JOURNAL OF

ELSEVIER Journal of Microbiological Methods 21 (1995) 83-96

An electrochemical method for assessing on stainless steel

biodeposition

Laurence Boulangt-Petermann”,*, Marie-Niielle Bellon-Fontaineb, Bernard BarouxavC

“Ilgine S.A., Research Center, 2 Avenue Paul Girod, 73403 Ugine cedex, France blnstitut National de la Recherche Agronomique, Laboratoire de Genie de I’Hygiene et des Procedes

Alimentaires, 25 Avenue de la Republique, 91300 Massy, France ‘Laboratoire de Thermodynamique et Physico-Chimie Metallurgique, UA CNRS 29,

38402 Saint Martin d’Heres, France

Received 3 April 1994; accepted 4 April 1994

Abstract

The interactions between stainless steel surfaces and some biological materials are investigated using electrochemical techniques. Two lactic bacteria were studied: Leuconos- tot mesenteroides, which is encapsulated and biosurfactant (dextran) producing and Streptococcus thermophilus, which is not encapsulated. Adding bacteria (or simply dextran) in a NaCl-containing electrolyte in contact with stainless steel, provokes some significant variations in the electrode potential. Conversely, in potentiostatic conditions, some anodic current variations are recorded. This electrochemical response is very sensitive to the considered biological material, the bacterial concentration and the electrochemical parame- ters as well. A decrease in the electrode potential lowers both the values of current variations and the number of deposited bacteria. Due to the presence of the capsular biopolymer (dextran), the adhesion of Leuconostoc mesenteroides, is expected to be independent of the solid surface properties when no polarization is applied. The present results sug,gest that the surface polarization acts as an inhibitor of the dextran adsorption. Finally, electrochemical techniques appear to be a powerful tool for investigating the interaction between bacteria and metallic surfaces.

Keywords: Bacterial adhesion; Bacterial deposition; Dextran; Electrochemical techniques; Leuconostoc mesenteroides; Stainless steel

* Corresponding author. Tel.: +33 79893301. Fax: +33 79893500.

0167-7012/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDZ 0167-7012(94)00037-8

84 L. Boulangt-Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96

1. Introduction

Biofouling onto metallic surfaces is commonly observed in food industry [1,2]. The adhesion of microorganisms is an important source of possible contamination for any material in contact with the surface [3], which leads to significant economic problems [4].

Bacterial adhesion depends on the physical chemistry of the solid surface. Recent studies have reported on bacterial adhesion to steel surfaces, dealing with the relationship between the number of adhering bacteria and the solid surface properties [5,6]. In the food industry, the assessment of microbial populations on food contact surfaces allows the evaluation of the food surface hygiene [7]. However, observations on steels and other opaque surfaces are more difficult than on glass or translucide polymers, needing the development of some specific techniques, namely epifluorescence [8,9], scanning electron microscopy [4,10], impedance measurement [ll], culture of fouled samples [12], determination of the bacterial removal by ultrasound or by sampling [13,14].

However, some difficulties arise from the use of all these techniques: (i) the impedancemetry is in fact a measurement of the variations of the suspending liquid conductivity induced by the bacterial growth: no information on the physico-chemical properties at the steel/bacteria interface is given; (ii) the microscopic techniques generally deal with the analysis of a small surface area (about few mm*); (iii) in culture medium counting techniques, it is impossible to recuperate all the microorganisms adhering to the solid surfaces; the number of removal bacteria depends on some surface characteristics such as roughness or surface free energy and on the technique employed for recovering the adhering bacteria [15,16].

In this paper, an electrochemical approach is proposed, which enables detection of the deposition of biological products such as bacteria or organic macro- molecules to stainless steel surfaces. It is convenient at first to recall some basic features of the surface reactivity of the stainless steels in aqueous media [17]. First, a metal immersed in water tends to convert to its oxidized form, the reaction involving the transfer of metallic cations from the metal into the solution. The intensity (iA) of this anodic reaction depends on the difference in potential (V) established between the metal and the solution. Moreover, the electrons are consumed by the so called ‘cathodic reaction’, leading to a cathodic current (ik), which also depends on the potential difference (V). In the steady state, the sum Zo = i, + i, is obviously zero, and this occurs at the ‘rest potential’ V,,,,. On departure from V,,,, , for example in the presence of an imposed positive over-potential, V-V,,,, , the anodic reaction becomes predominant and the current density markedly depends on the physical chemistry of the passive film. However, it may happen that the metal is not oxidized to soluble cation, but rather forms a stable oxide or hydroxide, known as the ‘passive film’. In the case of stainless steel, this passive film is found to be a (Fe, Cr) oxi-hydroxide [17]. Its thickness (L) is of the order of some nanometers. A potential drop V, takes place in this film, leading to an electrochemical field F = V,IL of the order of lo8 V/m. The

L. Boulang&Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96 85

potential difference between the metal and the electrolyte is then V= V, + V,, (where V,, is the potential drop in the double layer) which differs form the case of ‘bare’ metals, where V, vanishes.

2. Experimental methods

2.1. Metallic surfaces

The material selected for the study was the AISI 304 stainless steel with a bright annealed finish (BA) which is commonly employed in industrial equipment manufacturing or domestic uses. Before experiment, all samples were soaked in an acetone/ethanol 95% (50:50 ratio) and air-dried.

2.2. Bioc’ogical materials

Two different bacteria Leuconostoc mesenteroides and Streptococcus ther- mophilw whose the surface properties are well characterized [18] and a polysac- charide (dextran) produced by Leuconostoc mesenteroides [19] were used as adhering materials.

Leucoivostoc mesenteroides NCDO 523 was originally isolated from a sugar refinery plant. Streptococcus thermophilus B was isolated from heat exchanger plates in the downward section of a pasteurizer and kindly provided by Dr. A.H. Weerkamp (NIZO , Ede , The Netherlands).

Freeze-dried cells were suspended in 1 ml MRS broth [20] supplemented with 1% sucrose. This suspension is inoculated fresh medium (9 ml), which is kept for 24 h at 22°C for Leuconostoc meseneroides and at 37°C for Streptococcus thermophilus.

This culture was used to inoculate a second culture which was grown for 16 h and then harvested by centrifugation for 15 min at 8400-X g and twice washed in a NaCl O,O2 M or 0.15 M solution (pH 6.6).

The dextran used in this study came from the industrial production (Sigma,M, 40,000-110,000). The following procedure was developed in order to compare the dextran production obtained in our experimental conditions to the usual rates of industrial strains published in the literature [21]. This technique was based on the dextran insolvability in ethanol. After a first centrifugation at 7500 x g for 15 min at 4°C the floating (containing dextran, proteins, saccharose and other macro- molecules with high molecular mass) was water mixed (v/v) and then centrifu- gated at 18,000 x g for 15 min at 4°C. A mixing of ethanol and the second floating (3 v/v) was centrifugated at 60,000 x g for 15 min at 4°C. The concentrate was then resuspended in ethanol (3 v/v) and centrifugated at 18,000 X g for 15 min at 4°C. The new concentrate was dried for 90 min at 100°C and weighted. A dextran concentration of 1.28 g/l was found for a 16 h culture of Leuconostoc mesen- teroides. Thus, for the further electrochemical assays, a dextran concentration of 1 g/l was chosen.

86 L. Boulangh-Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96

2.3. Electrochemical assays

The electrolyte used in this study was an aerated 0.15 M NaCl (pH 6.6) solution at 22°C for Leuconostoc mesenteroides and 37°C for Streptococcus thermophilus.

The electrochemical reactor is presented in Fig. 1. The area of the tested samples was equal to 4.15 cm’. The reference calomel electrode (SCE) was protected by an extension, the auxiliary electrode was made in platinum. The electrochemical reactor possessed double walls in order to control the tempera- ture in assays. The metallic sample was put horizontally in the reactor in order that the bacteria deposition to steel surfaces could occur. The electrochemical cell was connected to a EGG/PAR potentiostat/galvanostat Model 273 interfaced with a computer. The metal/solution potential difference was expressed in mV/SCE (standard calomel electrode) and the current densities measured with the auxiliary electrode were expressed in nA/cm*.

Two types of electrochemical experiments were conducted. First, the sample was left at rest potential V,,,, (i, = 0) and the bacteria introduced in the

Electrochemical reactor

Reference eleclrode

Electrode __

protection

Thermostable jacket 1

- 4 Platinum electrode

J Wring

Sample /q _ Sample holder

Samale holder

Sample

Sample contact

Joint SC&

Fig. 1. Schematic representation of the electrochemical reactor.

L. Boulang&Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96 87

electrochemical reactor induced a rest potential jump. Secondly, after 30 min at the rest potential, the samples were polarized for 3 h at a potential (VP) either higher (+200, +300 and +400 mV/SCE) or lower (-200 mV/SCE). The electrochemical current (i, > 0 when VP > V,,,, and i, < 0 when VP <V,,,,) is recorded as a function of the polarisation time $,. The bacteria were introduced at time t,,,, + t, = 45 min (leading to t, = 15 mm) and the variation of i, were recorded. It should be noticed that the standard error for current density measurements did not exceed 2 nA/cm*.

2.4. Adhesion assays

Immediately after the electrochemical experiment, the steel surfaces were washed for 15 min with a sterile water flow system (Filtron Apparatus) and then dehydrated in an ethanol solution. Samples were coated with a layer of gold by vacuum evaporation. A Zeiss DSM 950 operated at 15 kVwas used in the study at a 3000 magnification, 30 successive fields were examined corresponding to 2.8 lo-* mm2. Results ere expressed as the number of adhering bacteria per mm2.

3. Resuks

3.1. Evor’ution of the rest potential in presence of bacteria

Fig. 2 presents the evolution of the rest potential before and after the introduction of Leuconostoc mesenteroides (C = 5 * lo8 cells/ml) at time t, = 6 min. In the absence of bacteria, V,,,, slowly decreased, indicating a reorganisation for the metal/electrolyte interface. The bacteria introduction at t = t, induced a sharp increase in V,,,, followed by a plateau. The amplitude of the rest potential

jump AL, was of the order of 120 mV. At a first glance, this jump could be due to some increases either in the potential drop V, in the passive film, or in the double layer potential difference V,,,. In the first hypothesis, and considering a reasonable passive film thickness of the order of 3 nm [17], this potential jump corresponds to an electrical field jump AF,,,, of the order of 4. lo7 V/m to be

Fig. 2. Evolution of the rest potential (mV/CSE) in absence (curve 1) or in presence of bacteria (curve

2) as a function of time (s).

88 L. BoulangC-Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96

compared to the accepted order of magnitude of F which is close to lo* V/m. Another possibility is that the potential jump is due to a variation in the double layer potential drop V,,. These points will be discussed with more details in a further paper, since detailed analysis of the rest potential variations (even in absence of bacteria) would need more advanced theoretical considerations. Anyway, the present paper findings suggest an electrochemical effect resulting from the interaction between the solid surface and the bacteria. For the following, we chose to evidence this effect by measurements at some constant potentials, differing from the rest potential at the time where bacteria were introduced in the electrochemical reactor.

3.2. Chronoamperometry: typical behavior

First, one compared the current density obtained for VP > V,,,, in the absence or and in the presence of bacteria. As shown on Fig. 3, the current density first decreased evenly up to the bacteria introduction (phase 1). This phenomenon is well known in passive film electrochemistry and possibly corresponds to the passive film growth [22]. Once the bacteria were introduced in the reactor (phase 2), a sharp decrease in anodic current was observed (during some few seconds), followed by a relatively smoother increase up to a value i,. Last, the current stabilized at a constant value (phase 3), contrasting with the behaviour observed in the absence of bacteria (phase 1’) where a slow current decrease was observed.

This typical behaviour was observed in all the cases which were considered in

Current densities

0’

0 3

I I I

0 900 ime (s)

Fig. 3. Schematic representation of current densities measured before phase 1 and after (phase 3) the addition of bacteria (phase 2). The grey line represents the current density when no bacteria are added (phase 1’).

L. Boulang&Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96 89

this study. For the following, the current densities i,, i, and i, were determined (see Fig. 3) and the relevant parameters were found to be

A, := i,-i,

A, := i,-i,

A, and A, will be respectively referred to the ‘cathodic pulse’ and the ‘anodic current variation’, the order of magnitude for the measured currents are for i, 6 to 40 nA/cm*, i,-174 to 37 nA/cm’, i,-2 to 37 nA/cm’, A, 1 to 220 nA/cm2 and A, 3 to ‘i!3 nA/cm’. Table 1 and Figs. 4 and 5 present the results obtained when the considered bacteria is Leuconostoc mesenteroides, for different cellular concentrations and different imposed potentials VP. It can be seen that A, increased with C whatever the concentration between 3 * lo6 and 6. lo7 cells/ml. At the opposite, the anodic variations A, became independent of C when the concentration exceeded 6. lo6 cells/ml.

The effect of the applied electrode potential (V) (200,300 and 400 mV/ECS) is shown in Fig. 4 for A, and in Fig. 5 for A,. It is found that A, and A, also increase with V. This effect was more pronounced between 300 mV and 400 mV for A,.

The case where VP <V,,,, (VP = -200 mV/SCE) is somewhat different. First, the curr’ent density before the bacterial introduction is negative and nearby constant (i, - -3200 nA/cm*). Then, a very large cathodic pulse is observed: typically A, > 200 nA/cm’ for Leuconostoc mesenteroides at C = 6 * lo7 cells/ml,

Table 1 Summary of current values i,, i,, i,, A, and A, (nA/cm*) obtained in different experimental conditions: “logarithmic or bdecline phases, with ‘Streptococcus thermophilus or din presence of the macromolecule

Electrode potential (mV/ SCE)

400 400

400

300

200

400 400

Bacterial concentration (cells/ml)

6.3. lo6 a 6.3.10” b

3. lo6 6.106 3.10’ 6.10’ 3. lo6 6.106 3.10’ 6.10’ 3. lo6 6.106 3.10’ 6.10’ 3. lo7 c

lgr/ld

i, lb i, A, A,

43 20 1 42 23 40 18 -4 44 22 41 38 27 16 3 40 22 -3 43 18 39 21 -161 200 18 41 21 -159 220 20 13 11 9 4 2 12 7 -18 31 6 13 5 -112 125 8 16 7 -164 180 9 6 6 5 1 0 6 0 -14 20 6 9 2 -54 63 7 8 0 -152 160 8

40 14 - 145 185 26 40 36 -42 80 2

90 L. Boulangk-Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96

250

200

150

100

50

0

l,ooE+06 1 ,OOE+07

Bacterial concentration

l,OOE+OS

Fig. 4. Cathodic pulse A, (nA/cm*) as a function of the bacterial concentration (cells/ml) and the

electrode potential (mV/SCE) in NaCl 0.15 M (pH 6.6) solution.

A2 lO--

20 ,

8 --

6 --

4 --

2 --

0 1

l,ooE+06 l,OOE+07 l,OOE+08

Bacterial concentration

Fig. 5. Anodic variations A, (10’ nA/cm*) as a function of the bacterial concentration (cells/ml) and

the electrode potential (mV/SCE) in NaCl 0.15 M (pH 6.6) solution.

L. Boulangk-Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96

followed by a very slow decrease of ]iG] which reached to a value lower to i, nA/cm*)s after several hours.

3.3. Further results

91

(-50

The preceding results were obtained for Leucunostuc mesenteroides harvested in a logarithmic phase. Now it is worthy to present the effect of the bacterial metabolism, as far as Leuconostoc mesenteroides is concerned and the results obtained for other biological materials.

Influence of the bacterial metabolism. We tested two bacteria cultures of Leuconostoc mesenteroides with different metabolic states at a concentration of 6.3 1 lo6 cells/ml: (1) bacteria harvested in the logarithmic culture corresponds to the bacterial growth phase with a high metabolic excretion of lactic acid, acetic acid and carbonic gas [23,24] and (2) bacteria harvested in a decline phase; in this phase, only one bacteria for lo4 cells is still alive, then no bacterial excretion is expected.

Fig. 6 shows the electrochemical responses A, and A, obtained with both cultures. It can be seen that for a same bacterial concentration, there are no significant differences in cathodic pulse A, and anodic variation A, between the two cultures.

Influence of the biological materials. A second lactic strain Streptococcus thermophilus which is a surface property well-characterized bacteria [18] has been studied too. We also tested the dextran, a macromolecule produced by Leuconos-

Logarithmic phase Decline phase

Fig. 6. Electrochemical responses A, and A, (nA/cm’) obtained with bacteria harvested in the logarithmi,: and decline phases in NaCl 0.15 M (pH 6.6) solution. The bacterial concentration was equal to 6.3. lo6 cells/ml.

92 L. Boulangk-Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96

1000 -

r f 100 --

3 z .Y

8

3 4 10 -- w

It i

st Lm Dextmn

Fig. 7. Electrochemical responses A, and A, @A/cm’) obtained in NaCI 0.15 M (pH 6.6) solution with two lactic strains Leuconostoc mesenteroides (Lm), Streptococcus thermophilus (St) and with the dextran, polymer produced by Leuconostoc mesenteroides. The bacterial concentration was equal to 4. lo7 cells/ml. The dextran concentration was equivalent to one harvested in a Leuconostoc mesenteroides culture [20,21].

tot mesenteroides [19,21]. The bacterial concentration for both strains was 4.10’ cells/ml. The electrochemical responses A, and A, are presented according to the bacteria and the macromolecule in Fig. 7.

It was found that no significant difference in A, and A, were observed between Leuconostoc mesenteroides and Streptococcus thermophilus in the tested con- ditions. Moreover, adding dextran also produces the typical electrochemical responses presented in Fig. 3, even if the measured values of A, and A, are not exactly the same that for Leuconostoc mesenteroides (A, is slightly smaller and A, is one order of magnitude smaller).

Correlation between the number of adhering bacteria and the imposed potential. The number of adhering bacteria (for C = lo7 cells/ml) depends on the electrode potential as shown in Table 2. This number increases with the electrode potential whatever the sign of (VP-V,,,,).

4. Discussion

It was shown in this work that adding lactic bacteria in aqueous electrolytes in contact with metallic surfaces produces an electrochemical response, what we believed to be typical of the metal-bacteria interaction.

Contrasting with some previous works in microbiologically induced corrosion

L. BoulangtCPetermann et al. I Journal of Microbiological Methods 21 (1995) 83-96 93

Table 2 Number of adhering Leuconostoc mesenteroides par mm* to stamless steel with a BA finish in a NaCl 0.15 M solution et the electrode potential (mV/SCE)

Electrode potential (mV/SCE)

Number of adhering bacteria

-200 85 200 140 400 280

[25-271, no effect of the bacterial metabolism was found. It is worth pointing out that Leuconostoc mesenteroides and Streptococcus thermophilus are lactic strains excreting metabolites such as lactic acid, acetic acid, and carbonic gas [23,24], much less corrosive for stainless steel than ones produced by the sulfate-reducing bacteria.

Furthermore, adding dextran instead of Leuconostoc mesenteroides produces the same qualitative effect, suggesting a form of chemical interaction between the bacteria or the biopolymer and the metal substrata. Summarizing the results obtained in potentiostatic conditions, a cathodic pulse is further observed on the anodic current versus time, followed by a stabilisation of this current. This cathodic pulse is believed to result from some modification occurring in the electrochemical double layer at the metal surface when the bacteria or biopolymer are added. When the macromolecule or microorganism approaches the steel surface, the ionic double layer associated to the dextran or bacterial cell will be able to disturb for a short while the electrochemical equilibrium at the passive film/solution interface and consequently the anodic or cathodic reactions.

It is worth pointing out that both bacterial strains presented in this study possess a different surface composition. The Streptococcus thermophilus surface is composed highly by proteins; whereas the Leuconostoc mesenteroides surface presents a higher polysaccharide content [18]. Therefore, a slightly higher variation A, was obtained with Leuconostoc mesenteroides. So, the double layer associated with the bacterial surface could be different in ionic composition. Then, the proposed technique is able to differentiate a deposed bacterial strain from another. So the variation A, depending of the bacterial concentration can constitute a good indicator for the detection of the biological material approach to steel surfaces.

Concerning AZ, one should not that the values obtained when adding dextran differs from the one measured with Leuconostoc mesenteroides. These results, in addition with the fact that the anodic current remains constant after a long time, suggest a diffusion limiting step throughout the bacteria or biopolymer deposit. The baa;erial deposition to steel surfaces should be denser and thicker than one created by the dextran and could explain the strong anodic reaction restriction as can be schematized in Fig. 8.

In comparison with the microscopic observations [4,7-111, the above-men- tioned method allows to measure the biocontamination deposition on all the surface area. This technique is much faster than culture counting [12] and

94 L. Boulangk-Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96

5 Deposition

1 Deposition

c {f@Jj+- Dextran

Fig. 8. Schematic representation of metal/macromolecule (A), metal/Streptococcus rhermophilus) (B) and metal/Leuconostoc mesenteroides (C) interactions.

microscopic observations [4,7-111. Another advantage is to measure in situ the number of deposing bacteria onto steel surfaces compared with the sampling techniques [13,14]. Finally, our technique supplies a sensitive signal as soon as the bacteria are approaching the metal surfaces. On the contrary, the impedance measurements [ll] are only available when the biological material deposition has been achieved.

In previous work [28], it has been shown that Leuconostoc mesenteroides was not a sensitive surface energy bacteria. Its adhesion behaviour was explained by a possible adsorbing of dextran on the solid substrata and masking the differences in physical chemistry of these surfaces. Generally, a conformational change occurs during the adsorption of macromolecules to surface [29-321. The surface polariza- tion could act as an inhibitor of the dextran effect described above, explained by a modification of dextran conformation induced by the electric field present in the passive film at the stainless steel surface. It would be possible that the macro- molecule stabilizes its new conformation by incorporation of sodium ions into its structure, displacing water [32]. In that case, the bacterial surface is not still masked by the dextran, and the own physical chemistry of the bacterial surface could also occur in the adhesion phenomena. Future work will be devoted to the study of the possible relations between the modifications of adhesion phenomena and the polarization effect, taking into account the proper electrochemistry of the underlying passive film.

5. Conclusion

In this study, we developed a new electrochemical and sensitive technique which is able to detect the approach and the deposition of microorganisms to steel surfaces. The electrochemical responses defined by the parameters A, and A, are dependent on the biological material used such as the bacterial strain or the macromolecule, on the potential imposed to the metal and on the bacterial

L. Boulan&Petermann et al. I Journal of Microbiological Methods 21 (1995) 83-96 95

concentration. A, and A, constitute relevant indicators to study the bacterial approach and deposition to the steel surface. Future works will be devoted to the understanding of the determining mechanism for the electrochemical effects. Lastly, it was shown that decreasing the electrode potential both lowers the value of A, and lthe number of deposited bacteria. This point will be also discussed in a forthcoming paper.

Acknowledlgement

L.B.-P. is greatly indebted to Miss S. Evallet for technical assistance.

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