International Journal of Adhesion & Adhesives 65 (2016) 70–78

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International Journal of Adhesion & Adhesives

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

Investigation of bacterial attachment and biofilm formation of two

different Pseudoalteromonas species: Comparison of different methods
Masoumeh Moradi, Zhenlun Song n, Xia Nie, Minsheng Yan, Fang Qin Hu
Key Laboratory of Marine New Materials and Related Technology, Zhejiang Key Laboratory of Marine Materials and Protection Technology, Ningbo Institute of
Material Technology & Engineering, Chinese Academy of Sciences, Ningbo 315201, PR China

art ic l e i nf o a b s t r a c t

Article history: Biological fouling in marine environments creates numerous problems for engineered structures.
Accepted 13 November 2015 Microbial attachment to a solid surface and biofilm formation initiates the process of biofouling.
Available online 24 November 2015 Therefore, detecting the initial bacterial attachment and understanding the mechanism of biofilm for-
Keywords: mation are important for controlling biofouling. In the present study, the mechanisms of bacterial
Titanium and alloys attachment and biofilm formation of two marine isolated bacteria, namely Pseudoalteromonas sp. and
Confocal microscopy Pseudoalteromonas flavipulchra on Ti-coated samples were examined through different electrochemical,
Microscopy surface analysis and thermodynamic methods. The results revealed that the rate of bacterial attachment
Biological adhesion and mechanism of biofilm formation varied for different species of bacteria. The amount of exopoly-
Biofilm formation
saccharide production could affect the bacterial attachment rate. Open circuit potentiometry has been
found to be a valid and simple technique for continuous real-time monitoring of the biofilm formation
compared to other electrochemical and thermodynamic techniques. Finally, two different models have
been suggested to explain initial adhesion and biofilm formation of bacteria of different species.
& 2015 Published by Elsevier Ltd.

1. Introduction
Bacterial attachment to surfaces is a complicated process that is
affected by numerous factors such as environment, bacterial
properties and material surface characteristics [1]. This process
affects various types of applications, such as biomedical, industrial,
marine and environmental, and results in expensive cleaning and
maintenance [2,3]. Furthermore, the localised corrosion failure of
industrial equipment can be due to bacterial adhesion to metal
surfaces and biofilm formation [4,5]. Therefore, a better under-
standing of the variables governing bacterial adhesion to metal
surfaces will contribute to finding solutions for these problems.
The two approaches for studying bacterial adhesion to surfaces
include; electrochemical interaction and thermodynamic meth-
ods. Some researchers have used electrochemical impedance
spectroscopy (EIS) to detect and characterise bacterial cell
attachment and biofilm formation using different resistive sur-
faces, such as platinum [6], indium-tin oxide [3,7], and graphite
[8]. Bacterial adhesion and biofilm maturation reduce double layer
capacitance (CPEdl) [6], but an increase in CPEdl has also been
reported [9]. On the other hand, there are many studies on the
surface thermodynamics of bacterial adhesion [10–13]. Some
parameters, such as surface tension [14], contact angle, surface
n
Corresponding author. Tel.: þ 86 574 87911131; fax: þ 86 574 86685159.
E-mail address: songzhenlun@nimte.ac.cn (Z. Song).
free energy [15] and surface roughness [16] have been used to
characterise microbial cell adhesion to surfaces. The results show
that the cell surface is hydrophobic and has poor wetting and
adhesiveness characteristics at higher contact angles ( 490) and
lower surface tension. The roughness of the surface aids in the
attachment process when the surface irregularities are compar-
able to the size of the bacteria [17].
According to the aforementioned techniques, different
mechanisms have been proposed to explain bacterial adhesion [9].
Attachment of bacteria to a material surface is a two-phase process
including an initial, instantaneous and reversible physical phase,
followed by a time-dependent and irreversible molecular and
cellular phase [15]. In addition, biofilm formation encompasses
several different stages; starting with the adsorption of macro-
molecules, such as proteins, lipids and polysaccharides, on the
metal surface. These molecules change the physical chemistry of
the interface, including the hydrophobicity and surface charge
[18]. The bacteria then move from the bulk to the solid surface
because of concentration gradients of diffusible chemical factors,
which can modulate bacterial growth on surfaces by regulating
cellular adhesion components [1,18]. The initial attachment is a
crucial step that depends on surface properties [19]. Thus, detec-
tion of initial attachment can help us control biofouling and
biodegradation.
On the other hand, while the mechanisms of bacterial attach-
ment and biofilm formation have been examined [15,20,21], the

http://dx.doi.org/10.1016/j.ijadhadh.2015.11.004
0143-7496/& 2015 Published by Elsevier Ltd.
 M. Moradi et al. / International Journal of Adhesion & Adhesives 65 (2016) 70–78
precise nature of this phenomenon remains unknown. In the
present study, we attempted to define the parameters that
determine the extent of initial adhesion of two different species of
bacteria from the same genus. We also attempted to introduce a
simple, cost-effective and valid technique to identify bacterial
attachment and biofilm formation. Different electrochemical and
thermodynamic techniques were used in this study, and the
results were compared with the data obtained through field
emission scanning electron microscopy (FESEM) and confocal
scanning laser microscopy (CSLM) in order to obtain images of the
cell distribution and information about the biofilm morphology.
Energy Dispersive X-Ray (EDS) analysis was also conducted to
detect the elements that attach to the surface. A new model to
explain bacterial attachment and biofilm formation stages on Ti-
coated surfaces is presented.
2. Materials and methods
2.1. Preparation of Ti coating
Physical vapour deposition was used to prepare a smooth and
pure Ti coating. Deposition is conducted under a vacuum to
deposit thin films by condensing a vaporised form of the desired
film material onto various work piece surfaces [22]. In this study,
deposition was conducted by using a magnetron sputtering
apparatus on silicone substrates. Substrates were ultrasonically
cleaned in acetone; followed by alcohol; and then dried. The
chamber was evacuated to a base pressure of 8  10  4 Pa. Before
deposition, the specimens were cleaned by Ar þ ion beams which
were provided by two end-Hall ion guns (Shilong Yahe Corp.,
Beijing, China 2008) with an energy of 150 V  1 A for 20 min [23].
The Ti coating was then prepared through DC magnetron sput-
tering with a bias voltage of  50 V and a temperature of 200 °C to
four Ti targets (99.99%). The power of each target was 100 W and
300 W for 30 min and 4 h, respectively. During deposition, the
magnets ran with both revolution and rotation at 5 rpm.
2.2. Isolation and identification
Marine Pseudoalteromonas sp. KJ569267 (PS) bacterium was
isolated from marine sludge collected from the East Sea (Ningbo,
China; GPS 29° 59‘50.59“ N, 122° 2‘ 34.99“ E). Sampling, strain
isolation and cultivation procedures have been described pre-
viously [24,25]. This bacterium was deposited as CPCC 100540 at
the China Pharmaceutical Culture Collection (CPCC), Institute of
Medicinal Biotechnology, Chinese Academy of Medical Sciences,
China. Pseudoalteromonas flavipulchra MCCC1A06 (PF) strain was
obtained from the Marine Culture Collection of China (MCCC),
China and grown under the same conditions. The cultures of both
strains were stored at  80 °C in marine broth containing 30%
glycerol.
2.3. Adhesion tests
Adhesion tests were conducted in an incubator shaker at 30 °C.
The tested substrate samples were placed in a sterilised petri dish
and covered with bacterial suspension. The inoculation con-
centration was 1  106 cells mL  1 in sterile artificial seawater with
3 g/L peptone. The bacteria were allowed to adhere to the surface
for different time periods. The tested substrate was gently washed
two to three times with PBS to eliminate loosely adherent bacterial
cells. The rinsing method consisted of pipetting and pouring of PBS
using a micropipette to minimise the applied rinsing force as
much as possible [14]. Each sample was examined under CLSM
which was connected to a digital camera. Image-Pro Plus software
71
(Model IPP6.0, Media Cybernetics, USA) was used to calibrate the
dimension of the field of view for each image to a scale bar so that
the pixels could be converted to square millimetres. The adhesion
tests were performed in triplicate for each substratum and bac-
terial strain. The mean number of bacterial cells in different fields
of view was then divided by the surface area of the field of view to
obtain the density (number of bacteria per square centimetre).
2.4. Thermodynamic properties
Contact angle measurements for bacterial strains were made
using the standard sessile drop technique [26]. To evaluate the
intrinsic cell surface hydrophobicity, each bacterial strain was
suspended in Millipore-Q water to form a concentrated suspen-
sion. The latter was then deposited on 0.45 mm pore-size cellulose
acetate membrane filters on a fritted glass support by filtration of
the suspension using negative pressure to produce a bacterial
lawn of stacked layers. The wet filters were placed carefully on a
metal support using double-sided sticky tape and air dried. For
each bacterial strain, at least three contact angle measurements
were made.
Surface tension measurements were performed by a tensi-
ometer (KRUSS K100, Germany) with the Wilhelmy plate method
[27]. In this method, a thin Pt plate is lowered to the surface of the
liquid and the downward force to the plate is measured.
2.5. Extraction and dry weight of the extracted exopolysaccharide
substances
The culture medium of the exopolysaccharide substances (EPS)
was prepared by inoculating 1  106 cells mL–1 of the PS and PF
strains into 100 mL sterile artificial seawater containing 5 g/L
peptone and 1.5 g/L yeast extract, which was then incubated at
30 °C on a rotary shaker at 130 rpm for different lengths of time up
to 5 days. After incubation, the broth culture was centrifuged at
14000  g, 4 °C for 15 min to obtain the supernatant. The super-
natant was then filtered through a 0.2 μm filter paper to remove
all the suspended particles. The filtered samples were mixed with
isopropanol at a ratio of 1:3 and retained for precipitation at 4 °C
overnight. The precipitated EPS was collected by centrifugation at
10,000  g, 25 °C for 5 min. After being dried by heating at 50 °C,
EPS was quantified by weighing.
2.6. Electrochemical tests
The open circuit potential (EOCP) was monitored for an exposure
period of 5 days using a voltmeter (Agilent 33210A) for Ti-coated
samples exposed in duplicate. A saturated calomel electrode (SCE)
was used as the reference electrode. EOCP was recorded at a fre-
quency of one reading every 30 s.
EIS measurements were performed in a corrosion cell with a
three-electrode system that consisted of the following: (1) a
saturated calomel reference connected to a reactor via a Luggin
capillary filled with a salt bridge; (2) a platinum sheet as a counter
electrode; and (3) a Ti-coated sample as a working electrode. EIS
was performed using a Potentiostat–Galvanostat (273A; Princeton)
with a steady state EOCP at amplitude of 10 mV AC sine wave at
frequencies ranging from 10 kHz to 10 mHz. In each case, duplicate
experiments were conducted; the differences between the dupli-
cates were mostly within 71%. If the differences were large, a
third test was conducted to confirm the data. ZSIMPWIN software
(version 3.22, 2010) was used for the analysis of the EIS data [28].
72 M. Moradi et al. / International Journal of Adhesion & Adhesives 65 (2016) 70–78
2.7. Surface observation/analysis
Bacterial attachment and biofilm formation on Ti-coated sur-
faces after different exposure times were examined through
FESEM (FEI Quanta FEG 250, USA) and CLSM (Leica TCS SP5 11,
Germany). The preparation of the samples for FESEM observation
was performed by immersion of the samples in 2% glutaraldehyde
solution for 4 h and dehydration by four successive ethanol solu-
tions (15 min each): 25%, 50%, 75% and 100%. This method can
immobilise bacterial cells and confirm bacterial adhesion. The Ti-
coated surfaces cultivated with bacteria were stained with a
fluorescent dye (FilmTracer™ LIVE/DEADs Biofilm Viability Kit,
Invitrogen, USA) before CLSM examination. EDS (Oxford, X-Max
EDX; UK) analysis was also conducted for chemical analysis at the
local regions of the Ti-coated samples.
3. Results
3.1. Surface analysis
Figs. 1 and 2 show FESEM images of bacterial attachment and
biofilm formation on Ti-coated surfaces after different time peri-
ods. The difference between the two strains was observed during
the cultivation times. The PF strain adhesion occurred on the Ti-
coated surface (Fig. 1a) after 1 h. After 24 h (Fig. 1b), a monolayer
of the PF strain had adhered on the entire metal surface, and after
72 h (Fig. 1c), a thin biofilm was visible. The cells then increased
continuously in number, and a multilayered biofilm was observed
over the following days (Fig. 1d). In the presence of the PS strain
(Fig. 2), the first adhesion occurred after 1 h (Fig. 2a) and a large
Fig. 1. FESEM images of bacterial adhesion and biofilm maturation of the PF strain on Ti-coated surfaces. The images were obtained after 1 h (a), 24 h (b), 48 h (c) and 6 days
(d) of exposure.
amount of EPS was produced on the surface (Fig. 2b) after 24 h.
The biofilm (Fig. 2c) was clearly observable after 48 h, although it
did not cover the surface completely. After 6 days (Fig. 2d), the
amount of bacteria significantly decreased which might be
attributed to biofilm detachment.
The CLSM images also confirmed the FESEM results. In the
presence of the PS strain, the biofilm was observed on the metal
surface after 24 h (Fig. 3a). After 6 days (Fig. 3b), the bacterial
colonies were noticeable. Compared with the presence of the PF
strain, the biofilm formed on the metal surface after 24 h (Fig. 3c)
and completely matured after 6 days (Fig. 3d).
EDS analysis revealed the chemical composition of the metal
surface after bacterial attachment and biofilm formation. Table 1
shows the EDS results of the PF and PS strains at different expo-
sure times. In the presence of the PF strain, some inorganic ions,
such as K þ , Na þ and Cl-, were deposited on the surface in the first
hours of exposure; bacterial attachment was associated with
inorganic ion deposition. After 72 h of exposure, high N con-
centration was observed, which increased as the exposure time
increased, and reached 42.41% after 6 days of cultivation. The
presence of N can be attributed to biofilm formation; the
maturation of biofilm resulted in increased amounts of N and the
appearance of P on the Ti-coated surfaces. These results confirmed
the SEM and CLSM images.
In the presence of the PS strain, N was observed from the first
hour of cultivation; longer exposure times led to an increase of N.
Inorganic ions, such as K þ , Na þ , Ca2 þ and Cl-, were also observed
on the surface. After 24 h of cultivation, some elements, such as P
and S, appeared due to EPS production on the metal surface. These
results also confirm that inorganic ions are deposited along with
bacteria.
 M. Moradi et al. / International Journal of Adhesion & Adhesives 65 (2016) 70–78 73

Fig. 2. FESEM images of bacterial adhesion and biofilm maturation of the PS strain on Ti-coated surfaces. The images were obtained after 1 h (a), 24 h (b), 72 h (c) and 6 days
(d) of exposure.

Fig. 3. CLSM images of attached bacteria and biofilm on the Ti-coated surface. PS and PF strains were obtained after 24 h (a, c) and 6 days (b, d) of cultivation. Magnification
250 mm.
74 M. Moradi et al. / International Journal of Adhesion & Adhesives 65 (2016) 70–78

Table 1
EDS elemental analysis of Ti-coating samples in sterile artificial seawater and in the presence of two strains after different exposure times (b).

Sample Ti C O N P S Na K Ca Cl

Sterile artificial seawater 98.88 1.12

PF strain 1h 84.43 0.61 3.7 — — — — 6.11 — 6.14
24 h 78.98 0.09 15.24 — — — 1.91 1.02 — 2.76
72 h — 1.56 40.26 28.03 — — 13.5 2.3 — 14.35
144 h — 0.93 50.25 42.41 1.43 — 0.75 4.23 — —
PS strain 1h 85.12 2.14 7.96 — — — 0.5 — — 1.69
24 h 67.01 2.76 22.46 4.18 0.67 0.56 0.64 — 1.81 —
72 h — 0.91 52.94 34.36 5.18 — 0.55 5.43 — —
144 h — 0.58 35.85 31.75 4.1 1.01 0.54 7.3 16.34 2.55

Fig. 4. Comparison of the adhesion of the PS and PF strains on the hydrophobic Ti-
coated surfaces during 300 min of cultivation. Fig. 5. Profile of EPS production by the PS and PF strains over exposure time at
30 °C on a rotary shaker at 130 rpm.

3.2. Bacterial adhesion 3.4. EPS production

Fig. 4 shows the bacterial adhesion of both the PF and PS strains
during the 300 min of cultivation. The results demonstrated that
the attachment rate of the PF strain was much faster than that of
the PS strain during the initial exposure up to 120 min; the bac-
terial adhesion then increased gradually. Meanwhile, the PS strain
regularly attached to the surface.
3.3. Thermodynamic parameters
Thermodynamic parameters, such as surface tension and con-
tact angle, were used to investigate bacterial attachment during
the initial exposure time. Bacterial hydrophobicity was deter-
mined by contact angle measurement on the bacterial lawns. The
contact angle of the Ti-coated surface without the bacteria was
approximately 96°. However both types of bacteria exhibited a
predominantly hydrophilic surface, with contact angles ranging
from 20° to 35°, which increased the degree of wetting. They
behaved as a monopolar surface, which strongly interacted with
water and decreased the interfacial tension values [29]. Thus, the
surface tension of the deionised water was about 72.61 mN/m, but
it decreased in the presence of bacteria and reached 39.12 and
45.95 mN/m for the PF and PS strains, respectively.
The most interesting result was the similarity of the contact
angles at the initial exposure and after 6 days of cultivation. This
similarity showed that the bacteria and their by-products had the
same effects on the surface, and the accumulation of the bacteria
or biofilm gradually increased the amount of wetting.
The EPS production capacity of the PS and PF strains were
evaluated; the results are presented in Fig. 5. For both strains, the
weight of the EPS increased with incubation time and reached its
maximum value at 3 days; however the weight of the EPS in the PS
strain (10 g/L) was 4.35 times higher than the weight of the EPS in
the PF strain (2.3 g/L). After 3 days, the EPS production decreased
for both strains, which confirmed that the EPS production is
growth associated.
3.5. Electrochemical results
Open circuit potentiometry (OCP) and EIS were used to observe
bacterial attachment and biofilm formation over the exposure
time. Fig. 6 shows the variations in an EOCP, with an exposure time
of 5 days for the Ti-coated surface in the sterile artificial seawater
and in the presence of bacteria at 25 °C. EOCP versus time exhibited
different trends in the presence of the two strains. In the presence
of PS, EOCP increased sharply over the first 24 h, reaching  0.08 V
(SCE) and then remaining constant. According to the FESEM ima-
ges, the increase of EOCP may be related to the high levels of EPS
production on the metal surface. In the presence of the PF strain,
an initial shift of potential towards the negative direction occurred,
and the potential reached  0.53 V (SCE) after 6 h. A small increase
in EOCP was then observed, and remained steady until 72 h. Finally,
it strongly shifted in the positive direction. In contrast, the EOCP
remained relatively constant in the sterile medium throughout the
exposure period.
Fig. 7 shows the Nyquist plots for Ti-coated surfaces in the
presence of PS (Fig. 7a) and PF strains (Fig. 7b) at different
 M. Moradi et al. / International Journal of Adhesion & Adhesives 65 (2016) 70–78
Fig. 6. Variations in open-circuit potential (EOCP) over exposure time for Ti-coated
surfaces in sterile artificial seawater and in the presence of two bacterial strains at
room temperature.
Fig. 7. Nyquist plots obtained for Ti-coated surfaces in the presence of the PS (2a)
and PF strains (2b) at different immersion times.
exposure times. The results demonstrated variation with the
impedance values when the samples were exposed for different
lengths of time. In the presence of the PS strain, the impedance
value increased in the first 2 h of exposure, then decreased and
75
Fig. 8. Trend of changes in impedance value and double-layer capacitance of Ti-
coated surfaces in sterile artificial seawater (a) and in the presence of the PS (b) and
PF (c) strains at different exposure times.
reached its lowest value after 24 h. Again, the impedance value
increased as the exposure time increased. After 24 h, the biofilm
that formed on the metal surface acted as a barrier layer and
increased the impedance value [30]. In the presence of the PF
strain, the first impedance value increased and reached its highest
value after 6 h, and then decreased. In this study, the PF strain
exhibited a different electrochemical behaviour than the PS strain,
and biofilm formation decreased the impedance value.
Fig. 8 demonstrates the variation of the impedance magnitude
and CPEdl as a function of time over 168 h of exposure in sterile
76 M. Moradi et al. / International Journal of Adhesion & Adhesives 65 (2016) 70–78
Fig. 9. Schematic model of the phases involved in bacterial adhesion and biofilm formation of the PS (a) and PF (b) strains.
artificial seawater (Fig. 8a) and in the presence of PS (Fig. 8b) and
PF (Fig. 8c) bacterial strains, respectively. CPEdl decreased in the
first 6 h in the sterile solution and in the presence of PF strain, but
it was almost stable in the presence of the PS strain. In the sterile
solution, the decrease in CPEdl was due to the TiO2 formation on
the metal surface; this oxide can form a protective layer and
increase the impedance value. Bacterial attachment of the PF
strain indicated the same trend and the capacitance decreased
until 72 h of exposure.
In the presence of the PS strain, CPEdl increased significantly
until 24 h of exposure and then remained approximately stable.
The increase of CPEdl is probably due to the high levels of EPS
production.
4. Discussion
The mechanisms of bacterial cell adhesion to solid surfaces and
biofilm formation are complicated, and different chemical and
physical parameters affect these processes [1,18–19]. In the pre-
sent study, various electrochemical and physical methods were
used to explain this phenomenon. The results indicated that the
potential of substrates changed with time when the bacteria were
exposed to artificial seawater. The initial decrease in EOCP observed
for the PF strain supports the activity and the growth of the bac-
teria and enhances the redox quality of the medium [31,32].
However, EOCP increased for both strains after a certain exposure
time. The FESEM images showed the presence of the EPS in PS
strain for 24 h of exposure and confirmed the presence of biofilms
for both the PS and PF strains after 48 and 72 h, respectively.
Therefore, the PS strain, with its higher levels of EPS production,
colonised the entire substratum very rapidly, and EOCP reached its
highest value in 24 h. However, the increase of EOCP in the pre-
sence of the PF strain was observed after 72 h of exposure because
of the formation of biofilm. According to these results, the OCP
technique is valid for continuous real-time monitoring of the
initial biofilm formation, and provides a simple and non-expensive
electrochemical method for in vivo assessment of the presence of
biofilms on metal surfaces [14].
Bacterial adhesion and biofilm maturation affect CPEdl [6]. In
this study, CPEdl was examined to monitor bacterial attachment
and biofilm formation. The attachment of cells and development of
a biofilm on the metal surface are expected to retard the interfacial
electron-transfer kinetics and increase the electron-transfer
resistance [32]. The results demonstrated that the CPEdl values of
the bacterial adhesion stage and the biofilm maturation stage were
different for the two strains. The increase in capacitance observed
in the presence of the PS strain at 24 h was due to the higher rate
of EPS production on the Ti-coated surface. The formation of the
biofilm resulted in an electron transfer barrier between the bac-
terial solution and the metal surface [14]. Charged groups were
present inside the bacterial cell wall, and the distribution of these
charges influenced the electric double layer interactions in the
bacterial adhesion [33]. CPEdl remained stable after 48 h; thus the
changes in the biofilm structure did not significantly influence the
electron-transfer barrier between the electrodes or capacitance.
In the presence of the PF strain, the resistance increased and
the capacitance gradually decreased with a culture time of up to
6 h. During the early cultivation periods, numerous cells were
thought to attach on the metal surface, which caused changes in
its electrochemical behaviour [34]. A decreasing trend in the
capacitance continued during the biofilm formation due to the
increase in the adsorption film area (which decreased the elec-
trode surface area) [35]. The attached bacterial cell bodies may not
obstruct the movement of the ionic charge in the electrolyte [6];
rather, other components accessible to the diffused layer may
affect the CPEdl [36].
However, the changes in the CPEdl trend for the PF strain were
similar to those for sterile artificial seawater at the initial exposure.
These results demonstrated that EIS is not a comprehensive
method used for identifying bacterial activity. Some materials can
be activated in an aggressive environment, and their degradation
also affects the variation in capacitance or resistance.
Surface tension provides information about the bacterial solu-
tion. Bacterial cells and substratum hydrophobicity are speculated
to be the key parameters controlling the initial interactions of the
adhesion process [14]. Adhesion to hydrophilic substrata (i.e.
substrata of relatively high surface tension) is more extensive than
to hydrophobic substrata, where the surface tension of the bac-
teria is higher than that of the suspending medium. When the
surface tension of the suspending liquid is higher than that of the
bacteria, the opposite pattern of behaviour prevails [12]. In the
present study, the surface tension of the bacteria was less than
that of the suspending liquid; thus, bacterial adhesion on hydro-
phobic surfaces was much easier. This phenomenon confirmed the
adhesion trend of the PF and PS strains, and explained the higher
rate of bacterial adhesion for the PF strain. Because the roughness
of the surface can affect bacterial adhesion, all the samples used in
this study had the same surface roughness.
With regard to the bacterial adhesion of the two strains at the
initial exposure and the thermodynamic results, the first adhesion
of these bacteria also resulted in physical changes. According to
 M. Moradi et al. / International Journal of Adhesion & Adhesives 65 (2016) 70–78
the DLVO (Derjaguin and Landau, Verwey and Overbeek) theory,
particle adhesion is governed by long-range interactions between
the adhering particle and the macroscopic substratum. These
interactions include Lifshitz–van der Waals interactions and
interactions resulting from overlapping electric double layers [34].
The bacterial cell surface carries a net negative charge under most
physiological conditions, with a few exceptions [35]. Considering
that most natural surfaces are also negatively charged, bacteria
generally experience electric double layer repulsion when
approaching these surfaces [37]. However, bacterial cell surfaces
are highly dynamic; and respond strongly to environmental
changes through the adsorption of ions and macromolecular
components [38]. The bacterial cell surface may be penetrable to
solvents and solutes, in particular ions, probably because of the
presence of a peptidoglycan layer [33]. Adsorption of some ions on
the metal activates the surface and facilitates bacterial adhesion.
EDS analysis showed high levels of K þ and Cl- ions in the first hour
of the cultivation period in the presence of the PF strains.
Adsorption of these ions is speculated to facilitate the adhesion
process of the PF strain. The existence of some cations, such as
Na þ , Ca2 þ and K þ , in the presence of the PS bacteria confirmed
that this strain is also ion penetrable, and that bacterial adhesion
aided the deposition of these ions.
The results from the present study suggest two different
models for the attachment of the PS and PF strains (Fig. 9), which
were confirmed during adhesion. The physicochemical properties
of the surfaces influenced the first stage of adhesion only. There-
after, their metabolic by-products covered the surfaces and
established contact with them. The difference in the results
between the two strains was due to the presence of the EPS and
detachment of the biofilm for the PS strain. Both strains were
suggested to have some cations, such as Na þ , K þ and Ca2 þ ,
deposited on the surface, which facilitated the adsorption of the
bacteria on the Ti-coated surfaces.
5. Conclusion
Bacterial adhesion and biofilm formation of two marine strains
were examined on Ti-coated samples in sterile artificial seawater.
This study used different electrochemical, surface analysis and
thermodynamic techniques. Electrochemical studies showed that
EOCP changed with exposure time in the presence of bacteria, and
reached its highest value when the EPS and biofilm were formed
on the metal surface; however the trends for these changes dif-
fered for the two strains. Thus, OCP was suggested to be a valid
technique for continuous real-time monitoring of the initial bio-
film formation.
The different electrochemical behaviours of the biofilm for-
mation for the two strains were also observed by EIS measure-
ments. CPEdl increased in the presence of the PS strain which
confirmed that the biofilm formation resulted in an electron
transfer barrier between the bacterial solution and the electrode
surface. On the other hand, bacterial adhesion of the PF strain
could not prevent the movement of the ionic charge in the elec-
trolyte; the diffusion process occurred in the presence of these
bacteria, and thus, CPEdl decreased during the biofilm formation.
EDS analysis showed the deposition of K þ , Na þ and Ca2 þ
cations on the metal surface in the presence of these two strains.
However, the concentrations of these ions were much higher in
the presence of the PF strain than for the PS strain. Thus, initial
adhesion of the PF strain exhibited a physical characteristic, which
was associated with surface tension and the deposition of ions on
the metal surface. The deposition of the PS strain affected the CPEdl
and influenced the electrochemical behaviour. Two different
models were suggested to explain initial adhesion and biofilm
77
formation of the PS and PF strains. The different behaviours of
these two strains were due to the higher amount of EPS produc-
tion in the PS strain.
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (Grant no. 51301193) and China Postdoctoral
Science Foundation (No. 2013M540503).
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