Corrosion Science 51 (2009) 1372–1385

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Corrosion Science
journal homepage: www.elsevier.com/locate/corsci

AFM study of microbial colonization and its deleterious effect on 304 stainless
steel by Pseudomonas NCIMB 2021 and Desulfovibrio desulfuricans
in simulated seawater
S.J. Yuan a, S.O. Pehkonen b,*
a
Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge 119260, Singapore
b
CEWIC (Center of Expertise in the Water Industry Cluster), Thule Institute, University of Oulu, P.O. Box 7300, FI-90014 Oulu, Finland

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

Article history: The biofilm colonization dynamics of Pseudomonas NCIMB 2021 and Desulfovibrio desulfuricans (ATCC
Received 24 November 2008 27774) on 304 stainless steels (304 SS) was evaluated using atomic force microscopy (AFM) in simulated
Accepted 18 March 2009 seawater-based media under aerobic and anaerobic conditions. Results showed that the biofilm formed
Available online 16 April 2009
on the coupon surface by the two strains of bacteria increased in the coverage, heterogeneity and thick-
ness with exposure time, thus resulting in the deterioration of the steel substratum underneath the bio-
Keywords: film in the form of pitting corrosion. The depth of pits induced by D. desulfuricans was larger than that by
A. Stainless steel
Pseudomonas NCIMB 2021, which was mainly attributed to the enhanced corrosion of 304 SS coupons by
B. AFM
B. Polarization
the biogenic sulfide ions, as revealed by the results of X-ray photoelectron spectroscopy (XPS) and Tafel
B. SEM polarization curves. AFM was also used to determine cell attachment/detachment processes of the Pseu-
C. Microbiological corrosion domonas and D. desulfuricans bacteria on the coupon surface by quantifying the tip–cell interaction forces.
The interactive forces between the tip and the bacterial cell surface were considerably smaller than those
between the tip and the cell–cell interface due to the accumulation of extra-cellular polymeric substances
(EPS) for both strains. Furthermore, the adhesion forces over the Pseudomonas cells were verified to be
more attractive than those of D. desulfuricans due to the former being a slime-producer.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The role of biofilm in the deterioration of steel structures in the

Metal surfaces are rapidly colonized by microorganisms in con-
tact with natural or industrial aquatic environments, giving rise to
a complex and strongly adhering microbial community, termed as
‘‘biofilm” [1]. The biofilm accumulation not only protects microbial
cells from the external environment, but it is also detrimental to
the underlying substratum, thereby causing physical degradation
or biodeterioration of the metal surface [2–4]. This phenomenon
is widely recognized as biocorrosion or microbiologically influ-
enced corrosion (MIC). Type 304 stainless steel, as a low grade
austenitic stainless steel, has been widely recognized to be vulner-
able to MIC as well as and to localized corrosion by chloride and
reduced sulfur compounds [5,6], although it exhibits excellent cor-
rosion resistance in abiotic aquatic environments due to the forma-
tion of a thin, compact and chromium-enriched oxide film [7]. MIC
is a serious problem in the marine environment and many indus-
tries, including power generation, petrochemical, pulp and paper
etc. [8], as approximately 20–30% of all corrosion is associated with
MIC, at a direct cost of $30–50 billion per year [9].
* Corresponding author. Tel.: +358 407697252; fax: +358 85533564.
E-mail address: Simo.Pehkonen@oulu.fi (S.O. Pehkonen).
industrial and marine aquatic environments has been of consider-
able worldwide interest. Thus, the microbial colonization and its
deleterious effect on the metallic substratum has been extensively
investigated to understand the involvement and the contribution
of microorganisms in the corrosion process of metals [10–17].
However, to date there is still uncertainty how large a role biofilm
has in the deterioration of stainless steel and by which of the sev-
eral possible mechanisms does the microbial colonization occur
[11–15]. Conventional microscopy techniques used for characteriz-
ing microbial colonization and assessing MIC, such as SEM, LM and
TEM etc., usually require extensive and time-consuming sample
preparation, which may dramatically affect the natural state of
the cells. Atomic force microscopy (AFM) is a powerful tool that al-
lows for the imaging of biological samples down to the nanometer
scale resolution and under physiological conditions [10], which
will be difficult or otherwise impossible to achieve using electron
or light microscopy. Another advantage of AFM is that it requires
minimum sample preparation and can operate under wet or dry
conditions. The AFM has, therefore, been useful in elucidating cor-
rosion phenomena related to biofilms or microbial colonization on
metal surfaces [11–14]. In addition to capturing topographic
information, a force–distance curve records the variation in the

0010-938X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.corsci.2009.03.037
 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385
interaction forces as the sample approaches the tip, makes contact,
and then retreats from the tip. Such curves provide valuable infor-
mation on the tip–sample interaction, which are useful in better
understanding the formation mechanisms of a biofilm [15,16].
Two categories of bacteria, i.e., aerobic and anaerobic bacteria,
are well-known to aggravate the deterioration of stainless steels
through microbial colonization. Aerobic bacteria, such as Pseudo-
monas and Bacillus sp., have been demonstrated to be detrimental
to the integrity of the passive oxide film, and facilitate the local
depassivation of the protective layer, thus promoting the ingress
of active ions (e.g., Cl) to the metal lattice [17,18]. Aerobic Pseudo-
monas species, most prevalent in industrial water and seawater
environments, are initially recognized be the pioneer colonizer in
the process of biofilm formation, and their primary role is to create
an environment to harbor sulfate-reducing bacteria (SRB). Never-
theless, these strains are subsequently found to be aerobic slime-
formers and commonly grow in a patchy fashion over the metal
surface to create oxygen concentration cells through the exclusion
of oxygen via bacterial respiration [19]. As for anaerobic bacteria,
sulfate-reducing bacteria (SRB) are widely recognized to be the
most destructive microorganisms in MIC, and they are the princi-
pal causative organisms to induce MIC of stainless steel
[3,18,20,21]. SRB are abundant in natural habitats, such as marine
and fresh water sediments or sludges, and play a key role in the
biogeochemical sulfur cycle [22]. They are a group of taxonomi-
cally diverse anaerobic microbes that perform dissimilatory reduc-
tion of sulfur compounds, such as sulfate, sulfite, thiosulfate and
even elemental sulfur to the fully reduced product of sulfide [23].
The purpose of this research herein seeks to gain a better under-
standing on the detrimental effect of aerobic Pseudomonas NCIMB
2021 and anaerobic Desulfovibrio desulfuricans on the passivity of
304 SS surface using atomic force microscopy (AFM). The depth
of pits induced by microbial colonization as a function of exposure
time were quantified via AFM sectional analysis, in conjunction
with the analyses of pitted areas by scanning electron microscopy
and energy dispersive X-ray spectroscopy (SEM–EDX), to assess the
corrosivity of the two bacterial strains to the 304 SS coupons. The
difference in corrosion behaviors of 304 SS coupons under the
influence of Pseudomonas and D. desulfuricans was further evalu-
ated by Tafel polarization curves. Furthermore, the AFM tip–cell
interaction forces over the various sections of a bacterial cell sur-
face and at various interfacial regions after the preliminary forma-
tion of a biofilm are also quantified by recording the force–distance
curves to understand the initial bacterial attachment. For a com-
parison purpose, the 304 SS coupons were also exposed to the ster-
ile culture medium under the same experimental conditions to
serve as controls.
2. Experimental procedures
2.1. Bacterial cell growth conditions
Both culture media were prepared based on a simulated seawa-
ter according to Burkhoder’s formulation B [24]. The medium con-
sists of 23.476 g/l NaCl, 3.917 g/l Na2SO4, 0.192 g/l NaHCO3,
0.664 g/l KCl, 0.096 g/l KBr, 10.61 g/l MgCl26H2O, 1.469 g/l
CaCl26H2O, 0.026 g/l H3BO3, 0.04 g/l SrCl26H2O. Pseudomonas
NCIMB 2021 (from NCIMB, Sussex, UK), a marine aerobic Gram-
negative bacterium, was cultured aerobically at 25 °C in a nutri-
ent-rich simulated seawater-based medium, which was prepared
by adding 3 g of bacteriological peptone and 1.5 of yeast extract
into one liter of the aforementioned simulated seawater. The bac-
terial cultivation and medium preparation have been described in
detail previously.[17] Gram-negative anaerobic D. desulfuricans
(ATCC, No. 27774) bacterium was obtained from the American
1373
Type Culture Collection. The bacteria were cultured under anaero-
bic conditions in a simulated-seawater based Modified Baar’s med-
ium (SSMB medium), which was prepared by adding 0.41 g of
MgSO47H2O, 0.1 g of NH4Cl, 0.1 g of CaSO4, 0.05 g of K2HPO4,
0.5 g of tri-sodium citrate, 3.5 g of sodium lactate and 1 g of yeast
extract into one liter of the simulated seawater. Details on the
medium preparation and bacterial cultivation have been described
previously [25]. The D. desulfuricans bacterium was grown at 25 °C
in an anaerobic chamber (Bactron IV Shel lab anaerobic environ-
mental chamber, Sheldon Manufacturing, Inc., Cornelius, OR,
USA), which was maintained with an atmosphere containing 5%
H2, 5% CO2 and 90% N2.
2.2. Coupon preparation
304 stainless steel, with the nominal composition of 71.376% Fe,
8.18% Ni, 0.053% C, 18.08% Cr, 0.06% Cu, 1.68% Mn, 0.05% Mo,
0.047% N, 0.037% P, 0.007% S and 0.43% Si, was purchased from
the Metal Samples Company (Alabama, USA). The rectangular cou-
pons with a dimension of 10  10  3 mm were ground sequen-
tially up to 1200 grit SiC paper to obtain a smooth surface. Disk-
shaped specimens with a diameter of 15 mm and thickness of
3 mm were used for electrochemical measurements. These cou-
pons were further polished to a mirror-finish surface with an alu-
mina suspension (about 0.3 lm in particle size). The polished
coupons were rinsed with deionized (DI) water thrice, followed
by degreasing with acetone, then sterilized by immersion in 70%
ethanol for eight hours, and dried aseptically in a laminar flow
hood.
2.3. Microbial colonization of steel coupons in the media containing
Pseudomonas and D. desulfuricans bacteria
The development of biofilms on the 304 SS coupon surface in
the Pseudomonas inoculated medium was similar to the procedures
described previously [17]. Briefly, a 1 ml aliquot of the 3-day old
Pseudomonas culture was introduced into 500 ml of the nutrient-
rich medium in conical flasks. The conical flask was subsequently
placed on a rotary incubator shaker at 150 rpm on 25 °C. After
the optical density (OD) value was close to 1.0, the prepared cou-
pons hung on the Nylon string were aseptically introduced into
the inoculated medium. All flasks were capped with Bug-stoppers
(Whatman, USA) to prevent contamination. To maintain the bacte-
rial density near the steady-state growth phase throughout the
experiment, a semi-continuous mode of a Pseudomonas culture
growth was employed, i.e.: 75% medium were drained and re-
placed with an equal amount of a fresh sterile medium once a
week.
For the inoculation of D. deuslufricians in the SSMB medium, a
1 ml aliquot of inoculums of D. desulfuricans was introduced into
500 ml of the SSMB medium in Duran bottles (1.0 l) and incubated
at 25 °C in the anaerobic chamber. The viable cell number was
determined using the most probable number (MPN) method
(3-tube) as described in the literature [26]. At the same time, the
concentration of the biogenic sulfide ions was detected with the
polarographic method by a Model 757 Voltammeter Computrace
(Metrohm, Switzerland) using procedures described previously
[25]. The 304 SS coupons hung on the Nylon strings were asepti-
cally introduced into the D. desulfuricans inoculated medium for
the assay of microbial colonization and corrosion behavior. After
the prescribed exposure times of 3, 7, 14, 28 and 42 days, separate
coupons were removed from the inoculated medium for AFM.
For biofilm imaging, the coupons were lightly rinsed in a sterile
phosphate buffered saline (PBS) solution to remove the dead and
loosely attached bacteria, left to air dry for 5 min as described in
previous studies.[11,13] To reveal the extent of surface biodeterio-
1374 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385
ration, biofilms were detached from the coupon surface by ultra-
sonication for 5 min using procedures described previously
[11,17]. The surfaces after the removal of biofilms were finally
rinsed with deionized water, and dried in the air.
2.4. AFM operation
A Nanoscope IIIa AFM (Digital Instruments, Santa Barbara, CA,
USA) operating in a contact mode in air was used to capture the
images of biofilms and pits on the coupon surface. The nanoprobe
cantilevers were made of silicon nitride (Si3N4) with a spring con-
stant of k = 0.06 N/m (Digital Instruments, USA). The radius of cur-
vature of the AFM tip is 50 nm. The scan rate was varied between
1.0 and 2.0 Hz depending on the image size. The relative humidity
was 50–60% and no capillary forces were observed during the AFM
operation. During the AFM measurement, a 100  100 lm image
was first captured in order to assess the exact position and the nat-
ure of bacterial cells or biofilms, with further scans being used to
zoom onto any interesting features.
The Digital Nanoscope software (Version 5.30) was used to
analyze the topographic images of the coupon surface, as well
as the force–distance measurements over the coupon surface.
The arithmetic mean of the surface roughness (Ra) was deter-
mined by AFM to show the roughness profile of the 304 SS sur-
face after exposure to various media. The force–distance
measurements were performed at the scanning rate of 0.5 Hz
and a scan size of 0 nm (i.e., a point measurement) to avoid
the effect of the lateral force. Thus, only the vertical adhesion
forces between the AFM tip and the cell surface were obtained.
Three force measurements were made at each location, and
three locations were chosen on the colonized bacterial cells,
thereby the average value of the force measurements was taken
as the adhesion force between the tip and the bacterial cells. As
described previously [14,15], a typical force–distance curve be-
tween the tip and the cell surface was commonly displayed as
the tip deflection (nm) versus the piezo position (nm). The inter-
active force between the tip and the surface can be calculated
from the deflection distance of cantilever and the cantilever
spring constant on the basis of Hooke’s law:
f ¼ k  Dl
where f is force (nN), k is the spring constant of cantilever, which
was equal to 0.06 N/m in this study, and Dl is the deflection dis-
tance (nm). The reference zero point of deflection is defined as
the set point when the tip is far away from the surface. A negative
deflection denotes an attractive force, whereas a positive deflection
corresponds to a repulsive force.
2.5. Surface analysis by SEM–EDX and XPS
The morphology and corrosion features for the corroded 304 SS
coupon surface were examined by SEM and EDX (JSM-5600, JEOL
Co., Tokyo, Japan). As described previously [25], the biofilm and
the corrosion product were removed from the coupon surface with
sterilized cotton swabs immediately after the coupons were re-
moved from the medium, followed by rinsing with sterilized DI
water thrice, dried with a stream of pure N2, and finally stored in
a vacuum desiccator prior to SEM imaging. The EDX analyses were
performed on the pitted areas to determine the implication of ac-
tive anions in the initiation of pitting corrosion. To further ascer-
tain the formation of sulfide film on the coupon surface, XPS
study was carried out on the 304 SS coupon surface after exposed
to the D. desulfuricans inoculated SSMB medium for 28 days. The
detailed procedures of the coupon preparation and XPS measure-
ments have been described previously [17,25].
2.6. Tafel polarization curves
A conventional three-electrode glass corrosion cell with a
capacity of 500 mL was used to obtain the Tafel polarization
curves. After 28 days of exposure in the various media, the 304
SS coupons were mounted on a PVDF holder, leaving a circular area
of 0.785 cm2, and fixed at the bottom of the corrosion cell to serve
as the working electrode. An Ag/AgCl electrode was used as the ref-
erence electrode, and a platinum rod was used as the counter elec-
trode. The Tafel polarization curves were recorded at a scan rate of
2 mV/s within the range of 250 to 250 mV versus the open-circuit
potential (OCP) using an Autolab PGSTAT30 (Ecochemie Co., The
Netherlands), controlled by GPES software. The Tafel polarization
curves were further analyzed by extrapolating the linear portions
of cathodic and anodic branches to their interactions to determine
the Tafel slopes (ba and bc), the corrosion potentials (Ecorr), the cor-
rosion current densities (icorr) and the corrosion rates.
3. Results and discussion
3.1. Images of a single bacterial cell on the steel surface
Fig. 1a illustrates a typical image of a Pseudomonas NCIMB 2021
cell on the 304 SS surface after exposure to the Pseudomonas inoc-
ulated medium for 7 days. The dimensions of the cell are 1.78 
0.84  0.16 lm (length  width  depth). The rod shape of the
bacteria is also clearly visible. Fig. 1b shows a typical image of an
anaerobic D. desulfuricans cell colonized on the steel coupon sur-
face after exposure to the D. desulfuricans inoculated SSMB med-
ium for 7 days. The dimensions of the cell are 3.05  0.73 
0.35 lm (length  width  depth). The curved rod shape of the
bacterial cell as well as corrosion products is also observed. The
two images of the bacterial cells also reveal that EPS are distributed
around the cell surfaces, especially at the cell-substratum periph-
ery. EPS secreted by bacterial cells usually facilitates an irreversible
cell attachment, thus leading to microbial colonization on the cou-
pon surface [1].
3.2. The biofilm colonization on the steel coupon surface
ð1Þ Fig. 2 illustrates the development of the biofilm of the Pseudomo-
nas bacteria on the steel coupon surface as a function of exposure
time. Each group of AFM images includes the two-dimensional and
three-dimensional topographical images. This result demonstrates
that the biofilm colonization is a dynamic and continuous process.
Some distinguishable bacterial cells, either individually or micro-
colonies, are distributed on the coupon surface after three days of
exposure (Fig. 2a), indicative of the initial attachment of the Pseudo-
monas bacterium on the coupon surface. Initial bacterial attachment
to a solid surface is a crucial step in biofilm formation [2]. Thereafter,
bacterial cells begin to cluster due to the binding force at the inter-
face, which results from the increased accumulation of EPS
(Fig. 2b). As a result, a patchy biofilm or large-size bacterial clusters
are observed on the coupon surface after 14 days of exposure
(Fig. 2c). EPS, which is known to play an important role in biofilm
development, can be observed to be associated with cells of different
growth stages. A thick and heterogeneous biofilm is formed on the
steel coupon surface after 28 days of exposure (Fig. 2d), and the Pseu-
domonas cells are encased in EPS. At the same time, the values of the
arithmetic mean roughness, Ra, increase noticeably from initially
48 nm to 150 nm with exposure time, indicative of the increase
in heterogeneity of the biofilm on the steel coupon surface
(Fig. 2a–d). The patchiness and heterogeneity of the biofilm are re-
ported to be very important in the initiation of localized corrosion
and the acceleration of corrosion rates, since they can give rise to lo-
cal differences in metabolic products, pH, or dissolved oxygen (i.e.,
 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385
Fig. 1. AFM images of (a) a single Pseudomonas NCIMB 2021 cell and (b) a D. desulfuricans cell on the steel coupon surface after 7 days of exposure.
differential aeration cells), all of which can generate active electro-
chemical corrosion cells [11,17,27,28]. As shown in Fig. 2e, the aging
biofilm formed on the coupon surface exhibits a high level of heter-
ogeneity after 42 days of exposure with the Ra value as high as
246 nm. Patches of cell aggregates (not monolayers) were inter-
spersed throughout an EPS matrix that varied in its density, thus cre-
ating open areas, where water channels were formed (Fig. 2e). It has
been demonstrated that the volume of bacterial biofilms is typically
occupied more by the EPS matrix (75–95%) than by bacterial cells (5–
25%), and that these cells may be concentrated either in the lower or
the upper regions of the biofilm [28]. As revealed by the three-
dimensional AFM images (Fig. 2b–e), the current results further con-
firm the previous findings that biofilms are composed of clusters of
microbial cells, EPS and interstitial voids [11].
Under anaerobic conditions, a layer of biofilm is also formed on
the steel coupon surface within days in the SSMB medium sustain-
ing the SRB culture. AFM images (including two-dimensional and
three dimensional images) of the steel coupons with a biofilm
formed by D. desulfuricans over different exposure periods are
shown in Fig. 3. The results reveal that the biofilm formation of
D. desulfuricans on the steel coupon surface undergoes a similar
clustering process to the aforementioned biofilm colonization
dynamics of the Pseudomonas bacteria. The Ra values of the 304
SS coupon surface also increase markedly with prolonging expo-
sure time, and reach as high as 246 nm after 42 days of exposure
in the D. desulfuricans inoculated SSMB medium, indicative of a
high heterogeneous surface due to the accumulation of bacterial
cells and corrosion products. For sulfate-reducing bacteria, it has
1375
been widely recognized that the accumulation of bacterial cells
and corrosion products (mainly iron sulfide) on the coupon surface
results in a thick and porous layer, and that the porous surface
layer acts as a barrier to diffusion, leading to the generation of gra-
dients of pH, sulfide and chloride, and thereby inducing the initia-
tion of pitting corrosion [11,13–15,20,27–29]. As shown in Fig. 3d
and e, a thick and porous layer can also be observed on the 304 SS
coupon surface after 28 and 42 days of exposure in the D. desulfu-
ricans inoculated SSMB medium.
To further elucidate the influence of bacterial colonization on
the surface features (such as roughness and heterogeneity), AFM
images were captured on the 304 SS coupon surfaces after different
exposure periods in the sterile culture medium. Fig. 4 shows the
representative AFM images of the 304 SS coupon surface in the
sterile nutrient-rich simulated seawater-based medium under aer-
obic conditions. The Ra values of the control coupon surface remain
small throughout exposure periods (Fig. 4), indicative of a uniform
and homogeneous surface, albeit with some scratches introduced
during polishing. On the other hand, a slight increase in Ra values
with exposure time probably has attributed, at least in part, to
the spontaneous adsorption of organic macromolecules (such as
proteins) from the bulk solution to form the conditioning layers.
The formation of conditioning layers on the solid surface is a ubiq-
uitous phenomenon in aquatic environments and is recognized to
be the initial step of biofilm formation [19]. Similar results can be
obtained on the steel coupon surface after various exposure peri-
ods in the sterile SSMB medium under anaerobic conditions (not
shown).
1376 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385

Fig. 2. Representative two-dimensional and three-dimensional topographical AFM images of 304 SS coupons with the Pseudomonas biofilm after (a) 3 days, (b) 7 days, (c)
14 days, (d) 28 days and (e) 42 days of exposure in the Pseudomonas inoculated medium.
 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385 1377

Fig. 3. Representative two-dimensional and three-dimensional topographical AFM images of 304 SS coupons with D. desulfuricans biofilm after (a) 3 days, (b) 7 days, (c)
14 days, (d) 28 days and (e) 42 days of exposure in the D. desulfuricans inoculated SSMB medium.
1378 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385
Fig. 4. Representative two-dimensional and three-dimensional topographical AFM images of the 304 SS coupons after (a) 7 days, (b) 28 days and (c) 42 days of exposure in
the sterile nutrient-rich simulated seawater-based medium under aerobic conditions.
3.3. Quantitative determination of the steel surface deterioration
induced by the Pseudomonas and D. desulfuricans bacteria
The deterioration of metallic materials induced by the activities
of microorganisms is usually shown as localized corrosion of metal
surfaces [30]. The majority of biocorrosion is widely recognized to
be of the pitting-type, and to be the root cause of many corrosion
failures. The microbes at the metal/biofilm interface create condi-
tions, in which incipient pitting leads to localized corrosion driven
principally by microbiological activity [31].
The deleterious impact of the bacterial colonization was as-
sessed in this study by profiling the pits on the coupon surface
after the removal of biofilms and deposits. Fig. 5 shows the AFM
images of the 304 SS coupons upon the removal of deposits and
biofilms after 28 days of exposure in the sterile nutrient-rich, the
Pseudomonas inoculated and D. desulfuricans inoculated media.
Each group of images includes the two-dimensional and three-
dimensional topographical AFM images, as well as the sectional
analyses of the pitted areas. As shown in Fig. 5a, no localized cor-
rosion can be observed on the control steel coupon surface after
28 days of exposure, indicative of the stability of passive film in
the sterile nutrient-rich medium under aerobic conditions. Some
distinguishable micropits are spotted over the Pseudomonas-colo-
nized coupons as well as the D. desulfuricans-colonized coupon,
indicating that both strains can trigger the breakdown of passive
films of stainless steel, in the form of micro-pitting corrosion
(Fig. 5b and c). Nevertheless, the degree of the micro-pitting in-
duced by D. desulfuricans seems much greater in comparison with
that by the Pseudomonas bacteria, as revealed by the AFM sectional
analyses of the pitted areas (Fig. 5b and c).
AFM has been widely recognized to have a higher resolution
and accuracy in the vertical dimension compared to other micro-
scopic techniques, and this offers an opportunity to quantify local-
ized corrosion [13,14]. The depth and the width of a pit are
therefore obtained by the sectional analysis using the standard
AFM software. Fig. 6 illustrates the representative AFM images of
 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385 1379

Fig. 5. Representative two-dimensional topographical, sectional analysis and three-dimensional AFM images of the 304 SS coupons after 28 days of exposure to the (a) sterile
nutrient-rich medium under aerobic conditions, and the 304 coupons with the biofilm removal after 28 days of exposure to the (b) Pseudomonas and (c) D. desulfuricans
inoculated media.

the pits on the corroded coupon surface after 21 and 42 days of 3.4. Surface analysis of the bacterial-colonized surface by SEM–EDX
exposure to the Pseudomonas and D. desulfuricans inoculated med- and XPS
ia. The two-dimensional images together with the sectional analy-
sis graphs are shown in each group of AFM images. As the To reveal the different activities of the two strains participating
corrosion proceeds under the effect of the two strains, the width in the steel deterioration, SEM images coupled with EDX spectra
and the depth of pits propagate with time, thus leading to deeper were captured on the pitted areas of the steel coupons induced
and wider pits on both the Pseudomonas-colonized and the D. by the Pseudomonas and D. desulfuricans, and are shown as
desulfuricans-colonized coupons. Through the sectional analysis Fig. 7a and b, respectively. The EDX spectrum in Fig. 7a reveals that
of six randomly-selected areas on the corroded coupons, the mean the ingress of active chloride anions (Cl) underneath the biofilms
depth of the pits induced by the two strains is shown in Table 1. promotes the initiation and the propagation of pitting corrosion
Herein, two distinguishable features are highlighted: (i) the depth due to the pH decrease at the anodic sites via the formation of
of pits induced by both strains increase noticeably with exposure FeCl3 [17,32]. The anodic reactions to describe the influence of
time, and reaches as high as 600 nm for the Pseudomonas-colo- Cl ions on the steel passive film are shown as follows:
nized coupons and 1000 nm for the D. desulfuricans-colonized
Fe ! Fe2þ þ 2e ð2Þ
coupons after 42 days of exposure, (ii) the depth of pits induced
2þ 
by D. desulfuricans is much larger than that caused by the Pseudo- Fe þ 2H2 O þ 2Cl ! FeðOHÞ2 þ 2HCl ð3Þ

monas bacteria, and this difference is greater with increasing expo- FeðOHÞ2 þ 3Cl ! FeCl3 þ 2OH þ e ð4Þ
sure time, indicating that anaerobic D. desulfuricans bacteria are FeCl3 þ 3H2 O ! FeðOHÞ3 þ 3HCl ð5Þ
more corrosive to the passivity of 304 SS surface than aerobic Pseu-
domonas NCIMB bacteria. The depth of pits induced by the two The Cl ions interact with the hydrous oxide layer to replace OH


strains seems to increase linearly with exposure time, which is in inside the oxide film to form a soluble FeCl3 product, and FeCl3 is
good agreement with previous studies [11,17]. further hydrolyzed to produce a very porous precipitate Fe(OH)3,
1380 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385

Fig. 6. Representative AFM images of the presence of pits on the corroded surface of 304 SS coupons after 21 days and 42 days of exposure in the (a and c) Pseudomonas and (b
and d) D. desulfuricans inoculated media. Each group of images includes two-dimensional topographical images of the corroded surface and sectional analysis determining the
depth of pits.
 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385 1381

Table 1
The mean depth of pits on the 304 SS coupon surface induced by the aerobic Pseudomonas NCIMB 2021 bacteria and anaerobic D. desulfuricans bacteria (mean ± SD*, nm).

Exposure time (days) 14 21 28 35 42

Pseudomonas 144.24 ± 28.67 304.78 ± 23.68 397.02 ± 48.40 520.46 ± 57.98 606.41 ± 90.52
D. desulfuricans 322.54 ± 80.64 512.64 ± 76.89 737.31 ± 147.9 887.89 ± 253.7 1123.4 ± 449.4
*
SD denotes standard deviation.

Fig. 7. SEM images and EDX spectra of representative pits after the removal of biofilms on the 304 SS coupon surface after 28 days of exposure in the (a) Pseudomonas and (b)
D. desulfuricans inoculated media.

together with the localized pH reductions at the initiation site [32]. study, similar to that reported previously [25]. It is generally agreed
The result is a self-propagating or autocatalytic mechanism of pit that the Cl ions can catalyze the dissolution by sulfide inclusions,
growth [32]. However, it has been reported by Hakkarainen [33] thus cause metastable pits on stainless steel [34]. The anodic disso-
that there are two other prerequisites to fulfill for the initiation of lution by the sulfide ions (S2) in the marine environments pro-
pitting corrosion besides the active Cl ions, one is the existence ceeds as the following reactions [35]:
of potential difference on the substrate surfaces, the other is that
the reaction temperature must be beyond a critical temperature. Fe þ H2 S ! FeSHads þ Hþ ð6Þ
The bacterial colonization of Pseudomonas gives rise to a potential FeSHads ! FeSHþads þ 2e ð7Þ
difference between the area under the biofilm, which serves as
the anodic sites, and the surrounding regions, which act as the FeSHþads ! FeS1x þ xHS þ ð1  xÞHþ ð8Þ
 þ  
cathodic sites. The combined effect of active Cl ions and the colo- Fe þ 2Cl þ H2 O ! ½FeðOHÞ þ Cl  þ HCl þ 2e ð9Þ
nized Pseudomonas bacteria can therefore trigger the partial loss of FeðOHÞþ þ HS ! FeS þ 2H2 O ð10Þ
passivity of the 304 SS coupons and the initiation of pitting corro-
sion. On the other hand, the EDX spectrum in Fig. 7b indicates that To further confirm the influence of biogenic sulfide ions on the
the synergistic interaction of bacteria cells, active biogenic sulfide passivity of surface film, XPS measurement was performed on the
and chloride anions are responsible for the initiation of micro-pit- 304 SS coupon surface after 28 days of exposure in the D. desulfu-
ting corrosion on the steel coupon surface in the D. desulfuricans rican inoculated SSMB medium, and the results are shown in Fig. 8.
inoculated medium. The concentration of biogenic sulfide ions The curve-fitted S 2p core-level spectrum is significantly compli-
was in the range of 30 and 40 mg L1 (about 0.94–1.25 mM) in this cated by contributions from three doublets with 2p3/2 at the bind-
1382 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385

the passive film formed on the control steel surface consists mainly
a S 2p of FeO, Fe2O3, Cr2O3, Cr (OH)3 and FeOOH after exposed to the ster-
ile nutrient-rich simulated seawater-based medium under aerobic
S2- conditions [17], the sulfidation of passive film by biogenic sulfide
2-
Organic S/SO4 ions results in a substantial loss of passivity of the steel coupons.
S22- Hence, the higher corrosivity of D. desulfuricans to 304 SS than that
SO3 2- of the Pseudomonas bacteria is mainly associated with the biogenic
Sn 2- S2 O 32- sulfide anions.
S0
3.5. Tafel polarization curves

To assess the corrosivity of the Pseudomonas and D. desulfuricans

bacteria in a more quantitative manner, Tafel polarization curves
were recorded on 304 SS coupons after 28 days of exposure in
158 160 162 164 166 168 170 172 the sterile nutrient-rich, the Pseudomonas inoculated and the D.
desulfuricans inoculated media, and are shown in Fig. 9. The analyt-
b Fe 2p
ical parameters are summarized in Table 3. In comparison with
Intensity (Arb. units)

FeS
FeS1+x FeOOH that of the control steel coupons, the corrosion potential, Ecorr, of
Fe 2+ the 304 SS coupons undergoes a negative shift of 30 and
FeS2 370 mV, respectively, by the effect of the Pseudomonas and D.
desulfuricans bacteria (Table 3). This phenomenon is mainly attrib-
uted to the enhanced anodic dissolution in term of the mixed po-
tential theory [38]. Furthermore, the Ecorr value of the D.
desulfuricans-colonized steel coupon is much more negative than
that of the Pseudomonas-colonized steel coupon, indicating that
the anodic dissolution process of the 304 SS coupons is substan-
tially accelerated by the synergistic effect of D. desulfuricans bacte-
rial cells, biogenic sulfide ions and active chloride ions. The point is
704 706 708 710 712 714 716 718 confirmed by the larger value of the corrosion current densities,
icorr, for the steel coupon in the D. desulfuricans inoculated SSMB
c Cr 2p medium, which reaches as high as 95 lA cm2 after 28 days of
exposure. However, the icorr value for the steel coupon in the Pseu-
Cr2S 3 domonas inoculated medium is 3.5 lA cm2 after 28 days of
Cr2O 3 exposure, which is only 2.5-fold larger than that of the control steel
coupons. The higher corrosivity of D. desulfuricans to the 304 SS
coupons than that of Pseudomonas is thus ascertained.

3.6. Quantification of interactive forces between the tip and bacterial

cells
Satellite
During the biofilm formation, the initial bacterial attachment is
a crucial step, which is governed by electrostatic attraction and
570 575 580 585 590
Binding Energy (eV)
Sterile medium
Fig. 8. High-resolution (a) S 2p, (b) Fe 2p and (c) Cr 2p core-level spectra of the Pseudomonas
sulfide film formed on the 304 SS coupon surface after 28 days of exposure in the D.
desulfuricans inoculated SSMB medium. 0.0 D. desulfuricans

ing energies (BEs) of 160.5, 162.1 and 163.3 eV, attributable to

E/V (vs. Ag/AgCl)

monosulfide (S2), disulfide (S22) and polysulfide (Sn2), respec- -0.2

tively, and four singlet peak components at the BEs of 165.6,
167.0, 168.3 and 169.7 eV, attributable to S0, SO32, organic-sul-
fide/SO42 and S2O32, respectively, [36] (Fig. 8a). The monosulfide, -0.4
S2, species originate mainly from the iron sulfides and chromium
sulfides. The disulfides and polysulfide species were previously
reported by Herbert et al. [37] at the surface of iron monosulfide -0.6
precipitated in the presence of sulfate-reducing bacteria. Corrobo-
rating evidence can be sought in the Fe 2p and Cr 2p for the sulfide
film formation on the steel coupon surface. A significant amount of
-0.8
iron sulfides is formed during the exposure to SRB, including FeS,
1E-9 1E-8 1E-7 1E-6 1E-5 1E-4 1E-3
FeS2 and FeS1-x (Fig. 8b). At the same time, a predominant peak
component at the BE of 575 eV, attributable to Cr2S3 [36], can be I/(A· cm-2 )
observed on the curve-fitted Cr 2p core-level spectrum, accompa-
Fig. 9. Tafel plots of the 304 SS coupons in the sterile nutrient-rich simulated
nied by a minor peak component corresponding to Cr2O3 at the seawater-based medium, the Pseudomonas inoculated medium, and the D. desul-
BE of 576.2 eV [36]. In comparison with the previous findings that furicans inoculated SSMB medium for 28 days.
 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385
other physical forces, e.g., van der Waals forces and hydrophobic
interactions [39]. AFM force–distance curves have been extensively
used to extract the tip-to-sample adhesion data, which offers an
opportunity to measure the irreversible attachment of cells to con-
ditioned metal surfaces [13–15,27,39]. Fig. 10a shows the image of
a developing Pseudomonas biofilm surface after seven days of expo-
sure. Individual bacterial cells are clearly visible. Fig. 10b depicts
the force–distance curves at two locations of the cell surface A
and B. The former location is on the cell surface and the latter is
at the cell–cell interface. In Fig. 10b, the maximum downward
deflections during the sample retraction are 83.6 and
116.5 nm at locations of A and B, respectively. The corresponding
interactive force between the tip and the cell surface at the respec-
tive locations are 5.02 and 6.99 nN, as calculated from the
spring constant of 0.06 N/m. This reveals that the adhesion force
at the cell–cell interface is considerably higher than that at the
Pseudomonas cell surface. The average forces of the Pseudomonas
cell surface and the cell–cell interface with the corresponding stan-
dard deviation are summarized in Table 2. It has been widely
recognized that the high adhesion force at the cell–cell interface
is due to the accumulation of EPS at the cell periphery, consider-
ably enhancing the bacteria binding to the substratum
[14,15,27,39,40]. This result suggests that the sticky EPS accumu-
late on the cell–cell interface to enhance the aggregation of bacte-
Fig. 10. (a) The representative AFM image of a developing Pseudomonas biofilm on the 304 SS coupon surface after 7 days of exposure and (b) force curves at location A and B.
1383
rial cells, thus leading to the spreading of biofilm over the coupon
surface.
Fig. 11 shows the images of the developing biofilm of D. desul-
furicans after seven days of exposure, together with the force–dis-
tance curves at the D. desulfuricans cell surface (location A) and the
cell–cell interface (location B). It is obvious that the maximum
downward deflections during sample retraction at the cell–cell
interface are much larger than those at the cell surface, indicative
of the more attractive forces at the cell–cell interface than on the
cell-surface. As shown in Table 2, the average force at the cell–cell
interface is 5.33 ± 0.79 nN and 3.79 ± 0.48 nN on the cell surface
in the 7-day biofilm. The considerably more attractive adhesion
force at the cell–cell interface is expected due to the accumulation
of EPS, as discussed above.
The marked difference in the adhesion force between the two
strains can be readily distinguished, since the forces over the
D. desulfuricans cells are about 25% less than those of the Pseudomo-
nas cells on both the cell surface and the cell–cell interface. This phe-
nomenon is mainly related to the different physiology of the two
strains. It has been reported that the Pseudomonas strains are
slime-producing bacteria, and usually act as the pioneer colonizer
in the process of biofilm formation through secreting copious EPS
to form a matrix of primarily polysaccharides [41]. The more attrac-
tive adhesion forces over the Pseudomonas cells are probably indica-
1384 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385

Table 2 tive of the presence of relatively abundant EPS on the Pseudomonas

Comparison of tip-surface adhesion forces on cell surface and cell–cell interface of the cell surface as well as near the cell periphery. EPS is generally consid-
Pseudomonas and D. desulfuricans bacteria on the 304 SS coupon surface.
ered to be vital in cementing bacterial cells together in the biofilm
Strains Interaction force values at two locations (mean ± SD) (nN) structure [42]. Cell-to-cell signaling has recently been demonstrated
Cell surface (A) Cell–cell interface (B) to play a role in cell attachment and detachment from biofilms. A
Pseudomonas 5.03 ± 0.64 6.98 ± 0.87
class of diffusible molecular N-acylated homoserine lactones (AHLs)
D. desulfuricans 3.79 ± 0.48 5.33 ± 0.79 released by the bacterial cells into the local environments has been
reported to interact with the neighboring cells [43].

Table 3
Analysis of Tafel plots of 304 SS coupons after exposure to various exposure media for 28 days.

Time (days) Medium bca (mV/dec) bab (mV/dec) Ecorr.c (V) icorr.d (lA cm2) Corrosion rate (mm y1)
28 Sterilee 172 327 0.208 1.42 0.00147
Pseudomonasf 235 304 0.241 3.46 0.00584
D. desulfuricansg 244 522 0575 95.35 0.0987
a
bc is the Tafel slope of the cathodic polarization curve.
b
ba is the Tafel slope of the anodic polarization curve.
c
Ecorr refers to the potential, where the current reaches zero under polarization.
d
icorr is the corrosion current density.
e
Sterile refers to the 304 SS coupons exposed to a sterile nutrient-rich simulated seawater-based medium under aerobic conditions.
f
Pseudomonas refers to the 304 SS coupons exposed to the Pseudomonas inoculated medium.
g
D. desulfuricans refers to the 304 SS coupons exposed to the D. desulfuricans inoculated SSMB medium.

Fig. 11. (a) The representative AFM image of a developing D. desulfuricans biofilm on the 304 SS coupon surface after 7 days of exposure and (b) force curves at location A and
B.
 S.J. Yuan, S.O. Pehkonen / Corrosion Science 51 (2009) 1372–1385
4. Conclusions
This study has demonstrated the power and versatility of atomic
force microscopy (AFM) in visualizing high-resolution topographical
images of bacterial cells, biofilms and corroded surfaces. Results
showed that the biofilm colonization on the steel surface by the aer-
obic Pseudomonas and anaerobic D. desulfuricans bacteria was a dy-
namic process with an increase in the coverage, heterogeneity and
thickness over the substratum surface. The microbial colonization
was demonstrated to be detrimental to the passive film on the steel
surface, and leading to the deterioration of the steel coupons in the
form of pitting corrosion. With the accurate definition in the vertical
dimension, AFM was used to quantify the pitting corrosion of mate-
rials by profiling the depth of pits on the coupon surface. The depth of
pits induced by the two strains both increased with exposure time,
whereas the anaerobic D. desulfuricans exhibited more corrosive
properties due to the production of copious amount of sulfide ions
(S2) (about 0.94–1.25 mM), as revealed by the results of XPS mea-
surement and Tafel polarization curves. The measurement of tip-
biofilm adhesion forces revealed that the magnitude of the interac-
tive force over the bacterial cells was dependant on the relative
abundance of EPS excreted by the bacterial cells, and that the EPS
were mainly distributed in the cell-substratum periphery or the
cell–cell interface in the biofilm.
Acknowledgments
The authors thank the National University of Singapore and
Tropical Marine Science Institute (TMSI) for the financial support
of this study.
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