Characterizing heterogeneous bacterial surface functional groups using discrete affinity spectra for proton binding

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 acid-base 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 near-neutral 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.