Microbial cell surface hydrophobicity The involvement of electrostatic interactions in microbial adhesion to hydrocarbons (MATH)

Journal of Microbiological Methods, 18 (1993) 61-68 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167 - 7012/93/$06.00

61

MIMET 00581

Microbial cell surface hydrophobicity The involvement of electrostatic interactions in microbial adhesion to hydrocarbons (MATH) G.I. Geertsema-Doornbusch, H.C. van der Mei and H.J. Busscher Laboratory for Materia Technica, University of Groningen, Groningen, Netherlands (Received 15 January 1993; revision received 5 March 1993; accepted 5 March 1993)

Summary Microbial adhesion to hydrocarbons (MATH) is the most commonly used method to determine microbial cell surface hydrophobicity. Since, however, the assay is based on adhesion, it is questionable whether the results reflect only the cell surface bydrophobicity or an interplay of hydrophobicity and surface charge properties. In order to demonstrate the involvement of electrostatic interactions in MATH, hydrophobicities by MATH (kinetic mode) were measured in 10 mM potassium phosphate solutions at different pH's and compared with the zeta potentials of the microorganisms and of hexadecane droplets in the same solution. Two oral, microbial strains were involved: Streptococcus salivarius HB (a hydrophobic strain by MATH) and Streptococcus salivarius HB-C12 (a hydrophilic strain by MATH). The initial removal rates of S. salivarius HB-C12 by hexadecane were zero over the entire pH range (pH 2-pH 9) and its zeta potentials were negative in this pH range. S. salivarius HB, however, had an isoelectric point (IEP) at pH 3.2 and accordingly a positive zeta potential below IEP. Correspondingly, the initial removal rates found for this strain were high (2.6 min-J) below and around IEP and much lower ( ~ 0.5 m i n - t) above IEP. Surprisingly, the hexadecane droplets also had highly negative zeta potentials above pH 4 and appeared uncharged in the pH range 2-3. Taking the product of the bacterial (b and hexadecane (h zeta potentials as a measure for electrostatic interactions, it was observed that the measured hydr0Phobicity of S. salivarius HB, but not of the hydrophilic strain S. salivarius HB-C12, depended on electrostatic interactions as well. The highest removal rates by hexadecane were found in the absence of electrostatic interactions, i.e. in the pH range close to the IEP's of the interacting particles. It is concluded that, in general, MATH does not measure cell surface hydrophobicity but an interplay of hydrophobicity and electrostatic interactions. The involvement of electrostatic interactions in MATH can be reduced by performing the test under ionic conditions in which either the cells or the hydrocarbon droplets (or both) are uncharged.

Key words: Hydrophobicity; Microbial adhesion; Zeta potential

Correspondence to: G.I. Geertsema-Doornbusch, Laboratory for Materia Technica, University of Groningen, Antonius Deusinglaan l, 9713 AV Groningen, Netherlands.

62 Introduction

The expression 'microbial cell surface hydrophobicity' is often considered as selfexplanatory. The word 'hydrophobicity' implies that the expression should refer to the dislike of microbial cells for water. However, of all experimental methods to assess microbial cell surface hydrophobicity, only water contact angle measurements on microbial lawns directly reflect what the word 'hydrophobicity' expresses [1,2]. Other methods that claim to measure microbial cell surface hydrophobicity, are indirect. These methods include hydrophobic partitioning in aqueous two phase systems [3], hydrophobic interaction chromatography [4], salt aggregation [5], polystyrene microsphere attachment [6], and microbial adhesion to hydrocarbons (MATH) [7]. As a disturbing consequence of the use of different indirect methods to measure microbial cell surface hydrophobicity, differently measured hydrophobicities do not generally correlate [8-10] and only for special collections of strains relationships between the outcomes of various methods have been observed [10]. From all indirect methods, M A T H is probably the most commonly used method [7]. The outcomes of M A T H hydrophobicity tests have indeed been helpful in gaining an understanding of a variety of microbial adhesion phenomena, including adhesion of Pseudomonas aeruginosa to stainless steel [11], staphylococcal adhesion to biomaterials [12], bacterial adhesion to meat [13] or oral microbial adhesion to salivary pellicles [14]. Despite the importance of hydrophobicity in microbial adhesion to surfaces, including to hydrocarbon droplets as in M A T H , adhesion is determined by a complicated interplay between hydrophobicity, Van der Waals forces and electrostatic interactions [15]. Since it has recently been shown that hexadecane droplets in aqueous suspensions are highly negatively charged [16], presumably due to anionic ion adsorption, it appears likely that the outcome of M A T H not only reflects the microbial cell surface hydrophobicity, but up to varying extents, also the electrical surface charge properties. The aim of this paper is to demonstrate the involvement of electrostatic interactions in MATH. To this end, the adhesion of a known, hydrophobic and a hydrophilic oral streptococcal strain to hexadecane will be measured in potassium phosphate solutions of different pH and compared with the zeta potentials of both the bacteria and the hexadecane droplets. The pH values of the suspending solutions were varied in order to have various degrees of protonation of surface groups, resulting in different zeta potentials. Materials and Methods

Bacterial strains and growth conditions S. salivarius HB is a hydrophobic (by M A T H ) oral streptococcal strain, which has been characterized in detail with regard to its surface characteristics [17]. Strain HBC 12 was a spontaneous, stable variant obtained after prolonged cultivation of strain HB in a chemostat at a high specific growth rate. S. salivarius HB-C12 is a hydrophilic strain [18]. Both strains were grown from a frozen stock in batch culture in Todd-Hewitt broth

63 (Oxoid, Basingstoke, UK) for 24 h at 37°C. This culture was used to inoculate a second culture which was grown overnight at 37°C. Cells were harvested by centrifugation, washed twice with demineralized water, and then suspended in 10 mM potassium phosphate buffer (pH 7.0). All experiments involving bacteria were performed with three separately grown cell cultures.

Preparation of hexadecane suspensions for micro-electrophoresis Hexadecane was emulsified in 10 mM potassium phosphate solutions with appropriate pH's by sonication (Sonics & Materials, Danbury, USA at 300 W) for 5 minutes at a concentration of 40000 ppm. The particle size of the hexadecane droplets varied from 1 to 1.5 ~m. After 10 min these emulsions were diluted to concentrations of 3000, 6500, 13 000 and 20000 ppm and zeta potentials were measured directly afterwards. All experimental results involving hexadecane droplets in suspension were done with three separately prepared suspensions.

Microbial adhesion to hexadecane ( MA TH) Since the originally described M A T H test [7] has been criticized for not being sufficiently quantitative due to the neglect of kinetic effects, the so-called kinetic M A T H test as proposed by Lichtenberg et al. [19] has been employed at room temperature. Briefly, bacteria were suspended to an optical density, Ao (at 600 nm), of between 0.4 and 0.6 in 10 mM potassium phosphate buffer, with pH adjusted to 2, 3, 4, 5, 7 or 9. Next, 150 #1 of hexadecane was added to 3 ml of bacterial suspension, and the two phase system was vortexed for 10 s, allowed to settle for 10 min and the optical density A t measured. The latter procedure was repeated until the total vortexing time amounted to 60 s and log (At/A o × 100) was plotted against the vortexing time. Linear least square fitting subsequently yielded the initial removal rate R0 (min-1) as a measure of the adhesion of the cells to hexadecane, i.e. of their hydrophobicity as by MATH.

Micro-electrophoresis Zeta potentials of the bacteria (b and of the hexadecane droplets (h in the 10 mM potassium phosphate solutions with the appropriate pH's were measured at room temperature with a Lazer Zee Meter 501 (PenKem, Bedford Hills, NY, USA) which uses scattering of incident laser light to enable detection of the bacteria and hexadecane droplets at relatively low magnifications. The absolute electrophoretic mobilities can be derived directly from the velocity of the particles in the applied electric field, the applied voltage, and the dimensions of the electrophoresis cell [20]. Although the conditions required to convert the measured electrophoretic mobilities into zeta potentials may not have been completely met in the present study [20], it is considered preferable here to use more appealing zeta potential values rather than more abstract electrophoretic mobilities. Zeta potentials were measured for the bacteria suspended to a concentration of l 0 7 cells.ml- 1. For hexadecane droplets a concentration range from 3000 to 20 000 ppm hexadecane was measured. The experimental reproducibility of all zeta potentials reported was _+ 2 mV.

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Fig. 1. Zeta potentials ~h of hexadecane droplets suspended in a 10 m M potassium phosphate solution as a function of the hexadecane concentration at different pH values. The bar represents the average S.D. over three separately prepared hexadecane suspensions and is valid for all data points in the graph.

Results

Fig. 1 shows the zeta potentials of the hexadecane droplets as a function of the hexadecane concentration. A minor concentration dependence is obvious. Since we performed the M A T H test at 50 000 ppm hexadecane concentration, a concentration too high to employ in micro-electrophoresis, data in Fig. 1 had to be extrapolated to 50 000 ppm in order to obtain the hexadecane zeta potentials relevant in MATH. These extrapolated hexadecane zeta potentials are plotted as a function of pH in Fig. 2. Note that above pH 3, the hexadecane zeta potentials become increasingly negative. Fig. 2 also presents the bacterial zeta potentials as a function of pH. Whereas S. salivarius HB-C12 has a negative zeta potential over the entire pH range 2-9, S. salivarius HB has a positive zeta potential below its isoelectric point (IEP) located at pH 3.2.

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Fig. 2, Zeta potentials ~h of hexadecane droplets suspended in a ]0 mM potassium phosphate solution extrapolated to a hexadecanc concentration of 50 000 ppm zeta potentials (b of S. sa/ivarius HB and of S, sa/ivarius HB-C]2 as a function of pH. Bars represent the average S.D. over three separately prepared (grown) hexadecane suspensions (bacterial cultures) and are valid for all data points on a line.

The initial removal rates by hexadecane of both strains are shown in Fig. 3 for the different pH values employed. The known hydrophilic strain S. salivarius HB-C12 has a zero removal rate, independent of pH, but the hydrophobic strain S. salivarius HB is removed faster by hexadecane at solution pH's below its IEP than at solution pH's above IEP. Removal rates in Fig. 3 are plotted as a function of the product between the bacterial ~b and hexadecane (h zeta potentials, which is a measure for the electrostatic interactions in MATH. Although adhesion of the hydrophilic strain S. salivarius HB-C 12 is not affected by electrostatic interactions, adhesion of S. salivarius HB to hexadecane is faster in the absence of significant electrostatic interactions ((b " (h < 100 mV 2) than in the presence of electrostatic interactions.

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pH Fig. 3. Initial removal rates by hexadecane of S. salivarius HB and of S. salivarius HB-CI2 in 10 m M potassium phosphate solution as a function of zeta potential product ~b " ~h. The separate axes denote the pH values at which zeta potentials and adhesion to hexadecane were measured. The bar represents the average S.D. over three separately grown bacterial cultures and is valid for all data points on the line.

Discussion

Microbial adhesion to surfaces involves a wide range of different types of interactions, including which hydrophobicity and electrostatic forces [21]. Hexadecane droplets in aqueous suspension have long been considered uncharged, and hence adhesion of microorganisms to hexadecane droplets in suspension considered a measure for microbial cell surface hydrophobicity [7]. The present study confirms previous findings by Medrzycka [16] concerning the zeta potentials of hexadecane droplets in aqueous suspensions. Both studies demonstrate a highly negative zeta potential for hexadecane droplets in aqueous suspensions, although the source of the negative charge is not exactly known. Since the concentration of hydroxyl ions in an aqueous suspension is, in general, too low to

67 yield a charge accumulation high enough to account for the highly negative zeta potentials [16], it is envisaged that the highly negative zeta potentials are due to oriented adsorption of water molecules to the hexadecane droplets by attractive Van der Waals forces and adsorption of miscellaneous anions. Fig. 3 demonstrates that adhesion of microorganisms to hexadecane in M A T H is governed by an interplay of hydrophobicity and electrostatic interactions. Clearly, the attractive Van der Waals forces between S. salivarius HB-C 12 and water are larger than between those cells and hexadecane, yielding a 'hydrophilic' outcome of M A T H for this strain. Moreover, these Van der Waals forces are relatively high compared to the electrostatic forces operating, as no influence of electrostatic interactions in M A T H for this strain is obvious. This is in contrast to the situation for S. salivarius HB, having a much stronger, Van der Waals attraction to hexadecane than to water, yielding a more ' h y d r o p h o b i c ' outcome of the M A T H test, which is strongly influenced by electrostatic interactions. Only in the absence of significant electrostatic interactions, i.e. in the p H region of the isoelectric points of hexadecane and/or of the bacteria, maximal adhesion ('hydrophobicity') of bacteria to hexadecane is found due to the undisturbed action of the attractive Van der Waals forces. Finally, we note that it was also attempted to demonstrate the involvement of electrostatic interactions in M A T H , when the test is conducted in the originally proposed way [7]. Although the experimental data indicated the involvement of the same mechanisms outlined above, the results were less convincing than when employing the kinetic M A T H test, as in Fig. 3. This, first of all, confirms that in a quantitative sense, the kinetic mode of the M A T H test is preferable, but secondly suggests that also the kinetics of microbial adhesion to hexadecane droplets in suspensions are affected by electrostatic interactions. We conclude by stating, that when M A T H is to be used as a 'hydrophobicity test' rather than as an 'adhesion test', it is best to work in buffer solutions with ionic conditions under which electrostatic interactions are minimal, i.e. close to the isoelectric points of the interacting particles. Elsewise, M A T H measures adhesion, a multifactorial process, and not simply hydrophobicity.

Acknowledgements The authors are greatly indebted to Mrs. Marjon Schakenraad-Dolfing for manuscript preparation.

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