CELL SURFACE CHARGE AND DEBRIS CHARACTERISATION.

An understanding of the surface properties of bacterial cell walls is essential if the properties are to be altered in order to bring about flocculation. The properties of interest include the zeta potential and the degree of hydrophobicity which arise from the chemical constituents which make up the cell walls. The zeta potential is the potential at the plane of shear around the particle, at the interface between the fixed and mobile phases, when a particle moves in an electrical field (Ives, 1978). The fixed and mobile phases are referred to as the Stem and Gouy-Chapman layers respectively. The former is the layer of ions attracted to the overall charge (normally negative on bacterial cell surfaces); the diffuse layer (oppositely charged) forms around the Stem layer. The size and magnitude of the Stem layer indirectly controls colloid stability, since it affects attachment of aggregating species. The repulsive force between two similarly charged species is proportional to the square of the zeta potential; it decreases exponentially with increasing distance between particles. Polymer adsorption reduces the charge intensity of the Stem layer and causes rearrangement of the counter ions in the diffuse layer. Bell et al, (1983) note that for a stable, non-aggregating system, particle zeta potentials in the range +/- 10 to 4-/- 40 mV have been reported. The mobility of a particle in an electrical field is related to the zeta potential by the Henry equation:

. ■ A Ï J (1.12)

where \i is the electrophoretic mobility, e is the dielectric constant of surrounding medium, Z is the zeta potential, V is the viscosity and, f(K^) is the screening function (a = particle radius). Commonly, the thickness of the double layer is small compared to the curvature of the particle, so the Henry equation reduces to the Smoluchowski form in which f(K ,)= 1.5, hence:

Mobilities of cell debris from Saccharomyces cerevisae and S.carlsbergensis over a range of pH 2 to 9 (at low ionic strength) were found to vary from +0.5 to -1.5 pm s'^ V'^ cm'^ or approximately equivalent to + 8 to - 25 mV (Eddy and Rudin, 1958). Intact cells and cell debris were reported to behave in a similar fashion and varied with yeast strain. Other variables included the growth conditions, the yeast age and the culture medium. The iso-electric point was generally around pH 4. Yeast grown on phosphate deficient media gave mobilities which were independent of pH, with cells having virtually no charge. Cultures limited in carbon, sulphur or nitrogen gave electrophoretic properties similar to those grown in full synthetic medium. It was concluded that the phosphate was an integral part of the cell wall and not simply bound to the exterior. The electrophoretic mobility was directly related to the phosphate content for various cultures of yeast. Alkali extraction showed that the cell wall protein also contributed to the zeta potential.

The electrophoretic behaviour of whole cells and partially ruptured cells of

Micrococcus lysodeckticus, E.coli, Bacillus megaterium, B.subtilis and S.cerevisiae have been studied under a variety of pHs, ionic strengths and electrolytes (Neihoff & Echols, 1973). The mobilities of the isolated cell walls were 5 to 15% lower for the bacteria and 25% lower for the yeast, compared with those of the intact cells. Also they exhibited the same species specific behaviour. The differences were attributed to the removal of fimbriae or extracellular polysaccharide material. The fixed charged groups were responsible for electrophoretic movement and electrostatic interactions with other moieties; the properties of the surface are typical of the average bulk properties of the wall. The gram negative bacteria were found to have isoelectric points of approximately pH 3. Electronegative charge dominated the surface of all the organisms studied except at low pHs. The authors noted that strong disruption techniques such as ultrasonication, would produce very fragmented cell walls, whose interior surfaces may be different from the exterior, however this was not examined. The pH/mobility behaviour was thought to arise from the presence of carboxyl groups on the surface; phosphodiester groups were also thought to be present. The positive mobilities at low pHs indicate that cationic groups were also thought to be present. Eriksson and Axberg (1981) reported that the electrophoretic mobility of washed

E.coli has an isoelectric point at approximately pH 4; at lower pHs the zeta potential was positive indicating the presence of cationic groups. The zeta potential was

negative throughout the growth phase of the culture. The authors viewed bacteria as electrostatically and/or sterically stabilised colloids. The presence of extra cellular polymers greatly influenced bacterial aggregation, especially when the distances between the cells is sufficient for bridging to occur.

The surface composition of several yeast brewery strains has been determined by X- ray photoelectron spectroscopy (Amoury & Rouxhet, 1988). Bottom fermenting strains were found to be more electronegative than top-fermenting ones (-35 +/-5 mV compared to -12 +/-9 mV). The zeta potential at pH 4 was correlated with the phosphate ion surface concentration and the contribution of carboxyl groups was thought to be small.

The zeta potentials for various model systems have been reported (Douglas & Shaw, 1957); the pH/mobility relationship was determined for protein, lipid, polysaccharide and polypeptide coated onto hydrocarbon droplets. These revealed the specific nature of the ionisable groups. The mobility of protein coated particles (ribonuclease and fibrinogen) gave zero mobilities close to the iso-electric points for the two proteins. The polysaccharide coated surfaces gave low mobilities consistent with neutral polysaccharides. Thymus nucleic acid gave negative mobilities except at low pHs expected of the phosphodiester groups on the ribose-phosphate backbone. The electrophoretic mobilities of model systems consisting of octadecanoic acid, octanol and octadecylamine have been reported (Mehrishi & Seaman, 1968) with a view to extending the electrokinetic data to complex biological systems. Increasing the ionic strength (0.02 to 0.145 M) caused a reduction in electro-negativity of the acid and amine. The electrophoretic behaviour could be related to the specific groups on the particle surface.

Information concerning the properties of cell debris is scarce, Agerkvist and Enfors (1990) have characterised the viscosity and particle size distributions of E.coli

homogenates after various mechanical methods of disruption. Although the protein release was similar for the different methods, the physical properties were not. Treatment of the homogenates with different nucleases showed that the DNA was responsible for the increase in viscosity on disruption. The homogenates exhibited pseudoplastic behaviour with some degree of hysteresis occurring. The particle size

distribution for whole cells gave a mean diameter of 1169 nm; disruption produced peaks corresponding to approximately 460 and 800 nm in diameter. The particle size distributions showed a considerable degree of polydispersity, which was masked to some extent by the method of measurement. The dynamic light scattering would tend to skew the particle size distribution toward the larger particle sizes, since the light scattered is proportional to th e , radius of the particles which increases to the sixth power.