Proton binding of bacterial exudates determined through potentiometric titrations

Bacterial exudates can play a role in metal reduction and mineral dissolution. Exudates were collected from non-metabolizing Gram-positive (Bacillus subtilis) and Gram-negative (Shewanella oneidensis) bacterial species by suspending and rotating 40, 80, or 100 g (wet mass)/L bacteria in NaClO4 solutions of 0.01, 0.1, or 0.3 M for 2.5 h, and removing the cells by centrifugation. Exudate solutions were analyzed for total organic carbon concentrations, which varied as a function of bacterial type and concentration. However, FTIR spectra of all exudate samples were essentially identical under all experimental conditions. Potentiometric titrations of the exudate solutions were conducted for each of the bacterial concentrations and ionic strength conditions examined, for both of the bacterial species. Both ‘up-pH’ and ‘down-pH’ titrations were conducted to determine the extent of reversibility of the proton binding reactions, and titration data were modeled using a discrete-site modeling approach to determine the number of discrete proton-active functional groups required to account for the observed buffering capacity and to place quantitative constraints on values of the acidity constants and site concentrations for the proton binding sites. A 3-site model fit the titration data the best, and the calculated acidity constant values did not vary significantly as a function of bacterial species studied, ionic strength, or quantity of biomass used to generate the exudate solution. The calculated site concentrations varied with ionic strength and bacterial species. B. subtilis exudates showed a decrease in total site concentrations with increasing ionic strength, and S. oneidensis showed an increase with increasing ionic strength; however, the range of total site concentrations was similar for both species. Total buffering capacities and site concentrations for the exudates were approximately one order of magnitude lower than those previously determined for bacterial cells per gram of bacteria but were within the ranges of those calculated for EPS and humic substances.