of
sulphate-reducing bacteria on the corrosion of Q235 steels in 3.5wt.% NaCl media,
Corros. Sci., 65 (2012) 405-413.
[24] S.J. Yuan, S.O. Pehkonen, AFM study of microbial colonization and its deleterious
effect on 304 stainless steel by Pseudomonas NCIMB 2021 and Desulfovibrio
desulfuricans in simulated seawater, Corros. Sci., 51 (2009) 1372-1385.
[25] G. Bitton, V. Freihofer, Influence of extracellular polysaccharides on the toxicity of
copper and cadmium toward Klebsiella aerogenes, Microbial Ecol., 4 (1978)
119-125.
[26] G.P. Sheng, J. Xu, H.W. Luo, W.W. Li, W.H. Li, H.Q. Yu, Z. Xie, S.Q. Wei, F.C. Hu,
Thermodynamic analysis on the binding of heavy metals onto extracellular
polymeric substances (EPS) of activated sludge, Water Res., 47 (2013) 607-614.
[27] J. Morales, P. Esparza, G.T. Fernandez, S. Gonzalez, J.E. Garcia, J. Caceres, R.C.
Salvarezza, A.J. Arvia, A comparative study on the passivation and localized
corrosion of α, β, and α + β brass in borate buffer solutions containing sodium
chloride-I. Electrochemical data, Corros. Sci., 37 (1995) 231-239.
[28] M. Fantauzzi, D. Atzei, B. Elsener, P. Lattanzi, A. Rossi, XPS and XAES analysis
of copper, arsenic and sulfur chemical state in enargites, Surf. Interface Anal., 38
(2006) 922-930.
[29] S.W. Gaarenstroom, N. Winograd, Initial and final-state effects in esca spectra of

15
 cadmium and silver-oxides, J. Chem. Phys., 67 (1977) 3500-3506.
[30] D. Brion, Study by photoelectron-spectroscopy of surface degradation of FeS2,
CuFeS2, ZnS and PbS exposed to air and water, Applications of Surface Science, 5
(1980) 133-152.
[31] R. Procaccini, W.H. Schreiner, M. Vazquez, S. Cere, Surface study of films formed
on copper and brass at open circuit potential, Appl. Surf. Sci., 268 (2013) 171-178.
[32] M.A. Fazal, A.S.M.A. Haseeb, H.H. Masjuki, Corrosion mechanism of copper in
palm biodiesel, Corros. Sci., 67 (2013) 50-59.
[33] E. Abelev, D. Starosvetsky, Y. Ein-Eli, Potassium sorbate - A new aqueous copper
corrosion inhibitor electrochemical and spectroscopic studies, Electrochim. Acta,
52 (2007) 1975-1982.
[34] X. Fu, M.J. Jenkins, G.M. Sun, I. Bertoti, H.S. Dong, Characterization of active
screen plasma modified polyurethane surfaces, Surf. Coat. Technol., 206 (2012)
4799-4807.
[35] X.X. Jiang, N. Ellis, Z.P. Zhong, Characterization of pyrolytic lignin extracted
from bio-oil, Chin. J. Chem. Eng., 18 (2010) 1018-1022.
[36] E.J. Perez, R. Cabrera-Sierra, I. Gonzalez, F. Ramirez-Vives, Influence of
Desulfovibrio sp biofilm on SAE 1018 carbon steel corrosion in synthetic marine
medium, Corros. Sci., 49 (2007) 3580-3597.
[37] O. Furlong, B. Miller, W.T. Tysoe, Shear-induced boundary film formation from
dialkyl sulfides on copper, Wear, 274 (2012) 183-187.
[38] S.J. Yuan, S.O. Pehkonen, Surface characterization and corrosion behavior of
70/30 Cu-Ni alloy in pristine and sulfide-containing simulated seawater, Corros.
Sci., 49 (2007) 1276-1304.
[39] J.Z. Duan, B.R. Hou, Z.G. Yu, Characteristics of sulfide corrosion products on
316L stainless steel surfaces in the presence of sulfate-reducing bacteria, Mater.
Sci. Eng. C-bio. Syst., 26 (2006) 624-629.
[40] J. Chen, Z. Qin, D.W. Shoesmith, Long-term corrosion of copper in a dilute
anaerobic sulfide solution, Electrochim. Acta, 56 (2011) 7854-7861.
[41] K. Rahmouni, M. Keddam, A. Srhiri, H. Takenouti, Corrosion of copper in 3%
NaCl solution polluted by sulphide ions, Corros. Sci., 47 (2005) 3249-3266.
[42] F. Mansfeld, B. Little, A technical review of electrochemical techniques applied to
microbiologically influenced corrosion, Corros. Sci., 32 (1991) 247-272.
[43] X.N. Liao, F.H. Cao, L.Y. Zheng, W.J. Liu, A.N. Chen, J.Q. Zhang, C.A. Cao,
Corrosion behaviour of copper under chloride-containing thin electrolyte layer,
Corros. Sci., 53 (2011) 3289-3298.
[44] Y.J. Li, J.J. Wu, D. Zhang, Y. Wang, B.R. Hou, The electrochemical reduction
reaction of dissolved oxygen on Q235 carbon steel in alkaline solution containing
chloride ions, J. Solid State Electrochem., 14 (2010) 1667-1673.
[45] H.A. Videla, L.K. Herrera, Understanding microbial inhibition of corrosion. A
comprehensive overview, Int. Biodeter. Biodegr, 63 (2009) 896-900.
[46] F. Kuang, J. Wang, L. Yan, D. Zhang, Effects of sulfate-reducing bacteria on the
corrosion behavior of carbon steel, Electrochim. Acta, 52 (2007) 6084-6088.
[47] Y. Wan, D. Zhang, H.Q. Liu, Y.J. Li, B.R. Hou, Influence of sulphate-reducing

16
 bacteria on environmental parameters and marine corrosion behavior of Q235
steel in aerobic conditions, Electrochim. Acta, 55 (2010) 1528-1534.
[48] Y.J. Tan, Wire beam electrode: A new tool for studying localised corrosion and
other heterogeneous electrochemical processes, Corros. Sci., 41 (1999) 229-247.
[49] J. Chen, Z. Qin, D.W. Shoesmith, Kinetics of corrosion film growth on copper in
neutral chloride solutions containing small concentrations of sulfide, J.
Electrochem. Soc., 157 (2010) C338-C345.
[50] Y.H. Wang, W. Wang, Y.Y. Liu, L. Zhong, J. Wang, Study of localized corrosion of
304 stainless steel under chloride solution droplets using the wire beam electrode,
Corros. Sci., 53 (2011) 2963-2968.
[51] F. Liu, J. Zhang, S. Zhang, W. Li, J. Duan, B. Hou, Effect of sulphate reducing
bacteria on corrosion of Al-Zn-In-Sn sacrificial anodes in marine sediment, Mater.
Corros., 63 (2012) 431-437.

17
Figure captions:
Fig. 1 (A) the digital photo of WBE and (B) the schematics of WBE set-up.
Fig. 2 SEM micrographs of copper (A, C, E and G) before and (B, D, F and H)
after removing corrosion products immersed in sterile medium for (A and B) 2 hours,
(C and D) 1, (E and F) 3 and (G and H) 13 days.
Fig. 3 SEM micrographs of copper before (A, C, E and G) and after (B, D, F and H)
removing biofilm and corrosion products immersed in SRB medium for (A and B) 2
hours, (C and D) 1, (E and F) 3 and (G and H) 13 days.
Fig. 4 The wide XPS spectra for surface of copper exposed to (a) sterile and (b)
SRB mediums for 14 days.
Fig. 5 (A) Cu 2p, (B) O 1s and (C) S 2p spectra of copper immersed in sterile
and SRB mediums for 14 days.

Fig. 6 The evolution of Eoc as a function of exposure time for copper in (a) sterile
and (b) SRB mediums, (c) changes of sulfide content (c) as function of time in SRB
medium.
Fig. 7 Nyquist plots of copper in (A) sterile and (B) SRB mediums.
Fig. 8 Three equivalent circuits used for fitting EIS in Fig. 7.
Fig. 9 Changes of Rct as a function of time for copper in (a) sterile and (b) SRB
mediums.
Fig. 10 Current distribution maps of WBE in sterile medium for (A) 1, (B) 3, (C) 5,
(D) 7, (E) 9, (F) 11 and (G) 13 days.
Fig. 11 Current distribution maps of WBE in SRB medium for (A) 1, (B) 3, (C) 5,
(D) 7, (E) 9, (F) 11 and (G) 13 days.
Fig. 12 The evolution of ratios of cathodic and anodic area as a function of time for
WBE in (a) sterile and (b) SRB mediums.

18
19
20
21
22
23
24
25
26
27
28
29
Figure
Highlights
Sulfate-reducing bacteria tolerate copper toxicity and adhere to form biofilm.
Sulfate-reducing bacteria promote localized corrosion of copper.
Corrosion of copper is related to metabolic activity of sulfate-reducing bacteria.
Mechanism of copper corrosion induced by sulfate-reducing bacteria is
illuminated.

30