Surface Charge And Hydrophobicity

As reviewed by Liss,6 methods used in the past to determine microbial surface charge include attachment to charge-modified polystyrene, fluorescent probe ion exchange resin, and electrophoretic mobility. At present, the most common and reproducible method to determine surface charge density of microbial aggregates is by a colloid titration.105,106 Mikkelsen107 recently compared surface charge determinations of sewage sludge by various methods in order to identify applications and limitations of the colloid titration method for analysis that tends to yield widely ranging values amongst different studies. The colloid titration method was found to be limited to conditions of low reactant doses and valid for charge determination of extracellular polymeric substances primarily. For determining whole floc or sludge surface charge, estimates from zeta potential titrations may be more reliable than that determined from colloid titration.

Numerous methods have been reported in the literature for determining hydrophobic interactions of cells and have been summarized by Liss.6 These include methods that measure actual binding to a hydrophobic ligand such as microbial adhesion to hydrocarbons (MATH) and those giving an estimate of an overall surface property, such as salt aggregation test (SAT) and contact angle measurement (CAM) of dry cell layers.

The MATH method is a simple method to rapidly quantify cell surface hydrophobicity.108,109 This method is based on the partitioning of cells possessing hydrophobic surface characteristics at the interface of a biphasic hydrocarbon-aqueous system after brief mixing. The relative hydrophobicity is calculated from:

where a is the absorbance of the aqueous layer after phase separation, and A is the initial absorbance of the aqueous phase at 400 nm before mixing with hydrocarbons. Attention must be given to the possibility that cell clumping may occur during the assay, which can result in a huge reduction in absorbance. Changes in the initial cell density can also affect the measurement.

Contact angle measurement is one of the most common techniques for the measurement of hydrophobicity of bacterial cell surfaces and flocs because the surface free energy of these cells can be estimated from the measurement (see Chapter 19). Although different apparatus may be used, all the measurements involve the preparation of a thin bacterial lawn through the vacuum filtration of a bacterial suspension and the determination of sessile drop contact angles on the bacterial lawn, either by using a telegoniometer or by projecting a magnified image system. The application of axisymmetric drop shape analysis (ADSA) may overcome some of the problems inherent in contact angle measurements on biological cells.110,111

Salt aggregation test is based on subjecting cells to increasing concentrations of salting-out agents (e.g., ammonium sulfate).112 The order in which cells are aggregated and settled is a measure of their surface hydrophobicity. The most hydrophobic cells are aggregated first at a low salt concentration. All tests are compared to the reaction at the highest molarity of salt (positive control). Bacterial suspensions mixed with a 0.002 M sodium phosphate (pH 6.8) are used as a negative control. A drop of methylene blue can be added to enhance the visualization of the aggregates.113 Urbain et al.114 used this method to study the internal hydrophobicity of sludge flocs. Limitations of SAT include the fact that many hydrophobic bacterial cells will clump in the absence of any added ammonium sulfate, and that it provides only a qualitative estimation of the relative rank of hydrophobicity. Electrostatic interactions may affect the results of SAT more than other hydrophobic measurement techniques.109

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