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Reversibility of Bacterial Adhesion at an Electrode Surface Albert T. Poortinga, Rolf Bos,† and Henk J. Busscher* Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands Received November 29, 2000. In Final Form: January 31, 2001 Deposition of four bacterial strains from a 1 mM potassium phosphate buffer (pH 7) to an indium tin oxide (ITO) electrode surface has been studied in a parallel plate flow chamber at three electrode potentials (-0.2, 0.1, and 0.5 V). Capacitance measurements demonstrated that the ITO surface was negatively charged with respect to the solution at the electric potentials applied, that is, bacteria deposited under repulsive electrostatic conditions. Initial deposition rates were independent of the electrode potential, except for Actinomyces naeslundii T14V-J1. Application of a more negative electrode potential yielded increased desorption of Streptococcus oralis J22, Staphylococcus epidermidis 3399, and A. naeslundii 147, whereas for A. naeslundii T14V-J1 desorption decreased. If a high (g65 µA) cathodic current was applied by adjusting the potential between -0.4 and -0.5 V, adhering bacteria were stimulated to desorb with desorption probabilities increasing with increasing current density. Bacterial desorption could be described on the basis of the Derjaguin-Landau-Verwey-Overbeek theory assuming secondary minimum adhesion, except for A. naeslundii T14V-J1. When bacteria were forced to adhere in the primary minimum by application of a high (1.8 V) positive electrode potential during deposition, adhering bacteria could hardly be stimulated to desorb, indicating strong, irreversible adhesion. The deviating behavior of A. naeslundii T14V-J1 was attributed to direct contact between its relatively long surface appendages and the electrode surface.
Introduction Bacterial adhesion can be described by the DLVO (Derjaguin-Landau-Verwey-Overbeek) theory in which adhesion is envisaged as an interplay of Lifshitz-van der Waals and electrostatic interactions between the interacting surfaces.1 In the so-called XDLVO theory, Lewis acid-base interactions are accounted for in addition to the classical DLVO interaction forces.2 Electrostatic interactions in bacterial adhesion have been mostly investigated by studying adhesion to substrata with different surface potentials3 or to electrode surfaces4,5 or by carrying out experiments as a function of ionic strength6,7 or pH.8 Generally, bacterial adhesion is observed also when electrostatic repulsion between the interacting surfaces causes a potential energy barrier calculated from the DLVO theory that must be considered insurmountable to bacteria.6,7 Consequently, several authors have concluded that initial bacterial adhesion is a secondary minimum phenomenon characterized by adhesive energies that can be derived from measurements of the bacterial desorption * Corresponding author. Tel: + 31 50 3633140. Fax: +31 50 3633159. E-mail:
[email protected]. † Present address: Friesland Coberco Dairy Foods, Corporate Research, Harderwijkerstraat 41006, 7418 BA Deventer, The Netherlands. (1) Bos, R.; Van der Mei, H. C.; Busscher, H. J. FEMS Microbiol. Rev. 1999, 23, 179. (2) Van Oss, C. J. Interfacial Forces in Aqueous Media; Marcel Dekker: New York, 1994. (3) Harkes, G.; Feijen, J.; Dankert, J. Biomaterials 1991, 12, 853. (4) Morisaki, H.; Nakagawa, K.; Shiraishi, H. Colloids Surf., B 1996, 6, 347. (5) Poortinga, A. T.; Bos, R.; Busscher, H. J. J. Microbiol. Methods 1999, 38, 183. (6) Sjollema, J.; Van der Mei, H. C.; Uyen, H. M. W.; Busscher, H. J. J. Adhes. Sci. Technol. 1990, 4, 765. (7) Poortinga, A. T.; Bos, R.; Busscher, H. J. Colloids Surf., B 2000, 20, 105. (8) Meinders, J. M.; Van der Mei, H. C.; Busscher, H. J. J. Colloid Interface Sci. 1994, 164, 355.
probability.9 The kinetics of bacterial adhesion to surfaces can be described through a decrease in bacterial concentration in suspension using10
(
)
d dc cD dVtot dc ) D + dt dh dh kT dh
(1)
in which c is the concentration of bacteria, t is the time, D is the bacterial diffusion coefficient, k is Boltzmann’s constant, T is the absolute temperature, Vtot is the potential energy of the bacteria, and h is the surface to surface distance between bacteria and the substratum. In equilibrium, when the concentration of bacteria is constant in time, desorption is counterbalanced by deposition and an equilibrium desorption probability βeq can be calculated from eq 1:11
βeq )
j0 ( cb
∫hh
2
1
exp(-Vtot(h)/kT) dh)-1
(2)
in which j0 is the initial deposition rate, cb is the bulk concentration of bacteria in suspension, and h1 and h2 represent the lower and upper limits for the separation distance between adhering bacteria and the surface. Meinders et al., assuming adhering thermophilic streptococci to reside in a rectangular well of 10 nm width and using eq 2, derived secondary interaction minima of on average -15 kT from measured desorption probabilities.9 Norde and Lyklema showed that the density of adhering Arthrobacter, Escherichia coli, Micrococcus, and Pseudomonas strains followed a Langmuir isotherm, and an average interaction energy of about -3 kT was calculated, which would typically be the depth of a secondary minimum and (9) Meinders, J. M.; Van der Mei, H. C.; Busscher, H. J. J. Colloid Interface Sci. 1995, 176, 329. (10) Dahneke, B. J. Colloid Interface Sci. 1975, 50, 194. (11) Van de Ven, T. G. M. Colloidal Hydrodynamics; Academic Press Ltd.: London, 1989.
10.1021/la001673y CCC: $20.00 © 2001 American Chemical Society Published on Web 04/03/2001
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allow reversible adhesion.12 Others have stimulated bacterial desorption by decreasing the bacterial concentration in suspension,13 applying high shear forces using fluid flow,14 passing air bubbles over the adhering bacteria,15 or lowering the ionic strength of the suspending fluid to increase electrostatic repulsion.16 The role of electrostatic interactions in bacterial adhesion and its reversibility can be elegantly studied at electrode surfaces, especially because the chemical composition of an electrode surface does not change while its surface potential can be readily varied during an experiment. By changing the electrode potential, bacterial adhesion can be studied under either attractive or repulsive conditions through which bacterial adhesion can be directed either in the primary or in the secondary minimum, respectively. Moreover, the electrostatic repulsion between negatively charged bacterial cell surfaces and an electrode surface can be increased to stimulate desorption of adhering bacteria by making the electrode surface more negative. Stimulated desorption implies the existence of nonequilibrium conditions, and from eq 1 a stimulated desorption probability βst can be calculated according to10
βst ) D∞ f(v)
∞ h exp(-Vtot(h)/kT) ∫h)h ∫>v)h f∞ 2
1
exp(Vtot(v)/kT) dv dh (3)
in which D∞ is the bulk bacterial diffusion constant which can be calculated from the Einstein equation,10,17 f is the bacterial friction coefficient that accounts for viscous interaction between the bacterium and the electrode,10 and f∞ is the bulk friction coefficient. The aim of this paper is first to study adhesion of four different bacterial strains at an electrode surface in a parallel plate flow chamber as a function of the applied electrode potential to investigate the role of electrostatic interactions in bacterial adhesion. As a second aim, the reversibility of bacterial adhesion will be studied by stimulating adhering bacteria to desorb by applying a negative electrode potential. Materials and Methods Bacterial Strains, Culturing, and Surface Characterization. Experiments were conducted using Streptococcus oralis J22, Staphylococcus epidermidis 3399, and the rod-shaped Actinomyces naeslundii T14V-J1 and Actinomyces naeslundii 147. Staph. epidermidis 3399 and Strep. oralis J22 were cultured at 37 °C in ambient air in brain heart infusion and in Todd Hewitt broth, respectively. A. naeslundii T14V-J1 and A. naeslundii 147 were cultured in Schaedler’s broth supplemented with 0.01 g/L hemin in an anaerobic cabinet (DW Scientific, West Yorkshire, U.K.) in an atmosphere of 10% H2, 85% N2, and 5% CO2 also at 37 °C. For each experiment, strains were inoculated from blood agar plates in a batch culture for 24 h. This culture was used to inoculate a second culture which was grown for 16 h prior to harvesting. (12) Norde, W.; Lyklema, J. Colloids Surf. 1989, 38, 1. (13) Busscher, H. J.; Uyen, M. H. M. J. C.; Weerkamp, A. H.; Postma, W. J.; Arends, J. FEMS Microbiol. Lett. 1986, 35, 303. (14) Lyklema, J.; Norde, W.; Van Loosdrecht, M. C. M.; Zehnder, A. J. B. Colloids Surf., B 1989, 39, 175. (15) Go´mez Sua´rez, C.; Noordmans, J.; Van der Mei, H. C.; Busscher, H. J. Langmuir 1999, 15, 5123. (16) Rijnaarts, H. H. M.; Norde, W.; Lyklema, J.; Zehnder, A. J. B. Colloids Surf., B 1999, 14, 179. (17) Van der Mei, H. C.; Meinders, J. M.; Busscher, H. J. Microbiology 1994, 140, 3413.
Bacteria were harvested by centrifugation (5 min at 10 000g), washed twice with demineralized water, and, for the deposition experiments, resuspended in phosphate buffer solution (1 mM, pH 7.0) to a concentration of 3 × 108 bacteria/mL for actinomyces or 6 × 108 mL-1 for streptococci and staphylococci. Streptococci and actinomyces were sonicated at 30 W for 30 s while cooling on an ice/water bath, to obtain single cells (Vibra Cell model 375, Sonics and Materials Inc., Danbury, CT) prior to resuspending. Electrophoretic mobilities of the bacterial strains were measured in potassium phosphate buffer as a function of ionic strength (1-75 mM, pH 7.0) at room temperature using a Lazer Zee meter (PenKem, Bedford Hills, NY) equipped with an image analysis option for tracking and ζ sizing.18 For ionic strengths above 1 mM, electrophoretic mobilities as a function of potassium phosphate concentration were fitted to a “soft” particle model according to19
µ)
λ 21 + λ/2κ zeN 1+ κ 1 + λ/κ ηκ2
[ ()
]
(4)
in which µ is the electrophoretic mobility, κ is the inverse Debye length, η is the solution viscosity, e is the electron charge, N is the density of charged groups inside the ion-penetrable bacterial surface layer, z is the valency of the charged groups, which in the present study is set equal to -1, and λ-1 is the electrophoretic “softness” of the bacterium, which is a measure for the ease with which fluid can flow through the ion-penetrable bacterial surface layer during electrophoresis. Equation 4 is valid as long as the electric potential inside the ion-penetrable layer does not exceed 25 mV, that is, in high ionic strength solutions. By use of the densities of charged groups obtained from eq 4, bacterial cell surface potentials in a low ionic strength solution of 1 mM potassium phosphate buffer were then calculated from19
ψ0 )
kT zN zN + 1+ ln ve 2vn 2vn
( (
2n zN ( ( ) ) ) + zN (1 - (1 + (2vn ) ) )) 2 1/2
2 1/2
(5) where for v and n the valency and concentration of the main counterion (i.e., the potassium ion) are substituted and e is the electron charge. Electrode Material and Characterization. Transparent indium tin oxide (ITO) coated glass plates produced by dc sputtering (Philips Components, Heerlen, The Netherlands) were used as electrodes. The ITO films (thickness of 60 nm) were n-type semiconductors and had a sheet resistance of 30 Ω/0. Each experiment was carried out with newly prepared ITO-coated glass, cleaned by sonication in methanol (2 min), followed by thorough rinsing with tap water and sonication in Millipore filtered deionized water (also 2 min). The capacitance of the ITO electrode-solution interface as a function of the applied potential was measured in 1 and 25 mM potassium phosphate buffer solutions in the parallel plate flow chamber as during bacterial deposition (see below). The working area of the electrode was decreased to about 2% of the area of the counter electrode to eliminate the contribution of the counter electrode to the impedance measured. Applied electric potentials were measured with respect to a Ag/AgCl reference electrode (Ref201, Radiometer Copenhagen, Lyon, France) and ranged from -0.3 to 1.2 V. Briefly, a sinusoidal potential difference (50 mV amplitude) was applied at frequencies ranging from 20 to 800 Hz over the electrodes in series with a potentiostat and a resistor (390 and 180 Ω for the 1 or 25 mM solutions, respectively) to obtain the impedance (assumed to be a capacitor and a resistor in series) of the system. The potential difference across the diffuse double layer at the electrode surface was calculated by fitting the measured capacitance as a function of the applied electrode potential to the model of Natarajan et al.,20 assuming a dielectric permittivity for the ITO surface of 150,21 in which 0 is the permittivity of (18) Wit, P. J.; Busscher, H. J. Colloids Surf., A 1995, 125, 85. (19) Ohshima, H. Adv. Colloid Interface Sci. 1995, 62, 189. (20) Natarajan, A.; Oskam, G.; Searson, P. C. J. Phys. Chem. B 1998, 102, 7793.
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a vacuum. Effects of specific adsorption of phosphate ions at the Stern plane and nonlinearity of the diffuse double layer capacitance22 were included in the model, and the diffuse double layer was assumed to consist of potassium ions only. Bacterial Deposition and Desorption Experiments. Deposition of bacteria was measured to the bottom plate (5.5 × 2.8 cm) of a parallel plate flow chamber with a channel height of 0.06 cm.23 Top and bottom plates of the flow chamber consisted of ITO-coated glass plates with the coated side in contact with the solution. Deposition was observed with a CCD-MXR camera (High Technology, Eindhoven, The Netherlands) mounted on a phase contrast microscope (Olympus BH-2) equipped with a 40× ultra long working distance objective (Olympus ULWD-CD Plan 40 PI). This system allowed direct observation of a field of view covering about 0.014 mm2. Measurements were conducted in duplicate with bacteria suspended in 250 mL of potassium phosphate buffer (1 mM, pH 7.0) at room temperature. A pulse free flow (0.02 mL/s) was created by hydrostatic pressure, which produced a wall shear rate of 12 s-1 (Reynolds number of 0.9, within the limits of laminar flow). Bacterial deposition was studied at three different electrode potentials, -0.2 V, 0.1 V (i.e., the open circuit potential of the ITO), and 0.5 V applied to the bottom ITO plate for 4 h, long enough to reach an equilibrium number of adhering bacteria. At the electrode potentials applied, the electric current was less than 1 µA. Also, for each bacterial strain, a short (5 min) deposition experiment was conducted while applying a high, positive potential of 1.8 V, which resulted in a much higher electric current of about 2 mA. During deposition experiments, the total number of deposited bacteria per unit area n(t) and the number of desorbed bacteria per unit area and time jdes(t) were directly measured using image sequence analysis.24 From the initial, linear increase in time of the number of deposited bacteria per unit area, the initial deposition rate j0 was determined. Also, the equilibrium number of adhering bacteria neq and the equilibrium desorption rate jdes,eq were calculated from images taken over the last hour of an experiment. The equilibrium desorption probability βeq was calculated from
βeq ) jdes,eq/neq
(6)
At the end of each deposition experiment, the flow was switched to buffer without suspended bacteria, and after about 5 min, electric currents ranging from 65 to 400 µA were applied by adjusting the potential and using the bottom plate as a cathode, while the number of adhering bacteria was monitored. Stimulated desorption probabilities βst were subsequently determined from the decrease in the number of adhering bacteria per unit time divided by the number of adhering bacteria at that time. Stimulated desorption probabilities were determined at several points in time, that is, when different numbers of adhering bacteria were left at the electrode, during the stimulated desorption.
Results Characterization of Bacteria and Electrode. Figure 1 shows the electrophoretic mobility of the bacterial strains as a function of potassium phosphate concentration. The electrophoretic softness λ-1 and density of charged groups N as obtained by fitting eq 4 to the data in Figure 1 are compiled in Table 1, together with the shape and size of the strains. The electrophoretic softness of the strains varies between 1.3 and 1.6 nm for three out of the four strains, indicating a relatively hard surface. Only Staph. epidermidis 3399 is considerably softer than the other (21) Albery, W. J.; O’Shea, G. J.; Smith, A. L. J. Chem. Soc., Faraday Trans. 1996, 92, 4083. (22) Hunter, R. J. Zeta Potential in Colloid Science; Academic Press Ltd.: London, 1981. (23) Sjollema, J.; Buscher, H. J.; Weerkamp, A. H. J. Microbiol. Methods 1989, 9, 73. (24) Wit, P. J.; Noordmans, J.; Busscher, H. J. Colloids Surf., A 1997, 125, 85.
Figure 1. Electrophoretic mobility as a function of potassium phosphate concentration for the bacterial strains used: (2) Strep. oralis J22, ([) Staph. epidermidis 3399, (b) A. naeslundii T14V-J1, and (9) A. naeslundii 147. The lines denote fits according to the soft particle model (eq 4). Table 1. Characteristics of the Bacterial Strains Used, Including the Electrophoretic Softness λ-1, the Density of Charged Groups N, Bacterial Dimensions, and Bacterial Cell Surface Potentials in 1 mM Potassium Phosphate Buffer ψ0 Obtained from Equation 5 strain
shape
dimensa λ-1 N ψ0 (µm) (nm) (mM) (mV)
Strep. oralis J22 Staph. epidermidis 3399 A. naeslundii T14V-J1 A. naeslundii 147
sphere sphere rod rod
0.5 0.6 3 × 0.7 3 × 0.7
1.6 3.2 1.4 1.3
22 21 22 26
-27 -22 -27 -30
a For spherical bacteria, radii are given, whereas for rod-shaped bacteria length × width is presented.
strains. Bacterial surface potentials in 1 mM potassium phosphate buffer as derived from eq 5 are also indicated in Table 1. Capacitance measurements on the ITO electrode could well be fitted to the described theoretical model using an ITO doping density of 2.7 × 1026 m-3, a Helmholtz capacitance of 6 µF/cm2, and a flatband potential of the ITO of 0.4 V and assuming specific adsorption of HPO42ions with a specific interaction potential of -7 kT and a maximum adsorbed charge density of -4.5 C/cm2. On the basis of these fit parameters, Figure 2 shows the calculated potential difference across the diffuse double layer present at the ITO surface as a function of the electric potential applied to the ITO. The diffuse double layer potential is negative with respect to the solution at open circuit and becomes more positively or negatively charged depending upon the potential applied to the electrode. Bacterial Deposition and Desorption. Figures 3a and 4a illustrate the increase in time of the number of adhering Staph. epidermidis 3399 and A. naeslundii T14VJ1, respectively, including the stimulated desorption after 4 h by increasing electric currents. Note that the number of adhering bacteria decreases linearly in time during the application of an electric current, whereas if the current is switched off, stable numbers are instantly observed (Figures 3b and 4b). Similar observations were made for the other two bacterial strains. Table 2 summarizes the initial deposition rates j0, the equilibrium number of adhering bacteria neq, and the equilibrium desorption probabilities βeq. Initial deposition rates are not significantly influenced by the electrode potential applied, except for A. naeslundii T14V-J1.
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Figure 2. Theoretically calculated dependence of the diffuse double layer potential difference on the electrode potential with respect to a Ag/AgCl reference electrode, according to Natarajan et al. (ref 20). The dashed line represents the open circuit potential of the ITO electrode.
Figure 4. Number of adhering A. naeslundii T14V-J1 as a function of time during deposition and desorption as stimulated by an applied electric current of 400 µA (panel a). In panel b, the time scale has been adjusted with t ) 0 corresponding to the onset of an electric current. 1-3 denote different episodes of electric currents, all equal to 400 µA. Table 2. Initial Deposition Rates j0, Equilibrium Desorption Probabilities βeq, and the Equilibrium Number of Adhering Bacteria neq Obtained by Image Sequence Analysis during Bacterial Deposition in a Parallel Plate Flow Chamber to an ITO Electrode Surface with Electrode Potentials V with Respect to an Ag/AgCl Reference Electrodea electrode potential (V vs reference)
Figure 3. Number of adhering Staph. epidermidis 3399 as a function of time during deposition and desorption as stimulated by different applied electric currents (panel a). In panel b, the time scale has been adjusted with t ) 0 corresponding to the onset of an electric current. 1 and 2 denote electric currents of 100 µA; 3, 4, 6, and 8 denote 200 µA; 5, 7, and 9 denote 400 µA.
However, equilibrium numbers of adhering bacteria neq and equilibrium desorption probabilities βeq depend significantly on the applied electrode potential, except for Staph. epidermidis 3399. For Strep. oralis J22 and A. naeslundii 147, the equilibrium desorption probability βeq increases when a more negative potential is applied to the electrode surface, whereas for A. naeslundii T14V-J1 decreased desorption is seen upon making the electrode potential more negative. Interestingly, for Staph. epidermidis 3399, desorption probabilities could be obtained with
j0 (cm-2 s-1)
βeq (10-4 s-1)
neq (106 cm-2)
+0.5 0.1 -0.2
Strep. oralis J22 880 ( 170 8.4 ( 2.7 1020 ( 120 11.0 ( 3.1 890 ( 140 13.5 ( 5.4
6.0 ( 0.4 4.5 ( 0.2 3.5 ( 0.1
+0.5 0.1 -0.2
Staph. epidermidis 3399 3450 ( 120 3.1 ( 0.6 3770 ( 320 3.2 ( 0.3 3570 ( 220 4.0 ( 0.5
21.3 ( 2.2 20.9 ( 2.0 21.1 ( 2.5
+0.5 0.1 -0.2
A. naeslundii T14V-J1 1410 ( 110 12.2 ( 2.7 2020 ( 300 6.3 ( 2.1 1390 ( 280 5.7 ( 1.9
3.1 ( 0.3 8.9 ( 1.0 7.0 ( 0.5
+0.5 0.1 -0.2
A. naeslundii 147 1200 ( 200 11.4 ( 3.5 1105 ( 120 15.4 ( 3.2 1094 ( 220 21.7 ( 3.8
3.5 ( 0.3 2.9 ( 0.2 2.5 ( 0.4
a Results of duplicate runs with separately cultured bacteria and newly prepared ITO electrodes.
exceptionally small standard deviations as compared with the other strains. Figure 5 summarizes the stimulated desorption probabilities as a function of applied current, ranging from 65 to 400 µA, corresponding with electrode potentials between -0.4 and -0.5 V. Stimulated desorption probabilities
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Figure 6. Energy of interaction between A. naeslundii 147 and an ITO substratum in 1 mM potassium phosphate as a function of the surface to surface separation distance h according to the DLVO theory, assuming the Hamaker constant A132 to be 1 kT (ref 1): (1) ITO potential of 0.1 V, (2) ITO potential of 1.8 V, and (3) ITO potential of -0.4 V.
Discussion
Figure 5. Measured stimulated desorption probabilities as a function of the electric current: (2) Strep. oralis J22, ([) Staph. epidermidis 3399, (b) A. naeslundii T14V-J1, and (9) A. naeslundii 147. The numbers indicate the number of adhering bacteria/cm2 left at the electrode at the time of measurement. The bars represent standard deviations over six separate experiments.
increase with increasing electric current. Stimulated desorption of Strep. oralis J22 and A. naeslundii 147 was studied only for low electric currents (65 and 100 µA, respectively) because of their high desorption. For Strep. oralis J22 and A. naeslundii 147, bacteria that desorbed first had a higher desorption probability than the ones that desorbed later during stimulated desorption (see Figure 5). For example, for Strep. oralis J22 the stimulated desorption probability decreased from 22.2 × 10-4 s-1 at the onset of stimulated desorption (6 × 106 bacteria/cm2 adhering) to 0.9 × 10-4 s-1 at the last stages of stimulated desorption (only 2 × 106 adhering bacteria/cm2 left). For Staph. epidermidis 3399 and A. naeslundii T14V-J1, the stimulated desorption probability was independent of the number of bacteria left adhering at the electrode surface and therefore a unique value of the stimulated desorption probability could be calculated for each applied current, as shown in Figure 5. Bacteria, directed to deposit in the primary minimum by the application of a relatively high voltage (1.8 V, i.e., a positively charged electrode, as can be seen from Figure 2), could hardly be stimulated to desorb afterward by subsequent application of a cathodic electric current. Even when the current was increased to 400 µA, stimulated desorption probabilities for all strains were 10- to 100fold lower than when bacteria had been deposited at lower electrode potentials of -0.2, 0.1, and 0.5 V.
In this paper, we studied deposition of four bacterial strains from a low ionic strength solution at an electrode surface as a function of the electrode potential applied. By application of a negative potential to the electrode, deposited bacteria could be stimulated to desorb. It is instructive to calculate the interaction energy between the ITO surface and adhering bacteria according to the DLVO theory. Examples of this are given in Figure 6 for A. naeslundii 147, assuming nonretarded Lifshitz-van der Waals interactions25 and electrostatic interactions according to the LSA method26,27 and accounting for the rodlike shape of the bacterium using Derjaguin’s approach while describing the bacterium as a spheroid.28 At electrode potentials of -0.2, 0.1, and 0.5 V, insurmountably high energy barriers exist, suggesting that bacteria can adhere only in the secondary minimum. This is in line with the observation that the initial deposition rates are independent of the electrode potential applied as the secondary interaction minimum is reached at a mass transport limited rate. Alternatively, the interaction energy at an elevated electrode potential of 1.8 V (see also Figure 6) does not show a potential energy barrier. Bacteria deposited under this condition adhere strongly to the electrode, and hence they can hardly be removed by subsequent application of a negative electric potential (curve 3 in Figure 6), as experimentally observed. The measured equilibrium desorption probabilities and stimulated desorption probabilities at the onset of stimulated desorption can be fitted to eq 2 or eq 3, respectively, to yield a single value of the Hamaker constant for each strain, except for A. naeslundii T14V-J1. The potential energy of bacteria Vtot in eqs 2 and 3, in addition to the DLVO interaction energy, includes the bacterial energy due to gravity and buoyancy7 and, in eq 3, the bacterial energy due to flow of the surrounding liquid (i.e., due to the Saffmann lift force29) and due to the electrophoretic force.11 The location of the maximum of the DLVO (25) Visser, J. Surf. Colloid Sci. 1976, 8, 3. (26) Bell, G. M.; Levine, S.; McCartney, L. N. J. Colloid Interface Sci. 1970, 33, 335. (27) Willemski, G. J. Colloid Interface Sci. 1982, 88, 111. (28) Adamczyk, Z.; Warszynski, P. Adv. Colloid Interface Sci. 1996, 63, 41. (29) Saffman, P. G. J. Fluid Mech. 1965, 22, 385.
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interaction energy and 500 nm were taken as the lower and upper integration boundaries in eqs 2 and 3. Hamaker constants thus obtained amounted to 4 kT for Strep. oralis J22, 5 kT for Staph. epidermidis 3399, and 2 kT for A. naeslundii 147, and consequently bacterial adhesion was stronger than that calculated using the literature value of the Hamaker constant employed in Figure 6. The fact that one single Hamaker constant could be obtained for each bacterial strain, describing the desorption probabilities at all electrode potentials used, indicates that the DLVO theory adequately describes the desorption of these strains. It must be noted at this point, however, that for A. naeslundii T14V-J1 no single value for the Hamaker constant (values ranged from 2 to 3 kT at electrode potentials of +0.5 and -0.5 V) could be obtained, likely because this strain has relatively long surface appendages with a distinct role in adhesion. The depth of the secondary minimum of bacteria adhering at the open circuit potential as obtained from fitting the measured stimulated desorption probabilities at the onset of stimulated desorption to eq 3 ranges from 8 to 12 kT for the four strains used. For Strep. oralis J22, stimulated desorption probabilities are markedly smaller at the later stages of stimulated desorption than at the onset, and correspondingly it can be calculated that the depth of the secondary minimum increases from 8 to 12 kT. Differential strengths of adhesive bonds have been described before, both for microorganisms and for polystyrene particles, and have been explained by heterogeneity of both the particle and the substratum surface.30 Electron microscopy has revealed the presence of long surface appendages31 on A. naeslundii T14V-J1. Long surface appendages are hypothesized to promote bacterial (30) Elimelech, M.; O’Melia, C. R. Langmuir 1990, 6, 1153.
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adhesion by penetrating the repulsive energy barrier toward adhesion,32 and indeed A. naeslundii T14V-J1 adheres more strongly (11 kT at open circuit potential) than its mutant A. naeslundii 147 (9 kT), for which only these long appendages have not been observed. Because A. naeslundii T14V-J1 was the only strain for which desorption probabilities at different electrode potentials did not yield a single value of its Hamaker constant, it is concluded that for strains with long surface appendages the DLVO theory as applied here is inadequate. Likely, the observation that the negatively charged A. naeslundii T14V-J1 desorbs less at more negative potentials (see Table 2) suggests that its long surface appendages carry a positive charge, as has been observed for other fibrillated strains.33 Summary of Conclusions We have found that the initial deposition rate of four bacterial strains to an ITO electrode surface under repulsive electrostatic conditions did not significantly depend on the electrode potentials applied, except when a strain with relatively long surface appendages was used. The equilibrium number of adhering bacteria and their equilibrium desorption probability did depend on the electrode potential used. Deposited bacteria could be stimulated to desorb by application of a cathodic electric current, except when bacteria had been forced to adhere in a primary minimum by application of a positive voltage during deposition. LA001673Y (31) Cisar, J. O.; Vatter, A. E.; Clark, W. B.; Curl, S. H.; HurstCalderone, S.; Sandberg, A. L. Infect. Immun. 1988, 56, 2984. (32) Hermansson, M. Colloids Surf., B 1999, 14, 105. (33) Handley, P. S.; Hesketh, L. M.; Moumena, R. A. Biofouling 1991, 4, 105.
