| Abstract
| - Acid−base titration experiments and electrostatic forcemicroscopy (EFM) were used to investigate the cell surfaceelectrochemical heterogeneity of the Fe(III)-reducingbacteria, Shewanella putrefaciens. The acid−base titrationsextended from pH 4 to 10, and the titration data were fitusing a linear programming pKa spectrum approach. Overall,a five-site model accounted for the observed titrationbehavior with the most acidic sites corresponding to carboxylicgroups and phosphodiester groups, intermediate sitesphosphoryl groups, and two basic sites equivalent to amineor hydroxyl groups. The pH for the point of zero chargeon the bacteria was 5.4. In EFM images of cells rinsed insolutions at pH 4.0, 7.0, and 8.0, a pronounced increasein small (≤100 nm diameter) high contrast patches wasobserved on the cells with increasing pH. The pH dependenceof EFM image contrast paralleled the pattern of cellsurface charge development inferred from the titrationexperiments; however, quantitative analysis of high contrastregions in the EFM images yielded lower surface chargevalues than those anticipated from the titration data. Forexample at pH 7, the calculated surface charge of highcontrast regions in EFM images of the bacterial cells was−0.23 μC/cm2 versus −20.0 μC/cm2 based on the titrationcurve. The differences in surface charge estimates betweenthe EFM images and titration data are consistent notonly with charge development throughout the entire volumeof the bacterial cell wall (i.e., in association with functionalgroups that are not directly exposed at the cell surface)but also with the presence of a thin structural layer of watercontaining charge-compensating counterions. In combination, the pKa spectra and EFM data demonstrate thata particularly high degree of electrochemical heterogeneityexists within the cell wall and at the cell surface of S.putrefaciens.
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