Electrochemical characterization of the bacterial cell surface

Bacterial cells are ubiquitous in natural environments and also play important roles in domestic and industrial processes. They are found either suspended in the aqueous phase or attached to solid particles. The adhesion behaviour of bacteria is influenced by the physico-chemical properties of their cell surfaces, such as hydrophobicity and cell wall charge. The charge in the bacterial wall originates from carboxyl, phosphate and amino groups. The degree of dissociation of these anionic and cationic groups is determined by the pH and the activity of the surrounding electrolyte solution. Almost all bacterial cells are negatively charged at neutral pH, because the number of carboxyl and phosphate groups is generally higher than that of the amino groups. The presence of the charged cell wall groups leads to the spontaneous formation of an electrical double layer. The purpose of the present investigation is to elucidate the structure of the electrical double layer of bacterial cell surface. Such a study serves at least two goals. It allows the quantification of electrostatic interactions in the adhesion process and it contributes to gain better insight into the availability of (in)organic compounds for bacterial cells.The characteristics of the electrical double layer of bacterial cell surfaces have been revealed by applying a combination of experimental techniques, which include: chemical cell wall analysis, potentiometric proton titration and electrokinetic studies such as micro-electrophoresis, static conductivity and dielectric dispersion measurements.For the present study five Gram-positive bacterial strains, including four coryneforms and a Bacillus brevis, have been selected. Cell walls of these bacterial strains have been isolated and were subsequently subjected to chemical analyses and proton titration studies. Both methods provide information on the number of carboxyl, phosphate and amino groups.The chemical analysis of isolated cell walls involves the quantitative determination of both peptidoglycan and protein content. These analyses indicate that the chemical composition of the walls of the coryneforms are very similar, but considerably different from that of Bacillus brevis. Peptidoglycan is an important cell wall constituent of the coryneform bacteria and determines about 23 to 31 % of the cell wall dry weight. The protein fractions are somewhat lower, between 7 to 14%. The cell wall structure of the Bacillus brevis strain is more complex and multi-layered. It contains a thin peptidoglycan layer, which only determines 5 % of the cell wall dry weight. On the other hand, the protein content of these walls is higher than 56%. These proteins most likely can be attributed to a so-called S(urface)-layer, which is the outermost cell wall layer.The surface charge density of the bacterial cells is assessed by proton titrations of isolated cell walls at different electrolyte concentrations. Rather high values, i.e. between 0.5 and 1.0 C/m 2are found at neutral pH. The absence of hysteresis in the titration curves leads to the conclusion that the charging process can be considered as reversible. It also implies that the cell wall charge is continuously in equilibrium with the surrounding electrolyte solution, at any pH and salt concentration. This observation considerably facilitates the interpretation of the titration curves, because it allows a rigorous (thermodynamic) analysis. The anionic and cationic groups in the bacterial wall could be identified and their numbers determined by representing the differential titration curves as functions of pH and cell wall charge. The carboxyl and phosphate groups are almost entirely titrated in the pH range accessible by proton titration, allowing precise estimation of their numbers. These numbers compare very well with those based on a chemical analysis of the isolated cell walls. Estimates for the number of amino groups were less accurate, because these groups are only partly titrated in the pH range were precise titration measurements are feasible. Nevertheless, it could be concluded that the number of amino groups in the bacterial wall are lower than those of the carboxyl groups.Information about the ionic composition of the countercharge has been obtained from Esin-Markov analysis of the titration curves and from estimates of the cell wall potential based on a Donnan-type model. The Esin-Markov analysis is purely thermodynamic and based on first principles, whereas the Donnan model requires several assumptions about the structure of the bacterial wall. Both approaches lead to the same conclusion that at salt concentrations below 0.01 M the cell wall charge is predominantly compensated by counterions, with the excluded co-ions hardly contributing to the countercharge. This observation has considerably facilitated the interpretation of the electrokinetic properties of bacterial cell suspensions.Electrophoresis, static conductivity and dielectric response are related (electrokinetic) techniques and therefore share common physical bases. This also implies that the physical and mathematical problems that have to be solved in order to interpret the experimental data are very similar. Analytical solutions only exist for colloidal particles for which the electrical double layer is very thin compared to the particle dimensions. Most bacterial cells are relatively large colloidal particles and therefore the largeKa theory may be of help in the evaluation of their electrokinetic properties. However, the original theories do not include surface conductance in the hydrodynamically stagnant layer. Therefore, they had to be extended to account for the finite conductivity of ions in the bacterial wall.Static conductivity and dielectric dispersion both show that the counterions in the bacterial wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the counterions in the bacterial wall are of the same order but somewhat lower than those in the electrolyte solution.Due to surface conductance the electrophoretic mobility may be strongly retarded compared to the classical Helmholtz-Smoluchowski theory, especially at low electrolyte concentrations. In 1 mM and 10 mM electrolyte solution, the Helmholtz-Smoluchowski equation underestimates the ζ-potential by approximately a factor of 2 and 1.3, respectively.Resolving the fundamentals of the electrochemical characteristics of bacterial cell surfaces is a key step towards a quantitative understanding of the electrostatic interactions of bacterial cells with their surroundings. The success of such an investigation depends on the state of the art of the disciplines involved. Both microbiology and colloid chemistry have the microscopically small particle as object of study. Until recently there has hardly been any exchange of scientific knowledge between these two disciplines, despite their common interest. Colloid chemists prefered to study relatively simple particles to test their basic theories and bacterial cells were considered far too complex to serve as model colloids. However, the progress that has been made during the last decades in both colloid chemistry and microbiology provide the right tools for a successful cooporation. The present study is born from such a symbiosis and shows that many physicochemical characteristics of bacterial cell surfaces are accessible with (classical) colloid chemical techniques. In fact, for testing more advanced colloid chemical theories bacteria may even be better model particles than the generally used ionorganic colloids, because of their ability to produce a homogeneous population of identical cells.For the time being only Gram-positive strains have been considered, because of their relatively less complex cell wall structures. Nevertheless, the techniques used may mutatis mutandis also be applied to Gram-negative cells. In fact, such a study would be highly interesting, because it would contribute to a more complete description of the composition of the electrical double layer of bacterial cell surfaces.