Using ozone to control biofilms

Abstract

The biocidal action of ozone on pure and mixed bacterial biofilms were assessed in laboratory experiments. The effect of ozone on the corrosion behavior of SAE 1020 carbon steel and on AISI type 304L stainless steel samples, as well as the possible synergistic effect of ozone/bromide mixtures, were also studied. Ozone was effective not only to kill bacteria but also to facilitate the detachment of bacterial biofilms formed on stainless steel. Preliminary experiments showed that ozone/bromide ion mixtures did not modify either the biocidal action or the corrosion behavior of the metals with respect to that obtained with ozone alone.

Full text

INTERNATIONAL BIODETERIORATION
&
BIODEGRADATION
International Biodeterioration
&
Biodegradation 43 (1999) 31-35
An innovative method for preventing biocorrosion through microbial adhesion
inhibition
Patricia S. Guiameta
,
*, Sandra G. Gomez de Saraviaa
,
Hector A. Videlab
"Bioelectrochemistry Section. INIFTA. C.C.16.Suc.4. 1900. La Plata, Argentina "Department
of"
Chemistry. Faculty
of
Pure Sciences, University
of
La Plata. 1900. La Plata, Argentina
Received 22 June 1998; received in revised form 7 October 1998; accepted
I
November 1998
Abstract
An immunonoglobulin solution containing IgA, IgG and IgM was used to prevent the formation of Pseudomol1asIluorescens (P.
fluorescens) biofilms on carbon steel and two different types of stainless steel (SS) of industrial grade. A marked inhibition of bacterial adhesion was found when
an immunoglobulin film was formed on the metal surface prior to the immersion of the sample in bacterial cultures. Microscopic techniques like scanning electron
microscopy (SEM) and environmental scanning electron microscopy (ESEM) \\"ere used for assessing adhesion inhibition. The electrochemical behavior of the
steels was evaluated by means of potentiodynamic runs, corrosion potential vs. time evolution and linear polarization resistance techniques applied in the presence
and the absence of the immunoglobulins. © 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction
The formation of a biofilm is the key to the alterations of conditions at a metal surface before the initiation of biocorrosion processes. Thus,
preventing the initial step of biofilm formation (bacterial adhesion) on metal surfaces would be one of the potential ways to avoid biocorrosion.
Otherwise, the adhesion of microorganisms to surfaces affects a wide variety of industrial systems and operations, such as cooling water systems,
water injection for oil recovery, reverse osmosis membranes, the permeability of oil reservoirs, etc.
Negative results of water systems biofilms are: i) a decrease in the system performance; ii) a lesser durability of the structural materials due to
localized corrosion; iii) important economic losses due to an increase in both fluid frictional resistance and heat transfer resistance (Videla,
1996a).
Microbial adhesion is also widely accepted as an important stage prior to the induction or initiation of biocorrosion (Characklis, 1981; Videla and
Characklis, 1992). Adhesion processes starting immediately after the immersion of a metal surface in an industrial or natural aqueous medium lead
to the formation of a biofilm. This is the result of an accumulation process, not necessarily uniform in time or space (Characklis and Marshall,
*
Corresponding author.
1990). The biofilm, containing ca. 95% of water in a matrix of expolysaccharidic substances (EPS), in which bacterial cells and inorganic detritus
are suspended, is the key factor to explain the physical, chemical and biological alterations at the metal/solution interface prior to the induction of
localized attack or to corrosion inhibition (Videla,1996b).
An attempt to inhibit microbial adhesion to metals by using corrosion inhibitor mixtures containing film formers, surfactants and demulsifiers has
recently been reported (Prasad, 1998).
It has been published (Michetti et aI., 1994) that monoclonal IgA is able to prevent the adhesion of Salmonella sp. to epithelial cells in the absence
of other immune or non-immune protective mechanisms.
IgA is one of the five major types of immunoglobulins in the human body. It is selectively present in seromucous secretions as a dimer stabilized
against proteolysis by combination with another protein (the secretory component) that has a single peptide chain of molecular weight 60.000
(Roitt, 1988). The IgA antibodies function in the organism is developed through its specific inhibition of the adhesion of microorganisms to the
surface of mucosal cells, thereby preventing their entry into body tissues.
A recent finding (US Patent 5505945, 1997) claims that directly applying an amount of an immunoglobulin solution containing a combination of
IgG, IgM and IgA is sufficient to prevent adhesion of a selected group of
0964-8305(99($ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PIT: S0964-8305(98)00065-1

32
P.S. Guiamet
el
al./International Biodeleriol'lltiol1
&
Biodegradation
43 (1999) 31-35
microorganisms consisting of Pseudon1onas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis to tissue surfaces or
biomaterials by preventing biofilm formation. In this respect, the factors that affect the adherence of a bacterial cell to the surface of a
biomaterial include the surface chemistry of the cell and material, as well as the composition of the adsorbed protein layer when the
material is exposed to organic fluids (Nagel et aI., 1996).
The present article preliminarily explores the capacity of immunoglobulin solutions of different concentrations to impede the adhesion of
P.fluorescens (a frequent filmforming microorganism of industrial water systems) to carbon steel and two different stainless steel surfaces
in an attempt to offer an innovative method for preventing the induction of biocorrosion on these metal surfaces.
2. Experimental
2.1. Bacterial cultures
An active bacterial culture of P.fluorescens (an industrial strain isolated from cutting oil emulsions) was prepared in Postgate C medium
(Postgate, 1984) from a 24 h old culture slant in Cetrimide agar, kept at 28°C in the laboratory. Purity of the stock strain was periodically
checked by means of Gram stains, oxidase test, other biochemical tests and plating, as has been described in a recent paper (Videla et aI.,
1996). The inoculum was prepared in an Erlenmeyer flask containing 150 ml of sterile medium. After 1 h of agitation in a rotary shaker,
the bacterial suspension containing ca. 106 cells/ml was ready for the immersion tests.
2.2. Metal samples
Samples of carbon steel (SAE 1020 type) and two different stainless steels (UNS $30400 and UNS $31600) were previously embedded in
an epoxy resin, leaving an exposed area of ca. 0.4 cm2 for the carbon steel and ca. 0.2 cm2 for the stainless steel. Later, metal specimens
were wet ground with increasing grits of silicon metallurgical paper (200, 400 and 500) and finally with alumina paste (l um grain size).
Prior to their use, the metal samples were cleaned and degreased with acetone and finally rinsed with ethanol and sterile distilled water.
2.3. Immunoglobulin solutions
A commercial solution of immunoglobulins (Igabulin, Immuno AG, Austria) was used. Each ml of the solution contained ca. 60 mg of
IgA, 30 mg of IgG and 2 mg of IgM, total protein (l00 mg) and glucose (100 mg) in a saline solution.
Although the experiments were generally made using
the commercial solution, occasionally, 1: 100 and 1: 1000 dilutions of the former solution were also used to check if the inhibition capacity
was diminished by dilution. To assess the specificity of immunoglobulins in the inhibition of bacterial adhesion, a commercial solution
containing 165mg/ml of gammaglobulin (Seroglobin-T, Gador, Argentina) was tested.
2.4. Immersion tests
The metal samples were immersed either in sterile medium or in P.fluorescens cultures. The metal surfaces were tested:
a) without immunoglobulin coverage;
b) after forming a film of immunoglobulins (exposing the surface to immunoglobulin solutions for 3 h) prior to the immersion in the
bacterial culture;
c) forming another film of immunoglobulins (exposing the surface to immunoglobulin solution for 30 min) after a previous exposure of the
conditioned metal surface to the bacterial culture for 3 h.
2.5. Sessile bacterial counts
Bacterial biofilms were removed from the metal coupons by scraping and later poured into 10 ml of sodium chloride isotonic solution. The
number of colony-forming units (CFU) was determined after 48 h of incubation of the plate count agar medium at 28°C, using the standard
plating method for viable counts. As recommended by Woolfson (1993), non-parametric statistical tests were used to assess the
significance of the results. Rank Sum and Median Tests were used, with a significance value of 5%.
2.6. Microscopic observations
Samples were examined using SEM after succesive fixation with 2% glutaraldehyde solution in phosphate buffer, a later dehydration
through an acetone series to 100% and finally critical-point drying. To avoid any interference of the sample treatments prior to SEM
microscopy, an environmental scanning microscope (ESEM) was also used. In this case, the sample remained humid until its observation.

ESEM was also used to verify that the immunoglobulin films were bound to the metal surfaces.
2.7. Electrochemical experiments
To assess any effect of immunoglobulins on the electrochemical behavior of the metals tested, potentiodynamic runs at 0.003 V Is,
corrosion potential evolution vs. time (for a period of 24 h) and linear polarization resistance experiments (at ±O.OlOV of the cor-
P.S.
Guiamet et al
. Int
ernational Biodeterioration
&
Biodegradation
43 (1999) 31-35
Fig.
I.
SEM micrograph of a type 31600 SS surface after exposure (24 h) to a P. fluorescens culture. Scale= 10 um.
Fig. 2. SEM micrograph of a SAE 1020 carbon steel surface after exposure (24 h) to a P. fluorescens culture. Scale= 10 um.
rosion potential) were made (Dexter et a!., 1991) with the three metals tested in this paper, using Postgate C medium +3% NaCI, as electrolyte. A
conventional double wall glass cell was used and the potentials were referred to the saturated calomel electrode (SCE).
3. Results and discussion
3.1. Immunoglobulin effects
all
bacterial adherence
In all cases, after 24 h of exposure to the bacterial culture, the coupons of the different metals tested showed a copious biofilm of P.fluorescens by
SEM. This biofilm was easier to observe on stainless steel surfaces (Figure I) than on carbon steel (Figure 2) due to the lack of corrosion products.
The average sessile population was
33
in the order of 109CFUjcm2 (Table 1). Metal coupons, previously conditioned with the immunoglobulin solution, revealed a dramatic decrease in
attached bacteria (Figs. 3 and 4) that, according to the plate counts, was in the order of 104 to 105 CFU jcm2 for the metal conditioned with the full
concentration immunoglobulin solution. Decreases in sessile bacterial numbers to the order of 106 CFU jcm2 for type 31600 SS and 104 (1: 100
dil.) and 105 (1:1000 dil.) CFUjcm2 for carbon steel were found. Variations to the standard procedure (exposure of the metal coupons to
immunoglobulin solution for 3 hand, thereafter, to bacterial cultures for 24 h) did not show different results. Those variations were:

a) shorter exposure times (1 min, 5 min and 10 min) of metal coupons to immunoglobulin solution and
b) longer exposures (72 h) of the pre-conditioned metal samples to bacterial cultures.
Stainless steel coupons, exposed to gammaglobulin solutions in the same experimental conditions as for immunoglobulin solutions, showed
markedly different results. In this case, biofilms were more copious than those of the control coupons exposed to the culture without any previous
conditioning of the surface (Figure 5). These results are sustained by a recent publication (Cook et a!., 1993) which reports an increase in the
number of adhered bacteria by about 45% when sorbed proteins were present on coated hydrogels. Our results are also a clear evidence that the
ability to avoid bacterial adherence to metal surfaces is specifically related to immunoglobulin fractions and, in particular, to IgA immunoglobulin.
This finding is also supported by several recent publications in the area of medical implants (Masinick et a!., 1997; Michetti et a!., 1994) and by a
widely documented knowledge on the specific effect of IgA against bacterial adherence to surfaces (Roitt, 1988; US Patent 5505945, 1997).
ESEM microscopy with hydrated samples ratified the SEM observations (Figs 6 and 7).
3.2. Immunoglobulin effects on the electrochemical behavior of steel
To determine if immunoglobulins were able to affect the electrochemical behavior of the steels tested in this paper, several electrochemical
experiments were made in the laboratory.
The evolution of the open circuit potential of the different metals in Postgate C medium + 3 % NaCl showed that the variation of the open circuit
potential with time was similar for samples with and without a coverage of immunoglobulins. In addition, the potential sequence of the open
circuit potential values (from noble to active) was ordered according to the corrosion resistant behavior of each steel tested (type 31600 SS > type
30400 SS> SAE 1020).
Potentiodynamic runs for stainless steel samples in
P.S. Guiamet et al. Intemational Biodeterioration
&
Biodegradation
43 (1999) 31-35
34
Table 1
Statistical evaluation of results of P.fluorescens growth on UN S31600 and SAE 1020 carbon steel surfaces with and without adsorbed immunoglobulins.
Metal Biofilm (CFUjcm2)After exposing to
immunoglobulin solution
(CFU/cm2)
After exposing to
immunoglobulin solution (1:100
dil.) (CFU/cm')
After exposing to
immunoglobulin solution (1:1000
dil.) (CFU/cm')
UNS 31600
SAE 1020
X: 4.2 x 109
SD: 3.2 x 109
X: 2.35 x 109
SD: 2.28 x 109
X: 1.1 x 105
SD: 1.1 x 105
X: 4.4 x 104
SD: 1.6 x 104
X: 1.0 x 106
SD: 3.6 x 105
X: 5.44 x
]04
SD: 6.5 x 104
X: 7.5 x 105
SD: 1.0 x 106
X: 1.8 x 105
SD: 1.0
x
105
Fig. 3. SEM micrograph of a type 31600 SS surface with a coverage of adsorbed immunoglobulins after exposure (24 h) to a P. fluorescens culture. Scale
=
10 um.

x.
Fig. 4. SEM micrograph of a SAE 1020 carbon steel surface with a coverage of adsorbed immunoglobulins after exposure (24h) to a
P.
fluorescens culture. Scale = 10
Um.
Postgate C medium + 3 % NaCl, showed no significant variations in the breakdown potentials
(E
b
of the two stainless steels, independently of the
presence or absence of immunoglobulin coverage of the samples. For both
Fig. 5. SEM micrograph of a type 31600 SS surface with a coverage of adsorbed gammaglobulin after exposure (24 h) to a P. fluorescens culture. Scale
=
10 um.
Fig. 6. ESEM micrograph of a type 31600 SS surface after exposure (24 h) to a P.fluorescens culture. Scale= 10 um.
35
P.S. Guiamet el a/./lnlernationa/ Biodelerioration
&
Biodegradation
43 (1999) 31-35

Fig. 7. ESEM micrograph of a type 31600 SS surface with a coverage of adsorbed immunoglobulins after exposure (24 h) to a P. fluorescens culture. Scale
=
10 um.
steels, the pre-conditioned samples showed the passive behavior region to be slightly more extended than for non-treated coupons.
Linear polarization measurements showed results in agreement with previous measurements, revealing no significant difference in current
densities for the treated and not-treated samples. More data on the immunoglobulin effects on the electrochemical behavior of steel can be found
in a recent publication (Videla et aI., 1998).
4. Conclusions
•The coverage of the different steels with an immunoglobulin film was effective for preventing bacterial adherence of P. fluorescens cells to the
metal surface under the different experimental conditions used in this paper.
•The inhibition of bacterial adherence is specifically attained with immunoglobulin IgA mixtures, containing lesser amounts of IgG and IgM,
whereas other globulin solutions increase the number of sessile bacteria adhered to the metal surface.
•These results, supported by recent findings published in the literature, show that immunoglobulin solutions could, potentially, be used to avoid
the attachment of bacteria to surfaces exposed either to organic fluids (biomaterials) or aqueous industrial environments to avoid the deleterious
effects of bacterial biofilms.
Acknowledgements
The authors wish to acknowledge lnmuno Argentina S.A. for the donation of the immunoglobulin solutions and to CONICET (National Research
Council of Argentina) and UNLP (University of La Plata) for financial support.
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