Here we report results from a quantitative investigation of the types and densities of proton binding sites on a bacterial surface, Bacillus subtilis, from replicate acidbase titrations on bacteria at two ionic strengths (0.025 and 0.1 M). In contrast to the surface complexation modeling (SCM) approach developed and widely used for mineral, e.g., iron oxides, and more recently bacterial surfaces; we fit the data using the linear programming method (LPM). Our results using LPM indicate five discrete binding sites occurring on the surface of B. subtilis likely corresponding to carboxylic sites at low pK a values, phosphoric sites at nearneutral pK a values, and amine sites at high pK a values. Replicate titrations on subsamples from the same bacterial population indicated less variability than has been suggested for bacterial surfaces. Both the pK a and site density values were found to be dependent on ionic strength. Comparing the pK a values determined here with LPM for B. subtilis to those determined independently by using a fixed three site SCM model shows excellent agreement with the common sites likely corresponding to carboxylic, phosphoryl, and amine groups. However, the LPM approach identifies a further two sites as compared to the SCM approach. These results have an important implication. Surfaces of a given bacterial strain have a quantifiable, characteristic geochemical reactivity reflecting discrete sites that can be traced back in terms of function to the underlying, cell wall structure, a well-characterized phenomenon for most bacteria. However, an important caveat of our findings is that the absolute densities of these sites are highly dependent on a suite of both microbiological and system chemical parameters.

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Acid-base titration experiments and electrostatic force microscopy (EFM) were used to investigate the cell surface electrochemical heterogeneity of the Fe(III)-reducing bacteria, Shewanella putrefaciens. The acid-base titrations extended from pH 4 to 10, and the titration data were fit using a linear programming pKa spectrum approach. Overall, a five-site model accounted for the observed titration behavior with the most acidic sites corresponding to carboxylic groups and phosphodiester groups, intermediate sites phosphoryl groups, and two basic sites equivalent to amine or hydroxyl groups. The pH for the point of zero charge on the bacteria was 5.4. In EFM images of cells rinsed in solutions at pH 4.0, 7.0, and 8.0, a pronounced increase in small (< or = 100 nm diameter) high contrast patches was observed on the cells with increasing pH. The pH dependence of EFM image contrast paralleled the pattern of cell surface charge development inferred from the titration experiments; however, quantitative analysis of high contrast regions in the EFM images yielded lower surface charge values than those anticipated from the titration data. For example at pH 7, the calculated surface charge of high contrast regions in EFM images of the bacterial cells was -0.23 microC/cm2 versus -20.0 microC/cm2 based on the titration curve. The differences in surface charge estimates between the EFM images and titration data are consistent not only with charge development throughout the entire volume of the bacterial cell wall (i.e., in association with functional groups that are not directly exposed at the cell surface) but also with the presence of a thin structural layer of water containing charge-compensating counterions. In combination, the pKa spectra and EFM data demonstrate that a particularly high degree of electrochemical heterogeneity exists within the cell wall and at the cell surface of S. putrefaciens.