Electrochemistry Communications 26 (2013) 101–104

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Electrochemistry Communications
journal homepage: www.elsevier.com/locate/elecom

Accelerated anaerobic corrosion of electroactive sulfate-reducing bacteria by

electrochemical impedance spectroscopy and chronoamperometry
Lin Yu a, b, Jizhou Duan a,⁎, Xiangqian Du a, Yanliang Huang a, Baorong Hou a
a
Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China
b
SunRui Marine Environment Engineering Co., Ltd., Qingdao 266101, China

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

Article history: Many microbial species can use cathodes as electron donors for metabolism. This direct electron transfer
Received 17 September 2012 (DET) pathway has rarely been proposed in biocorrosion processes. DET from Q235 carbon steel to the
Received in revised form 3 October 2012 sulfate-reducing bacteria (SRB) Desulfovibrio caledoniensis and its effect on the corrosion behavior of carbon
Accepted 12 October 2012
steel were investigated in the present study. Electroactive SRB biofilm was found to play a key role in the en-
Available online 18 October 2012
noblement of the corrosion potential (Ecorr) and in the acceleration of the corrosion rate, indicating that SRB
Keywords:
mainly affected the cathodic reaction of low carbon steel corrosion. In addition, SRB biofilm obtained
Biocorrosion electrons from carbon steel electrode polarized at −0.74 V. These findings present new evidence for DET
Sulfate-reducing bacteria between SRB biofilm and carbon steels.
Direct electron transfer © 2012 Elsevier B.V. All rights reserved.
Carbon steel

1. Introduction from a carbon electrode polarized at −0.8 V to −1.0 V to catalyze H2

Sulfate-reducing bacteria (SRB) belong to a heterogeneous group of
anaerobic heterotrophic bacteria responsible for numerous cases of
biocorrosion [1,2]. Several theories have been proposed to explain the
role of SRB in biocorrosion. Kühr et al. [3] states that SRB metabolism in-
volves hydrogen consumption to accelerate cathodic reactions. Howev-
er, this theory holds true only if hydrogen removal is the rate-limiting
step. By contrast, hydrogen evolution on steel has been demonstrated
to be an irreversible process [4–6]. The role of bacteria in cathodic depo-
larization is minimized when the metabolic products H2S and FeS can
enhance a cathodic reaction [7–9]. These findings also indicate that
the efficiency of depolarization by a metabolic product alone decreases
with immersion time in the absence of bacteria. Therefore, the study on
biocorrosion caused by SRB reverts to the role of biotic SRB cells and
their enzymes. Silva et al. [10,11] report that hydrogenase induces
an effective hydrogenase-catalyzed cathodic reaction on carbon and
stainless steel surfaces. Dihn et al. [12] propose that SRB can directly
obtain electrons from iron when zero-valent iron serves as the only
electron donor. H2 then becomes a by-product of an imbalance between
the electron donor (zero-valent iron) and acceptor (sulfate). Neverthe-
less, the electron transfer process in biocorrosion has not been well
established, and new pathways have yet to be explored.
Recent studies on microbial electrolysis cells have shown that hydro-
gen evolution reactions occur at the biocathode in the absence of oxygen
and other alternative electron acceptors [13,14]. Geobacter sulfurreducens
is a well-known current-producing microorganism that obtains electrons
⁎ Corresponding author. Tel.: +86 532 82898851; fax: +86 532 82880498.
E-mail address: duanjz@ms.qdio.ac.cn (J. Duan).
evolution [15]. A Desulfitobacterium-enriched culture can catalyze H2 pro-
duction without mediators at cathode potentials lower than −0.9 V [16].
Methanogens are also reported to catalyze methane production from car-
bon dioxide reduction using carbon cathodes as electron donors, which
are polarized at negative potentials lower than −0.8 V [17]. Interestingly,
the polarization potentials mentioned above are similar to the corrosion
potential (Ecorr) of carbon steels in marine environments. Thus, carbon
steels may function as natural electron sources for electroactive biofilms.
A previous study described the corrosive SRB strain Desulfovibrio
caledoniensis isolated from two-year-old inner rust layers of Q235
carbon steel [18]. This SRB strain was also reported to have catalytic
effects on hydrogen formation on graphite electrodes [19]. Therefore,
we further studied its performance on a carbon steel surface, particu-
larly the electron transfer process, using electrochemical techniques.
2. Experimental
2.1. Materials and bacteria
The Q235 carbon steel coupons used in the present study are com-
posed of the following: 0.54 Mn, 0.05 Si, 0.01 S, 0.01 P, 0.16 C, and the
remaining was Fe. Cylindrical metal specimens (diameter = 10 mm)
were used as working electrodes. To create these electrodes, an elec-
trical contact to each sample was provided through a copper wire
connected to the back side of each coupon and mounted in epoxy
resin. The surface was polished using P120 to P800 grit SiC papers
and cleaned with ethanol. The working electrodes were subjected to
ultraviolet light for 30 min before use. Purification and identification
of D. caledoniensis were conducted as previously explained [18]. For

1388-2481/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.elecom.2012.10.022
102 L. Yu et al. / Electrochemistry Communications 26 (2013) 101–104

the enrichment culture, we used a modified Postgate C (PGC) medi- The sampling frequency was 0.02 Hz. The electrochemical reactors were
um containing 0.5 g KH2PO4, 1 g NH4Cl, 0.06 g CaCl2·6H2O, 0.06 g maintained at 30 °C for optimum bacterial growth. All potentials de-
MgSO4·7H2O, 6 mL 70% sodium lactate, 1 g yeast extract, and 0.3 g scribed in the present study were versus SCE. Electrochemical measure-
sodium citrate in 1 L of aged seawater from the Qingdao offshore ments were detected in two assays to analyze the role of SRB biofilm:
area. PGC was de-aerated by high-purity N2 purging for 30 min and Q235 carbon steel specimens in an SRB-inoculated PGC culture and
then autoclaved at 121 °C. Three-day-old bacteria (5% v/v) were Q235 carbon steel specimens in an SRB broth where the SRB cells were
injected into the electrochemical reactors, where the working elec- removed by filtration through a 0.22 μm polycarbonate membrane.
trode was hung for biocorrosion experiments. SO42 − was detected to
study the growth of SRB in the assay using a DX-120 ion chromatog- 3. Results and discussion
raphy system (Dionex Corporation) with a carbonate-selective
anion-exchange column (Dionex AS9-HC). 3.1. Corrosion behavior of Q235 carbon steel in SRB-inoculated culture

2.2. Electrochemical experiment
Electrochemical tests were carried out using three electrode arrange-
ments, with a saturated calomel electrode (SCE) as the reference elec-
trode. The SCE was connected to the reactor through a Luggin capillary
filled with a salt bridge. A platinum foil was used as the counter elec-
trode. Electrochemical impedance spectroscopy (EIS) was conducted
on a steady-state open circuit potential disturbed with an amplitude of
10 mV on Parstat 2273 (Princeton Applied Research). The frequency
ranged was from 105 Hz to 10−2 Hz. Data were collected and fitted
with different equivalent circuits using ZSimpwin software. The fitted
results were judged by χ2-value, and the measured and simulated
data were compared. The Ecorr and chronoamperometry (CA) data of
the working electrodes were detected using CHI 760C (CH Instruments).
Non-destructive electrochemical techniques Ecorr and EIS were
conducted with time under anaerobic conditions favorable for bacteri-
al growth. Meanwhile, SO42 − concentration was detected to study
the growth condition of SRB. Two equivalent circuit models were
used to simulate the measured EIS plot. A double-layer equivalent cir-
cuit Rs(Qdl(Rdl(CcpRct))) was used to estimate Q235 carbon steel in the
medium with SRB metabolic products (without SRB). For Q235 carbon
steel in the SRB-inoculated medium, the contribution of the corrosion
product layer and SRB biofilm was not resolved. Therefore, a single
layer equivalent circuit Rs(QbfRct) was used. The results of EIS analysis
are shown in Fig. 1b and d, where the symbols represent the experi-
mental EIS data and the lines represent their fitted data.
Ecorr and Rct as functions of immersion time in SRB-inoculated culture
are shown in Fig. 1a (Rct was calculated from Fig. 1b). Three-day-old SRB

a c 30000
0.016 15000 -0.64
-0.57 E corr SO 2-
4 Rct Ecorr 27000
13500 Rct
-0.60 0.014 -0.66 24000
Ecorr vs.SCE/V
Ecorr vs.SCE/ V

12000 21000

2
Rct/Ωcm 2

-0.63 -0.68

Rct / cm
0.012
so 42-/mM

10500 18000
-0.66 -0.70
0.010 9000 15000
-0.69 Inoculation
7500 -0.72 12000
0.008
-0.72 9000
6000
nd -0.74
1 st 2 3 rd stage
-0.75 0.006 4500 6000
0 2 4 6 8 10 -1 0 1 2 3 4 5 6
t/d t/ d

b d 10000
0d
0.5d
6000 1d
2d
1.5d
3d 8000
5000 2.5d
4d
3.5d
4d
4000
Zim/ohm cm2

6000 5.5d
Zim/ohm cm2

3000
1500 6d
1200 8d 4000
2000
900 10d
600
1000
300 2000
0
0
0 100 200 300 400

-1000 0
0 2000 4000 6000 8000 10000 0 3000 6000 9000 12000 15000 18000
Zre/ohm cm2 Zre/ohm cm 2

Fig. 1. EIS and Ecorr against immersion time in the presence and absence of SRB. (a) Ecorr, Rct, and SO42 − versus immersion time in the presence of SRB. (b) Nyquist plot for Q235
carbon steel in the medium with SRB. The equivalent circuit model is Rs(Qbf Rct), where Qbf and Rct represent the capacitance and resistance of the SRB biofilms, respectively.
(c) Ecorr and Rct versus immersion time in the medium with SRB metabolic products. (d) Nyquist plot for Q235 carbon steel in the medium with SRB metabolic products. The equiv-
alent circuit model is Rs(Qdl(Rdl(CcpRct))), where Qdl and Rdl represent the capacitance and resistance of the double layer at the electrode surface, respectively, and Ccp and Rct
represent the capacitance and resistance of the corrosion products (e.g., ferrous sulfide layers), respectively.
 L. Yu et al. / Electrochemistry Communications 26 (2013) 101–104
broth (15 mL) was inoculated to 500 mL sterilized PGC culture when
Ecorr became stable (black arrow). The time course was divided into
three stages after inoculation based on the changes in SO42−. During the
first stage, SO42− concentration remained stable, indicating that SRB did
not start to grow. Therefore, the inoculated-SRB metabolic product
imported in the assay by inoculation induced changes in Ecorr and Rct.
Ecorr dropped sharply, and Rct decreased after inoculation. Rct increased
as Ecorr shifted subsequently toward more positive values. During the sec-
ond stage, SRB were in the logarithmic phase because SO42− sharply
decreased, where ΔEcorr =140 mV±18 mV (average and standard devi-
ation from three reproducible experiment results), and Rct significantly
decreased meanwhile. During the third stage, the concentration of
SO42− remained unchanged, which indicated that SRB were in the declin-
ing phase, and Ecorr finally remained stable at approximately −0.62 V. Rct
increased as a function of immersion time. Hence, the activity of SRB
determined the corrosion rate of Q235 carbon steel.
During this process, using most probable number method, we deter-
mined the bacterial count to be 4 × 103, 6 × 107, and 7 × 102 cells/mL at
the time of inoculation, logarithmic phase, and at the end of the exper-
iments, respectively.
The same amount of SRB broth filtered through a 0.22 μm polycar-
bonate membrane was injected to the reactor to analyze the effect of
SRB metabolic products on the corrosion rate of Q235 carbon steel. Ecorr
and Rct as functions of immersion time are shown in Fig. 1c. Rct was cal-
culated from the Nyquist plot (Fig. 1d). Ecorr decreased immediately and
Rct decreased after injection (first arrow). Another injection of the meta-
bolic product also induced a rapid decrease in Ecorr (second black arrow).
Thereafter, Ecorr increased from − 0.73 V (3 d) to − 0.64 V (5.5 d);
Rct also increased. The sharp decrease in Ecorr after inoculation may
be attributed to S 2– in SRB metabolic products. It is likely that the
addition of S 2– may have accelerated the anodic reaction of Q235
carbon steel corrosion as follows:
2þ 2−
Fe þS →FeS ð1Þ
Positively charged Fe 2+ ion left the Q235 carbon steel electrode sur-
face by forming FeS or rust, whereas the cathodic reaction (H+ depolar-
ization) remained stable, which caused the accumulation of electrons
on the electrode surface. This reaction decreased Ecorr according to the
mixed potential theory.
Comparison of the Rct and Ecorr data obtained in the presence and
absence of SRB revealed two main differences. First, the addition of
SRB metabolic products caused a decrease in Ecorr (Fig. 1b), whereas
Ecorr continuously increased during the SRB logarithmic growth
phase when the Q235 carbon steel electrode was exposed to a high
amount of metabolic products. Second, the Rct of Q235 carbon steel
increased with increasing Ecorr (3 d to 6 d) in SRB metabolic products
(Fig. 1c), whereas Rct decreased with increasing Ecorr (3 d to 5 d) in
the presence of active SRB cells (Fig. 1a). These results suggested
that SRB played key roles in the ennoblement of Ecorr and in the accel-
eration of the corrosion rate. Furthermore, they indicated that SRB
mainly catalyzed the cathodic reactions of corrosion. Thus, SRB accel-
erated the consumption of accumulative electrons on the Q235
carbon steel electrode surface.
3.2. Electron transfer process between D. caledoniensis biofilm and the
cathodic-polarized Q235 carbon steel
The Q235 carbon steel electrode was polarized at −0.74 V (where
Ecorr began to increase (Fig. 1a)) to identify whether SRB biofilm can
obtain electrons from the carbon steel surface. Electron transfer can be
observed in the i–t curve (Fig. 2). SRB cells were injected into the assay
when the polarization current became stable (arrow 1). Polarization cur-
rent increased after approximately 0.5 d of immersion, reached −10.7 μA
at approximately 3 d, and then decreased. Current production increased
almost immediately when the depleted medium was replaced with
103
-2 Current production
-3 2
-4
-5
Current/mA
-6
1
-7
-8
3
-9
-10
-11
-12
-13
0 1 2 3 4 5 6 7 8 9 10 11
t/d
Fig. 2. Current production when Q235 carbon steel was polarized at −0.74 V in the
medium with SRB (1: inoculation; 2 and 3: medium replacement with fresh PGC).
fresh PGC at 5.5 d (arrow 2). This finding indicated that the decrease
in current may be attributed to the depletion of the organic carbon
source. Additionally, the formation of SRB biofilm likely increased the
efficiency of electron transfer because current production required a
shorter incubation time compared with initial inoculation. Furthermore,
the D. caledoniensis cells that were attached to the electrode, and not
the planktonic cells, were primarily responsible for current production.
The medium that surrounded the biofilm-coated electrodes was
removed and replaced with fresh anaerobic PGC medium when a sta-
ble oxidation current was observed (arrow 3). Polarization current
decreased slightly and then increased sharply. This result indicated
that the electron transfer process did not require any soluble
electroactive element mediator and that SRB biofilm likely played a
key role in the electron transfer process. These findings are consistent
with the characteristics of a biocathode in anaerobic environments.
Q235 carbon steel had a negative Ecorr value in aqueous environment
and acted as an electron donor for SRB.
Thus, SRB biofilms can obtain electrons directly from carbon steel.
However, the detailed electron transfer pathway from the carbon
steel to SRB should be further studied.
4. Conclusion
The effect of SRB biofilm on Q235 carbon steel was experimentally
demonstrated in this study. In the non-destructive electrochemical
study, SRB biofilm rather than their metabolic product were directly
related with the ennoblement of Ecorr and the acceleration of the cor-
rosion rate. SRB biofilm mainly influenced the cathodic process of car-
bon steel corrosion. Based on the CA results, a stable cathodic current
was produced after the electron transfer from the − 0.74 V polarized
electrode to the grown SRB biofilm. Our results indicated that SRB
biofilms can obtain electrons directly from Q235 carbon steel to accel-
erate the biocorrosion rate of carbon steel.
Acknowledgments
The present work was supported by the Chinese Academy of Sciences
key knowledge innovation project (Grant No. KZCX2-EW-205) and the
National Natural Science Foundation of China (Grant Nos. 40676048
and 40976046).
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