Biofouling

The Journal of Bioadhesion and Biofilm Research

ISSN: 0892-7014 (Print) 1029-2454 (Online) Journal homepage: https://www.tandfonline.com/loi/gbif20

Biofouling of stainless steel surfaces by four

common pathogens: the effects of glucose
concentration, temperature and surface
roughness

Katja Bezek, Damjan Nipič, Karmen Godič Torkar, Martina Oder, Goran
Dražić, Anže Abram, Janez Žibert, Peter Raspor & Klemen Bohinc

To cite this article: Katja Bezek, Damjan Nipič, Karmen Godič Torkar, Martina Oder, Goran Dražić,
Anže Abram, Janez Žibert, Peter Raspor & Klemen Bohinc (2019): Biofouling of stainless steel
surfaces by four common pathogens: the effects of glucose concentration, temperature and surface
roughness, Biofouling, DOI: 10.1080/08927014.2019.1575959

To link to this article: https://doi.org/10.1080/08927014.2019.1575959

Published online: 26 Apr 2019.

Submit your article to this journal

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=gbif20
BIOFOULING
https://doi.org/10.1080/08927014.2019.1575959

Biofouling of stainless steel surfaces by four common pathogens: the effects

of glucose concentration, temperature and surface roughness
Katja Bezeka, Damjan Nipi
cb, Karmen Godi
c Torkarb, Martina Oderb, Goran Dra
zicc, An
ze Abramd,


Janez Zibert b
, Peter Raspore and Klemen Bohincb
a
Faculty of Health Sciences, University of Primorska, Izola, Slovenia; bFaculty of Health Sciences, University of Ljubljana, Ljubljana,
Slovenia; cDepartment of materials chemistry, National Institute of Chemistry, Ljubljana, Slovenia; dDepartment for nanostructured
materials, Jozef Stefan Institute, Ljubljana, Slovenia; eDepartment of Food Science and Technology, Biotechnical Faculty,
University of Ljubljana, Ljubljana, Slovenia

ABSTRACT ARTICLE HISTORY

There is a wide range of factors affecting bacterial adhesion and biofilm formation. However, Received 19 May 2018
in both food processing and medical settings, it is very hard to obtain suitably controlled Accepted 22 January 2019
conditions so that the factors that reduce surface colonisation and biofouling can be studied.
KEYWORDS
The aim of this study was to evaluate the effect of glucose concentration, temperature and
Biofouling; stainless steel
stainless steel (SS) surface roughness on biofouling by four common pathogens (Escherichia coli, surface; glucose
Staphylococcus aureus, Pseudomonas aeruginosa and L. monocytogenes). Among the tested varia- concentration; temperature
bles, the untreated SS surface (3C) was shown to be fouled more than 3D polished, brushed or
electropolished SS surfaces. Although an array of parameters influenced biofouling, the most
promising control measure was the influence of low temperature (4
C) that reduced biofouling
even in the case of the psychrophilic Listeria monocytogenes. The study findings could signifi-
cantly contribute to the prevention of SS surface contamination and consequential biofouling
by food and healthcare associated pathogens.

Introduction medical field, biofilms are formed on almost all types

Bacteria can readily adhere to a wide variety of
solid materials and form organized communities
of phenotypically altered cells, surrounded by a self-
excreted matrix (Flemming 2011; Vickery et al. 2013).
Interactions between microorganisms and contact
material surfaces lead to stable microbial formations
and subsequent biofouling, representing one of the
most hazardous risk factors for the food, pharmacy
and service industries (Raspor and Jevsnik 2008; Srey
et al. 2013). In such formations embedded bacteria
are significantly less susceptible to unfavourable
conditions compared to their planktonic counterparts,
which significantly contributes to their environmental
persistence (Donlan and Costerton 2002; Carpentier
and Cerf 2011; Lourenço et al. 2013). Because of their
increased tolerance to cleansers and disinfections,
pathogenic and spoilage microorganisms within bio-
films are often causative agents of post-processing
contamination (Møretrø and Langsrud 2017).
Microbial biofilms can be found on food processing
equipment as well as on food surfaces such as meat
or fresh produce (Verran and Jones 2000). In the
of devices, such as contact lenses, urinary and cardio-
vascular catheters, surgical instruments, prostheses
and implants (Martinuzzi and Salek 2010). Moreover,
adhered microorganisms do not respond to anti-
microbial treatment as expected and are responsible
for a high percentage of hospital acquired infections,
along with increased morbidity and health care costs
(Salgado et al. 2003; Vickery et al. 2004). Factors
affecting bacterial biofouling have been linked to the
surface topography, the roughness and the physico-
chemical properties of the contact surface, the sur-
rounding suspension liquid (type of suspension, tem-
perature, pH and ionic composition), and microbial
cell properties (surface charge, surface hydrophobicity
and appendages) (Absolom et al. 1983; Boulane-
Petermann 1996). Due to its chemical and mechan-
ical/physical stability, cleanability and corrosion
resistance, stainless steel (SS), in particular austenitic
AISI (American Iron and Steel Institute) 304 and 316
grades, are widely used as contact surface materials in
the food industry and medicine (Cvetkovski 2012).
Surface roughness is one of the most important

CONTACT Klemen Bohinc bohinck@zf.uni-lj.si

ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 K. BEZEK ET AL.
features of these materials as diminished shear forces
and higher surface areas can increase microbial
retention (Whitehead and Verran 2006; Ammar et al.
2015). Surfaces are commonly treated with disinfec-
tants and cleaning agents such as peroxides,
chloramines or hypochlorites that can alter surface
characteristics (Schmidt et al. 2012). However, bacter-
ial cell properties, such as the presence of fimbriae,
flagella, production of extracellular polymeric substan-
ces (EPS), specific protein adhesins and cell size are
also an important factor in active bacterial adhesion
to surfaces of various materials (Donlan 2002; Van
Houdt and Michiels 2010; Limoli et al. 2015). These
properties can be affected by the surrounding envir-
onment; hence, alterations in these conditions may
alter the adhesion and biofilm formation of diverse
bacterial species in a different way (Garrett et al.
2008; Van Houdt and Michiels 2010). It has also been
shown that temperature can affect the physical prop-
erties of the compounds within and around the cells
as well as their surface chemistry, including the
composition of surface polymers and appendages. In
combination with other factors, such as changes in
pH, this can have a noticeable effect on bacterial
growth, aggregation and production of flagella
(Herald and Zottola 1988; Barker and Bloomfield
2000; Andersen et al. 2010).
Many foodborne pathogens and medically import-
ant bacteria including Escherichia coli, Staphylococcus
aureus, Pseudomonas aeruginosa and Listeria monocy-
togenes are capable of attaching to and forming bio-
films on various abiotic surfaces (metal, glass, plastic,
rubber) (Verran et al. 2001; Naves et al. 2008). E. coli
is a commensal member of the intestinal microflora
of animals and humans and some serotypes can cause
serious food poisoning and are occasionally respon-
sible for product recalls (Vogt and Dippold 2005).
The pathogenesis of biomaterial-associated staphylo-
coccal infections, caused mainly by S. aureus and
S. epidermidis, is often conditioned by a multilayered
biofilm, where bacteria are significantly less
susceptible to antibiotics compared to planktonic cells
(Chuard et al. 1991; Knobloch et al. 2002). P. aerugi-
nosa is an opportunistic pathogen that exhibits
a number of virulence factors, among which biofilm
formation plays a major role in infection and anti-
biotic resistance (Rasamiravaka et al. 2015). The food-
borne pathogen L. monocytogenes is known for its
ubiquitous distribution in nature and survival under
adverse environmental conditions such as refrigerator
temperatures, low pH and high salt concentration
(Gandhi and Chikindas 2007). Increased retention of
this pathogen in food processing environments has
been linked to its ability to attach to surfaces and
form biofilm (Carpentier and Cerf 2011). The
emergence and spread of microbial resistance has led
to an increased interest in the development of new
strategies to combat bacterial biofilms (Blackledge
et al. 2013; Akbas 2015; Nan et al. 2015). Some anti-
biofilm strategies are focused on molecular mecha-
nisms and other on surface material modifications
(Chen et al. 2013; Sadekuzzaman et al. 2015).
The aim of this study was to provide information
which may help reduce the contamination of surfaces
in food and healthcare facilities by determining
the parameters impacting on biofilm formation.
In this context the effect of selected parameters such
as glucose concentration, temperature and SS surface
roughness on biofouling of four common bacterial
pathogens was tested. The surface roughness and top-
ography of AISI 304 stainless steel (SS) were charac-
terized by mechanical profilometer and scanning
electron microscopy (SEM), respectively. To quantify
the biofouling as defined by the amount of bacterial
cells and debris present, a spectrophotometric method
using crystal violet dye was applied.
Materials and methods
Bacterial strains and growth conditions
To test the effect of glucose concentration, tempera-
ture and SS surface roughness on biofouling, a set of
four common bacteria strains with biofilm forming
potential was used. Selected strains were L. monocyto-


genes ZM58 (IHM, W€ urzburg, Germany), S. aureus
ATCC 25923 (clinical isolate), E. coli ATCC 35218
(canine isolate) and P. aeruginosa ATCC 27853 (blood
culture isolate). Prior to experiments, the strains
were sub-cultured on tryptic soy agar (TSA, Oxoid,
Basingstoke, UK) or nutrient agar (NA, Biolife,
Milano, Italy) at 37
C. For the experiments, overnight
cultures were prepared by inoculating nutrient broth
(NB, Biolife) with biomass of one bacterial colony
and incubated overnight at 37
C. The overnight
cultures were then diluted in fresh NB in the ratio
1:300 to the final concentration of 107 CFU ml1.
Stainless steel surfaces
Biofouling by bacteria was studied on square SS
coupons (1  1 cm; 2 mm) of AISI 304 grade of four
different surface roughnesses. For the roughness char-
acterization a mechanical profilometer Form Talysurf
Series 2 was used (Taylor-Hobson Ltd, Leicester, UK).
On each sample three-line measurements in the

length of 5 mm were made. To separate roughness
from waviness, a Gaussian cut-off filter of 0.25 mm in
accordance with the ISO 4288-1996 was applied
before final results of the arithmetic average rough-
ness (Ra), average maximum peak to valley (Rz) and
root mean squared roughness (Rq) were obtained.
The untreated SS surface was designated 3C, the
surface exposed to 3D polishing as 3D; the brushed
surface as BR and the electropolished surface as EL
(Bohinc et al. 2016). The edges of all coupons were
polished in order to minimize bacterial adhesion on
the edges and side surfaces. Prior to use, SS coupons
were degreased in ethanol and sterilized by autoclav-
ing at 121
C for 15 min.
For the study of preferential locations of bacterial
adhesion on the SS surfaces, scanning electron
microscopy (SEM; JSM-7600F, Jeol, Peabody, USA)
was used. The topography of the highest (EL) and
lowest (3D) root mean squared roughness values of
surfaces was investigated after incubation for 24 h at
37
C in NB medium without glucose.
The effect of glucose concentration and
temperature on biofouling
To study the effect of glucose concentration on bio-
fouling of the selected bacterial strains, NB medium
was supplemented with 0%, 1% or 5% of glucose
(Sigma-Aldrich, Darmstadt, Germany). For the experi-
ment, four sterile SS coupons of the same roughness
were aseptically transferred into one well of a six-well
microtitre plate (Techno Plastic Products AG,
Trasadingen, Switzerland ) and covered with 4 ml of
diluted overnight bacterial culture. The coupons were
then incubated at 37
C for 24 h and 48 h. As negative
controls, four SS coupons were incubated in a sterile
medium, under the same conditions.
The effect of temperature on biofouling was deter-
mined using NB medium without glucose addition.
The incubation conditions were 22
C (room tempera-
ture), 37
C (body-physical temperature) for 24 and
48 h or 4
C (refrigerator temperature) for 192 h. After
incubation, the biofouling on the SS coupons was
quantified using the crystal violet (CV) staining assay.
In the case of L. monocytogenes the temperature effect
was tested also in NB medium supplemented with 1%
and 5% glucose.
Quantification of biofouling
The term biofouling in this study combines both bac-
terial biofilm formation and subsequent loading of
dead cell debris and growth medium residues on the
BIOFOULING 3
Table 1. Roughness of four different stainless steel surfaces.
AISI 304 grade of different finishes were used: not treated
(3C); polished (3D); brushed (BR) and electropolished (EL). The
data analysis includes the following roughness parameters:
arithmetic average roughness (Ra), average maximum peak to
valley (Rz) and root mean squared roughness (Rq).
Surface Ra (lm) Rz (lm) Rq (lm)
AISI 304 3C 0.043 ± 0.015 0.355 ± 0,148 0.060 ± 0.025
AISI 304 3D 0.028 ± 0.010 0.198 ± 0.083 0.038 ± 0.015
AISI 304 BR 0.060 ± 0.000 0.340 ± 0.014 0.078 ± 0.005
AISI 304 EL 0.208 ± 0.034 1.370 ± 0.272 0.280 ± 0.048
SS surface. For quantification, a modified CV method
was used, as described previously (O’Toole and Kolter
1998; Kubota et al. 2008). Briefly, after each incuba-
tion time bacterial suspensions were aspirated and
non-adhered bacteria removed by gentle rinsing with
5 ml of sterile phosphate-buffered saline (PBS, Oxoid)
three times. The biofouling material on the surface of
SS coupons was fixed using a hair dryer for 10 min at
60
C and stained with 3 ml of 0.1% CV solution
(Merck KGaA, Darmstadt, Germany) for 5 min. The
SS coupons were then washed three times with 5 ml
of sterile PBS to remove the excess dye. In the next
step, coupons were dried and separately transferred
into one well of a 24-well microtitre plate. To release
the bound dye, 200 ml of 96% ethanol (Merck) were
added to a single coupon and shaken for 5 min.
Afterwards the dye solution was transferred into the
wells of a fresh 96-well microtitre plate and spectro-
photometrically measured (Infinite 200 VR PRO,
Tecan, Gr€ odig, Austria) at a wavelength of 620 nm
(A620). The measurements were corrected by subtract-
ing the mean values of the negative controls for each
individual SS surface (DA620) and discussed in terms
of biofouling.
Statistical analysis
Univariate analysis of variance (ANOVA) and post
hoc analysis with the Tukey HSD criterion were
employed for the statistical analysis of the differences
in the optical density values for the tested parameters
(Hirotsu 2017).
Results
Surface roughness and topography measurements
The surface roughness was calculated from the profil-
ometry measurements (Table 1). The latter includes
three roughness parameters as follows: arithmetic
average roughness (Ra), average maximum peak to
valley (Rz) and root mean squared roughness (Rq)
(ISO 1997). The highest values of all three parameters
4 K. BEZEK ET AL.

Figure 1. The effect of glucose concentration (0%, 1%, 5%) and temperature (22
C, 37
C) on bacterial biofouling (DA620) after
incubation for 24 h (A, C, E) or 48 h (B, D, F). AISI 304 grade SS with different finishes was used: (3C) untreated; (3D) polished;
(BR) brushed and (EL) electropolished.
(Ra, Rz and Rq) were measured for electropolished SS significantly influenced (p < 0.001) the optical density
surface. The lowest roughness was obtained for the values (bacterial biofouling rate), while temperature
3D polished surface. The topographies of all surfaces was not a significant factor (p ¼ 0.273). The interac-
were also examined by SEM (Figure 3). The brushed tions between factors were also significant, except the
surfaces showed pronounced stripes and lines in one interaction between SS surface type and glucose con-
direction and the 3C surfaces showed a granu- centration (p ¼ 0.512). Additional post hoc analysis
lar structure. showed significant differences between all the SS sur-
In the terms of topography more surface features face types, except between 3D polished and EL surfa-
such as cracks, scratches and grooves were observed ces as well as between BR and EL surfaces. The
on the 3D surface. Also, the amount of attached bac- results overview showed that the untreated (AISI 304
terial cells was higher on the AISI 304 3D surface 3C) SS surface was the most common site of biofoul-
where clusters of bacteria were often aligned with sur- ing for all the bacterial strains (Figures 1 and 2). The
face irregularities (Figure 4). The EL surface topog- summary of measured bacterial biofouling on SS AISI
raphy was shown to be less diverse with a lower 304 grade with different finishes for different temper-
number of surface features and a more randomized atures and glucose concentrations are given in Tables
surface distribution of bacteria. 2 and 3.

Biofouling of SS surfaces
The effect of glucose concentration
The statistical analysis showed that the type of SS sur- The biofouling of E. coli ATCC 35218 after incuba-
face, the bacterial strain and the glucose concentration tion for 24 h on 3C and 3D surfaces was shown to be

Figure 2. The effect of glucose concentration (0%, 1%, 5%) and temperature (22
C, 37
C) on L. monocytogenes biofouling
(DA620) after incubation for 24 h (A, C) and 48 h (B, D). AISI 304 grade SS with different finishes was used: (3C) untreated; (3D)
polished; (BR) brushed and (EL) electropolished.
higher in NB without glucose addition (p < 0.05)
(Figure 1A). After 48 h incubation, the biofouling was
significantly lower in NB supplemented with 1% glu-
cose (p < 0.05) and there were almost no differences
in biofouling among the various SS finishes (Figure
1B). The biofouling of S. aureus ATCC 25923 was
greatly influenced by glucose concentration after 48 h
incubation, with statistically significant higher values
in NB with 5% glucose (p < 0.05), for all the surfaces
tested (Figure 1D). On the other hand, the glucose
concentration had different effects on P. aeruginosa
ATCC 27853 biofouling – after both time points at
37
C it was higher in NB without glucose addition
(p < 0.05) and decreased conversely with glucose con-
centration (Figure 1E and F). L. monocytogenes had
the highest biofouling potential among the tested
strains and under the conditions used. Therefore, all
the experimental conditions of this study (different SS
surfaces, glucose concentration and temperature) were
BIOFOULING 5
tested. After both incubation times at 22
C the bio-
fouling was the highest in NB without glucose add-
ition (p < 0.05), followed by lower values in NB
supplemented with 1% or 5% of glucose (Figure 2A
and B). A similar effect of glucose concentration was
observed also after incubation at 37
C, with the high-
est biofouling potential of individual surface in NB
without glucose and with significantly lower values in
NB supplemented with 5% glucose (Figure 2C
and D).
The effect of temperature
To evaluate the effect of temperature on biofouling,
the CV assay was performed after incubation in NB
medium without glucose addition for all the tested
strains. Incubation temperatures were 22
C and 37
C
for 24 and 48 h (Figures 1 and 2), and for 192 h in
the case of incubation at 4
C (data not shown). For
the BR and EL surfaces, the biofouling of E. coli
6 K. BEZEK ET AL.
Figure 3. SEM micrographs of the surface features of untreated (3C), polished (3D), brushed (BR) and electropolished (EL) surface
finishes of AISI 304 grade.
ATCC 35218 was higher at 37
C and reached the
highest values after 48 h incubation (p < 0.05)
(Figure 1A and B). By contrast, the biofouling of S.
aureus ATCC 25923 after 24 h was higher at 22
C
than at 37
C (p < 0.05), with the highest values on
the 3C surface (Figure 1C). After 48 h the biofouling
difference between the incubation temperatures was
not so obvious (Figure 1D). Among the tested
strains, P. aeruginosa ATCC 27853 reached the high-
est biofouling levels at 22
C after 24 h incubation
(p < 0.05) on almost all surfaces (Figure 1E).
Nevertheless, after 48 h the biofouling at 22
C
decreased so that the values were higher after the
incubation at 37
C, with no differences between the
surface finishes. The highest level of biofouling by
the strains at 22
C after 48 h incubation was shown
by L. monocytogenes. Nevertheless, the biofouling of
L. monocytogenes was higher after incubation at
37
C than at 22
C (p < 0.05) for all surfaces and
glucose concentrations tested (compare Figure 2A
with C and Figure 2B with D). Despite its psychro-
philic nature the biofouling on SS coupons was
strongly inhibited at refrigerator temperature (4
C).
Moreover, after a prolonged incubation time (192 h)
at 4
C, statistically significant differences in bacterial
adhesion and subsequent biofouling on any of the SS
coupons was not observed for any of the tested
strains (p > 0.05).
 BIOFOULING 7

Figure 4. SEM micrographs of polished (3D) and electropolished (EL) surface finishes of AISI 304 grade after incubation for 24 h
at 37
C with adhered bacterial cells.

Table 2. Bacterial biofouling of AISI 304 grade SS of different finishes.

24 h 48 h
192 h


37 C 37

Temperature 4
C 22
C 22
C
Glucose [%] 0 0 0 1 5 0 0 1 5
E. coli AISI 304 3C 0.03 ± 0.04 0.32 ± 0.03 0.44 ± 0.13 0.21 ± 0.02 0.19 ± 0.04 0.38 ± 0.07 0.41 ± 0.09 0.07 ± 0.03 0.41 ± 0.08
AISI 304 3D 0.03 ± 0.01 0.33 ± 0.06 0.32 ± 0.04 0.21 ± 0.02 0.19 ± 0.02 0.37 ± 0.07 0.27 ± 0.06 0.10 ± 0.05 0.33 ± 0.11
AISI 304 BR 0.01 ± 0.01 0.08 ± 0.02 0.25 ± 0.06 0.15 ± 0.04 0.15 ± 0.02 0.17 ± 0.04 0.40 ± 0.10 0.05 ± 0.02 0.31 ± 0.02
AISI 304 EL 0.03 ± 0.01 0.09 ± 0.01 0.21 ± 0.01 0.26 ± 0.02 0.27 ± 0.09 0.14 ± 0.04 0.51 ± 0.11 0.07 ± 0.02 0.65 ± 0.14
S. aureus AISI 304 3C 0.01 ± 0.00 0.39 ± 0.03 0.03 ± 0.02 0.05 ± 0.02 0.18 ± 0.04 0.18 ± 0.07 0.10 ± 0.02 0.03 ± 0.01 0.97 ± 0.12
AISI 304 3D 0.01 ± 0.00 0.10 ± 0.05 0.01 ± 0.00 0.06 ± 0.03 0.07 ± 0.02 0.08 ± 0.02 0.18 ± 0.07 0.08 ± 0.04 0.57 ± 0.05
AISI 304 BR 0.02 ± 0.01 0.08 ± 0.01 0.00 ± 0.01 0.05 ± 0.01 0.06 ± 0.02 0.09 ± 0.02 0.11 ± 0.02 0.06 ± 0.05 0.14 ± 0.01
AISI 304 EL 0.02 ± 0.01 0.07 ± 0.01 0.01 ± 0.01 0.04 ± 0.03 0.04 ± 0.03 0.05 ± 0.02 0.07 ± 0.02 0.04 ± 0.01 0.63 ± 0.11
P. aeruginosa AISI 304 3C 0.02 ± 0.03 1.16 ± 0.20 0.49 ± 0.11 0.61 ± 0.06 0.12 ± 0.02 0.07 ± 0.04 0.50 ± 0.19 0.39 ± 0.07 0.18 ± 0.04
AISI 304 3D 0.02 ± 0.01 0.99 ± 0.06 0.64 ± 0.18 0.14 ± 0.03 0.08 ± 0.02 0.07 ± 0.03 0.64 ± 0.17 0.30 ± 0.04 0.18 ± 0.06
AISI 304 BR 0.02 ± 0.02 0.86 ± 0.15 0.59 ± 0.10 0.15 ± 0.03 0.13 ± 0.04 0.08 ± 0.03 0.64 ± 0.21 0.29 ± 0.06 0.18 ± 0.06
AISI 304 EL 0.06 ± 0.02 1.04 ± 0.21 0.67 ± 0.19 0.13 ± 0.02 0.10 ± 0.02 0.13 ± 0.04 0.59 ± 0.16 0.19 ± 0.05 0.11 ± 0.01
Note: The effect of glucose concentration (0%; 1%; 5%), temperature (4
C; 22
C; 37
C) and surface roughness (not treated (3C); polished (3D); brushed
(BR) and electropolished (EL)). The results are presented as means ± SDs of the optical density readings (DA620) for the crystal violet assay.

Discussion strains. The bacterial strains in this study were

The control and prevention of biofouling on food end
medically related surfaces is of concern worldwide. In
spite of many studies conducted with a selection of
conditions and microorganisms, a general insight into
the biofilm formation process and its persistence in
different environments is still unclear. In many man-
made environments, where selected species are not
exposed to environmentally regulated restrictions, this
can lead to an advantage for one of the species and
consequently generates difficulties for the remainder
of the population in a particular niche. Since this is a
very complex field, researchers are forced to limit the
parameters to an array that can be handled. In this
study, the same assay of biofouling quantification was
performed for all of the selected strains, so the adap-
tive response to set factors could be evaluated prop-
erly. Biofouling on the SS surfaces, assessed by the
use of CV assay, was shown for all of the tested
selected on the basis of previous research work, where
the influences of surface roughness, hydrophobicity
and the zeta potential of glass on adhesion and bio-
film formation were studied (Bohinc et al. 2014;
Kurin
ci
c et al. 2016). Bohinc et al. (2014) showed that
the roughness of a glass surface has an important
influence on the amount of biofouling. The latter was
confirmed by SEM imaging of S. aureus adhering to a
P220 graduation where after the inoculated bacterial
cells were initially observed mainly in the cracks and
gaps they spread after 8 h over the whole flat surface.
It was assumed that the number of adhered cells was
affected by surface roughness in the first few hours
but this had little or an unexplainable effect after
incubation for 24 or 48 h (Bohinc et al. 2014).
Based on surface roughness measurements and
topography imaging the importance of both factors in
influencing bacterial adhesion and subsequent
 8 K. BEZEK ET AL.

biofouling was observed in the present study.

0.61 ± 0.11
0.67 ± 0.14
0.46 ± 0.10
0.81 ± 0.18
Note: The effect of glucose concentration (0%; 1%; 5%), temperature (4
C; 22
C; 37
C) and surface roughness (not treated (3C); polished (3D); brushed (BR) and electropolished (EL)). The results are presented as
Although the EL surface had the highest roughness

5
values, the surface with the most biofouling was the
untreated (3C) SS surface (Figures 1 and 2). As

0.72 ± 0.10
0.83 ± 0.17
0.80 ± 0.11
1.16 ± 0.10
shown by SEM imaging the greater affinity of bacter-
37

1

ial retention on the 3D compared to the EL surface of

AISI 304 grade was also observed (Figure 4). The
0.94 ± 0.01
1.20 ± 0.12
0.98 ± 0.27
0.97 ± 0.20
lower number of surface features on the EL surface
0

resulted in a more random distribution of bacteria.

As reviewed by Crawford et al. (2012) the roughness
48 h

parameters that measure height variation of the sur-

0.38 ± 0.06
0.20 ± 0.07
0.21 ± 0.05
0.19 ± 0.05

face do not offer an insight into the spatial distribu-

5

tion of surface features. Therefore, the visualisation of

the surface should be considered when identifying
0.27 ± 0.09
0.14 ± 0.02
0.18 ± 0.02
0.22 ± 0.06

correlations between bacterial adhesion and substra-

22
1

tum properties.
In addition to the genetic predisposition of bacteria
0.74 ± 0.12
0.69 ± 0.15
0.67 ± 0.12
0.92 ± 0.10

to form biofilms, this process can be affected greatly

0

by numerous environmental factors such as tempera-

ture, pH and the composition of the growth medium
(Shi and Zhu 2009; Zeraik and Nitschke 2012) or the
0.26 ± 0.09
0.47 ± 0.11
0.34 ± 0.09
0.31 ± 0.12

cell and contact surface properties (Belessi et al. 2011;

5

Kova
cevic et al. 2016). In this study the biofouling

represented as the optical density of bound dye was
0.76 ± 0.09
0.86 ± 0.11
0.82 ± 0.20
0.64 ± 0.17

significantly (p < 0.001) influenced by the bacterial

37
1

strain and glucose concentration. In the case of E.

coli, the addition of glucose (1%) after 48 h incubation
at 37
C inhibited biofouling (Figure 1B), which could
1.22 ± 0.07
0.94 ± 0.06
0.75 ± 0.07
0.97 ± 0.13
0

be due to catabolic repression as suggested by Jackson

Table 3. L. monocytogenes biofouling of AISI 304 grade SS of different finishes.

et al. (2002). Conversely, higher levels were observed

24 h

in NB supplemented with 5% glucose (Figure 1B),

0.34 ± 0.09
0.26 ± 0.09
0.27 ± 0.04
0.39 ± 0.02

while non-starved E. coli cells were shown to be more

mean ± standard deviation of optical density readings (DA620) for crystal violet assay.
5

adhesive (Haznedaroglu et al. 2008) and biofilm for-

mation on polystyrene was promoted by higher glu-
0.27 ± 0.05
0.23 ± 0.03
0.25 ± 0.01
0.39 ± 0.08

cose concentrations (Moreira et al. 2013). The study

22
1

of the effect of glucose concentration on S. aureus

biofouling showed higher levels in a nutrient richer
0.74 ± 0.12
0.61 ± 0.10
0.59 ± 0.03
0.80 ± 0.18

medium (Figure 1D) which was also demonstrated on

polystyrene surfaces by Zeraik and Nitschke (2012).
0

On the other hand, the addition of 1% and 5% glu-

cose inhibited P. aeruginosa biofouling after both
0.01 ± 0.03
0.01 ± 0.01
0.05 ± 0.04
0.03 ± 0.02
192 h

incubation times (Figure 1E and F). Independent

4
C
0

findings have shown that initial adhesion of

Pseudomonas spp. could be favoured by a lower level
Temperature
Glucose [%]

AISI 304 3D
AISI 304 BR
AISI 304 3C

AISI 304 EL

of nutrients due to an increase in bacterial surface

hydrophobicity (Chen et al. 2005). As shown by
Kyoui et al. (2016) the surface structure of L. monocy-
togenes biofilms on SS surface was also affected by
L. monocytogenes

glucose concentration. At lower concentrations a

rougher biofilm surface was observed, predictably as a
survival strategy to increase nutrient uptake (Kyoui
et al. 2016). The latter could be supported by the

present results, where a higher (5%) glucose concen-
tration resulted in a decrease in biofouling after 24 h
and 48 h incubation at both temperatures (Figure 2).
Temperature is one of the main critical control
points in the food processing industry which affects
microbial survival and therefore is of great import-
ance also in the medical field. As suggested by Herald
and Zottola (1988) the initial interaction between bac-
teria and substratum may be increased at a lower
temperature because of the more uniform properties
of polysaccharides. Even a short change in growth
temperature can affect protein synthesis and presum-
ably influences the surface characteristics that might
alter bioadhesive behaviour. It has also been proposed
that temperature stress could induce adhesion and an
adaptive response of the strains to low temperatures
(Zeraik and Nitschke 2012). It has also been observed
that the high temperature effect can even increase the
adherent nature of the biofilm to the surface due to
its ‘baking effect’ (Garrett et al. 2008). Although tem-
perature was not a significant factor (p ¼ 0.273) affect-
ing biofouling in this study, there were some
observed differences due to incubation temperature.
In the case of E. coli and S. aureus it was shown by
Pompermayer and Gaylarde (2000) that reduced tem-
peratures had little or no effect on biofilm formation,
whereas total adherent cell numbers were the same at
12
C and 30
C regardless of incubation time. In this
study similar results were obtained after 48 h incuba-
tion, showing no significant differences in biofouling
values between 22 and 37
C (Figure 1B and D).
Equal biofouling at the temperatures tested may indi-
cate that adhesion- or other surface-colonization fac-
tors are more effective than a lower temperature
(Andersen et al. 2010). Biofilm formation by P. aeru-
ginosa was shown to be inversely temperature corre-
lated, with higher levels at 22
C compared to 37
C
(Constantin 2009). In this study the latter was
observed only after 24 h incubation (Figure 1E), while
biofouling after 48 h was generally higher at 37
C
than at 22
C (Figure 1F). For L. monocytogenes bio-
fouling levels were increased by temperature (37
C)
(compare Figure 2A with 2C and Figure 2B with 2D)
that could be due to modified cell surface hydropho-
bicity as shown previously (Di Bonaventura et al.
2008). In the case of L. monocytogenes where all con-
ditions were tested the great complexity of the param-
eters influencing biofouling was shown. From the
array of tested parameters, refrigerator temperature
was shown to be one of the most effective ways to
reduce surface biofouling.
BIOFOULING 9
Processing industries (food, pharmaceuticals) as
well as the medical sector are constantly searching for
new solutions regarding contact materials, techniques
and surface design for particular applications. In this
endeavour, new methods for surface testing after
exposure to microbial agents are needed for studying
their intrinsic and extrinsic growth parameters. For
this reason, a comparison of results between different
publications is difficult or even impossible, since cru-
cial experimental data such as temperature and pH
values and information about material characteristics,
such as roughness and topography are missing. In
addition, most importantly, type strains which are
used as standards are neither recognized nor applied
in these analytical processes. All this makes research
and development rather demanding. There is a wide
range of variables that can be influenced and taken
into consideration when setting the key control points
of pathogen surveillance. To the authors’ knowledge,
this paper summarizes for the first time the response
of different biofilm forming bacteria when exposed to
a selection of the most common SS materials used in
medical and processing industries which can be influ-
enced by a wide range of variables. Furthermore, the
unexpected results such as biofouling fluctuations
through time and higher values on a surface that was
not the roughest requires further research work. To
obtain a better understanding of the observed com-
plexity of biofilm formation will require studies at a
molecular level as well as more information of how
bacterial physiology and cell wall topography is
affected by the residual surrounding materials.
Acknowledgements
The authors would like to thank Dr Barbara Jer
sek
(Biotechnical Faculty of the University of Ljubljana,
Slovenia) for providing the L. monocytogenes strain.
Disclosure statement
The authors declare no conflict of interest.
Funding
The authors thank the Slovenian Research Agency for sup-
port through grant L1-4067 and Iskra Pio d.o.o. We also
thank ARRS programmes P4-0092 and P3-0388.
References
Absolom DR, Lamberti FV, Policova Z, Zingg W, Oss CJ.
V, Neumann AW. 1983. Surface thermodynamics of bac-
terial adhesion. Appl Environ Microbiol. 46:90–97.
10 K. BEZEK ET AL.
Akbas MY. 2015. Bacterial biofilms and their new control
strategies in food industry. In: Mendez-Vilas A, editor.
The battle against microbial pathogens: basic science,
technological advances and educational programs.
[accessed 2017 Jun 6]. http://www.microbiology5.org/
microbiology5/book/383-394.pdf
Ammar Y, Swailes D, Bridgens B, Chen J. 2015. Influence of
surface roughness on the initial formation of biofilm. Surf
Coat Technol. 284:410–416. doi:10.1016/j.surfcoat.2015.07.062
Andersen TE, Kingshott P, Palarasah Y, Benter M, Alei M,
Kolmos HJ. 2010. A flow chamber assay for quantitative
evaluation of bacterial surface colonization used to inves-
tigate the influence of temperature and surface hydro-
philicity on the biofilm forming capacity of
uropathogenic Escherichia coli. J Microbiol Methods. 81:
135–140. doi:10.1016/j.mimet.2010.02.009
Barker J, Bloomfield SF. 2000. Survival of Salmonella in
bathrooms and toilets in domestic homes following sal-
monellosis. J Appl Microbiol. 89:137–144. doi:10.1046/
j.1365-2672.2000.01091.x
Belessi C-EA, Gounadaki AS, Psomas AN, Skandamis PN.
2011. Efficiency of different sanitation methods on Listeria
monocytogenes biofilms formed under various environmen-
tal conditions. Int J Food Microbiol. 145:S46–S52. doi:
10.1016/j.ijfoodmicro.2010.10.020
Blackledge MS, Worthington RJ, Melander C. 2013.
Biologically inspired strategies for combating bacterial
biofilms. Curr Opin Pharmacol. 13:699–706. doi:10.1016/
j.coph.2013.07.004
Bohinc K, Dra
zic G, Fink R, Oder M, Jev
snik M, Nipi
c D,
Godi
c-Torkar K, Raspor P. 2014. Available surface dic-
tates microbial adhesion capacity. Int J Adhes Adhes. 50:
265–272. doi:10.1016/j.ijadhadh.2014.01.027
Bohinc K, Dra
zic G, Abram A, Jev
snik M, Jer
sek B, Nipi
c
D, Kurin
ci
c M, Raspor P. 2016. Metal surface character-
istics dictate bacterial adhesion capacity. Int J Adhes
Adhes. 68:39–46. doi:10.1016/j.ijadhadh.2016.01.008
Boulane-Petermann L. 1996. Processes of bioadhesion on
stainless steel surfaces and cleanability: a review with spe-
cial reference to the food industry. Biofouling. 10:
275–300. doi:10.1080/08927019609386287
Carpentier B, Cerf O. 2011. Review-Persistence of Listeria
monocytogenes in food industry equipment and prem-
ises. Int J Food Microbiol. 145:1–8. doi:10.1016/
j.ijfoodmicro.2011.01.005
Chen MJ, Zhang Z, Bott TR. 2005. Effects of operating con-
ditions on the adhesive strength of Pseudomonas fluores-
cens biofilms in tubes. Colloids Surf B Biointerfaces. 43:
61–71. doi:10.1016/j.colsurfb.2005.04.004
Chen M, Yu Q, Sun H. 2013. Novel strategies for the pre-
vention and treatment of biofilm related infections. Int J
Mol Sci. 14:18488–18501. doi:10.3390/ijms140918488
Chuard C, Lucet JC, Rohner P, Herrmann M, Auckenthaler
R, Waldvogel FA, Lew DP. 1991. Resistance of
Staphylococcus aureus recovered from infected foreign
body in vivo to killing by antimicrobials. J Infect Dis.
163:1369–1373.
Constantin OE. 2009. Bacterial biofilms formation at air
liquid interfaces. Innov Romanian Food Biotechnol. 5:
18–22.
Crawford RJ, Webb HK, Truong VK, Hasan J, Ivanova EP.
2012. Surface topographical factors influencing bacterial
attachment. Adv Colloid Interf Sci. 178-182:142–149.
Cvetkovski S. 2012. Stainless steel in contact with food and
beverage. Metall Mater Eng. 18:283–293.
Di Bonaventura G, Piccolomini R, Paludi D, D’Orio V,
Vergara A, Conter M, Ianieri A. 2008. Influence of tem-
perature on biofilm formation by Listeria monocytogenes
on various food-contact surfaces: relationship with motil-
ity and cell surface hydrophobicity. J Appl Microbiol.
104:1552–1561. doi:10.1111/j.1365-2672.2007.03688.x
Donlan RM. 2002. Biofilms: microbial life on surfaces. Emerg
Infect Dis. 8:881–890. doi:10.3201/eid0809.020063
Donlan RM, Costerton JW. 2002. Biofilms: survival mecha-
nisms of clinically relevant microorganisms. Clin Microbiol
Rev. 15:167–193. doi:10.1128/CMR.15.2.167-193.2002
International Organization for Standardization Geneva
(ISO). 1997. EN ISO 4287:1997. Geometrical product
specifications (GPS) - surface texture: profile method -
terms, definitions and surface texture parameters.
Flemming H-C. 2011. Microbial Biofouling: unsolved prob-
lems, insufficient approaches, and possible solutions. In:
Flemming H-C, Wingender J, Szewzyk U, editors.
Biofilm highlights [Internet]. Springer Berlin Heidelberg;
p. 81–109. [accessed 2017 Jun 1] http://link.springer.
com/chapter/10.1007/978-3-642-19940-0_5.
Gandhi M, Chikindas ML. 2007. Listeria: a foodborne patho-
gen that knows how to survive. Int J Food Microbiol. 113:
1–15. doi:10.1016/j.ijfoodmicro.2006.07.008
Garrett TR, Bhakoo M, Zhang Z. 2008. Bacterial adhesion
and biofilms on surfaces. Prog Nat Sci. 18:1049–1056.
doi:10.1016/j.pnsc.2008.04.001
Haznedaroglu BZ, Bolster CH, Walker SL. 2008. The role
of starvation on Escherichia coli adhesion and transport
in saturated porous media. Water Res. 42:1547–1554.
doi:10.1016/j.watres.2007.10.042
Herald PJ, Zottola EA. 1988. Attachment of Listeria mono-
cytogenes to stainless steel surfaces at various tempera-
tures and pH values. J Food Science. 53:1549–1562. doi:
10.1111/j.1365-2621.1988.tb09321.x
Hirotsu C. 2017. Advanced analysis of variance. 1st ed. New
York: John & Wiley Series in Probability and Statistics.
Jackson DW, Simecka JW, Romeo T. 2002. Catabolite
repression of Escherichia coli biofilm formation. J
Bacteriol. 184:3406–3410. doi:10.1128/JB.184.12.3406-
3410.2002
Kova
cevic D, Pratnekar R, Godi
c-Torkar K, Salopek J,
Dra
zic G, Abram A, Bohinc K. 2016. Influence of poly-
electrolyte multilayer properties on bacterial adhesion
capacity. Polymers. 8:345. doi:10.3390/polym8100345
Knobloch JK-M, Von Osten H, Horstkotte MA, Rohde H,
Mack D. 2002. Minimal attachment killing (MAK): a ver-
satile method for susceptibility testing of attached bio-
film-positive and negative Staphylococcus epidermidis.
Med Microbiol Immunol (Berl). 191:107–114. doi:
10.1007/s00430-002-0125-2
Kubota H, Senda S, Nomura N, Tokuda H, Uchiyama H.
2008. Biofilm formation by lactic acid bacteria and resist-
ance to environmental stress. J Biosci Bioeng. 106:
381–386. doi:10.1263/jbb.106.381
Kurin
ci
c M, Jer
sek B, Klan
cnik A, Mo
zina SS, Fink R,
Dra
zic G, Raspor P, Bohinc K. 2016. Effects of natural

antimicrobials on bacterial cell hydrophobicity, adhesion,
and zeta potential. Arh Hig Rada Toksikol. 67:39–45.
doi:10.1515/aiht-2016-67-2720
Kyoui D, Hirokawa E, Takahashi H, Kuda T, Kimura B.
2016. Effect of glucose on Listeria monocytogenes
biofilm formation, and assessment of the biofilm’s
sanitation tolerance. Biofouling. 32:815–826. doi:10.1080/
08927014.2016.1198953
Limoli DH, Jones CJ, Wozniak DJ. 2015. Bacterial extracel-
lular polysaccharides in biofilm formation and function.
Microbiol Spectr. 3.
Lourenço A, de Las Heras A, Scortti M, Vazquez-Boland J,
Frank JF, Brito L. 2013. Comparison of Listeria
monocytogenes exoproteomes from biofilm and plank-
tonic state: Lmo2504, a protein associated with biofilms.
Appl Environ Microbiol. 79:6075–6082. doi:10.1128/
AEM.01592-13
Martinuzzi RJ, Salek MM. 2010. Numerical simulation of fluid
flow and hydrodynamic analysis in commonly used bio-
medical devices in biofilm studies. In: Angermann L, editor.
Numerical simulations - examples and applications in com-
putational fluid dynamics. InTech; [accessed 2017 Jun 1].
http://www.intechopen.com/books/numerical-simulations-
examples-and-applications-in-computational-fluid-dynamics/
numerical-simulation-of-fluid-flow-and-hydrodynamic-
analysis-in-commonly-used-biomedical-devices-in
Moreira JMR, Gomes LC, Ara ujo JDP, Miranda JM, Sim~oes
M, Melo LF, Mergulh~ao FJ. 2013. The effect of glucose
concentration and shaking conditions on Escherichia coli
biofilm formation in microtiter plates. Chem Eng Sci. 94:
192–199. doi:10.1016/j.ces.2013.02.045
Møretrø T, Langsrud S. 2017. Residential bacteria on surfa-
ces in the food industry and their implications for food
safety and quality. Compr Rev Food Sci Food Saf. 16:
1022–1041. doi:10.1111/1541-4337.12283
Nan L, Yang K, Ren G. 2015. Anti-biofilm formation of a
novel stainless steel against Staphylococcus aureus. Mater
Sci Eng C. 51:356–361. doi:10.1016/j.msec.2015.03.012
Naves P, del Prado G, Huelves L, Gracia M, Ruiz V, Blanco
J, Rodrıguez-Cerrato V, Ponte MC, Soriano F. 2008.
Measurement of biofilm formation by clinical isolates of
Escherichia coli is method-dependent. J Appl Microbiol.
105:585–590. doi:10.1111/j.1365-2672.2008.03791.x
O’Toole GA, Kolter R. 1998. Flagellar and twitching motil-
ity are necessary for Pseudomonas aeruginosa biofilm
development. Mol Microbiol. 30:295–304. doi:10.1046/
j.1365-2958.1998.01062.x
Pompermayer DMC, Gaylarde CC. 2000. The influence of
temperature on the adhesion of mixed cultures of
Staphylococcus aureus and Escherichia coli to polypropylene.
Food Microbiol. 17:361–365. doi:10.1006/fmic.1999.0291
Rasamiravaka T, Labtani Q, Duez P, El Jaziri M. 2015.
The formation of biofilms by Pseudomonas aeruginosa:
BIOFOULING 11
a review of the natural and synthetic compounds
interfering with control mechanisms. BioMed Res Int. 2015:
1. doi:10.1155/2015/759348
Raspor P, Jevsnik M. 2008. Good nutritional practice
from producer to consumer. Crit Rev Food Sci Nutr. 48:
276–292. doi:10.1080/10408390701326219
Sadekuzzaman M, Yang S, Mizan MFR, Ha SD. 2015.
Current and recent advanced strategies for combating
biofilms. Compr Rev Food Sci Food Saf. 14:491–509. doi:
10.1111/1541-4337.12144
Salgado CD, Calfee DP, Farr BM. 2003. Interventions to
prevent methicillin-resistant Staphylococcus aureus transmis-
sion in health care facilities: what works? Clin Microbiol
Newsl. 25:137–144. doi:10.1016/S0196-4399(03)80042-9
Schmidt RH, Erickson DJ, Sims S, Wolff P. 2012.
Characteristics of food contact surface materials: stainless
steel. Food Prot Trends. 32:574–584.
Shi X, Zhu X. 2009. Biofilm formation and food safety in
food industries. Trends Food Sci Technol. 20:407–413.
doi:10.1016/j.tifs.2009.01.054
Srey S, Jahid IK, Ha S-D. 2013. Biofilm formation in food
industries: a food safety concern. Food Control. 31:
572–585. doi:10.1016/j.foodcont.2012.12.001
Van Houdt R, Michiels CW. 2010. Biofilm formation and the
food industry, a focus on the bacterial outer surface: biofilm
formation and the bacterial outer surface. J Appl Microbiol.
109:1117–1131. doi:10.1111/j.1365-2672.2010.04756.x
Verran J, Jones M. 2000. Problems of biofilms in the food
and baverage industry. In: Walker J, Surmann S, and Jass
J. editors. Ind biofouling detect prev control. Chichester:
Wiley; p. 145–173.
Verran J, Rowe DL, Boyd RD. 2001. The effect of nano-
meter dimension topographical features on the hygienic
status of stainless steel. J Food Prot. 64:1183–1187. doi:
10.4315/0362-028X-64.8.1183
Vickery K, Pajkos A, Cossart Y. 2004. Removal of biofilm
from endoscopes: evaluation of detergent efficiency. Am J
Infect Control. 32:170–176. doi:10.1016/j.ajic.2003.10.009
Vickery K, Hu H, Jacombs AS, Bradshaw DA, Deva AK.
2013. A review of bacterial biofilms and their role
in device-associated infection. Healthc Infect. 18:61–66.
doi:10.1071/HI12059
Vogt RL, Dippold L. 2005. Escherichia coli O157:H7
outbreak associated with consumption of ground beef,
June-July 2002. Public Health Rep. 120:174–178. doi:
10.1177/003335490512000211
Whitehead KA, Verran J. 2006. The Effect of surface
topography on the retention of microorganisms. Food
Bioprod Process. 84:253–259. doi:10.1205/fbp06035
Zeraik AE, Nitschke M. 2012. Influence of growth media
and temperature on bacterial adhesion to polystyrene
surfaces. Braz Arch Biol Technol. 55:569–576. doi:
10.1590/S1516-89132012000400012