A modified regularized least squares pK(a) spectrum approach is applied to determine disassociation constants and proton binding site concentrations on bacteria, hydrous ferric oxide (HFO), and bacteria/HFO composite surfaces. This involves fitting experimental acid-base titration data to a continuous binding site model for a chemically heterogeneous surface with a variety of reactive groups yielding a pKa spectrum. The modified parameter fitting method optimizes simultaneously for both smoothness of the pK(a) spectrum and goodness of fit, whereas other methods optimize for goodness of fit given a fixed smoothness factor. Uncertainty estimates in pKa spectra were made by taking the mean and standard deviation of the spectra from replicate titration data. Titration of Shewanella putrefaciens strain CN32, a facultative iron-reducing bacterial species, demonstrate five types of binding sites consistent with known cell surface groups on bacteria, with mean pK(a), values of 3.62, 4.97, 6.92, 8.22, and 9.97. Composite surfaces formed by precipitation of HFO onto bacteria surfaces were also titrated. These surfaces no longer yielded low pK(a), sites in pK(a), spectra, indicating that ferric iron interacts with the bacteria via carboxylic (low pK(a)) sites during precipitation. In addition, mechanically mixed HFO bacterial samples also showed removal of carboxylic binding sites, suggesting that solid phase HFO interacts directly with carboxylic sites on bacterial cells. Moreover, the pK(a), spectra for HFO bacterial composites were not dependent on how the composite was formed; the mechanically mixed or surface-precipitated samples exhibited very similar binding site distributions. The determined pK(a), spectra imply that the overall binding mechanism for bacteria interactions with HFO involve carboxylic groups on the bacteria binding to the most basic sites on the HFO surface in approximately 1:1 stoichiometry. (C) 2003 Elsevier Inc. All rights reserved.