Surface chemistry and acid-base activity of Shewanella putrefaciens: Cell wall charging and metal binding to bacterial cell walls

Abstract

To gain insight into the surface chemistry of live microorganisms, pH stat experiments are combined with analyses of the time-dependent changes in solution chemistry using suspensions of live cells of Shewanella putrefaciens. The results of this study illustrate the complex response of the live cells to changes in pH of the aqueous medium. In particular, the observed acid-base activity of the organism cannot be attributed solely to the protonation or deprotonation reactions of functional groups in the cell wall. Time-dependent acid and base consumption curves by the bacterial cells are interpreted to consist of the following three contributions. (1) The near-instantaneous (time scale of minutes) buffering capacity associated with the functional groups present in the cell wall, (2) the short-term (< 1 hour) utilization of the intracellular buffering capacity, and (3), under basic conditions, the long-term (1-5 hours) release of (acidic) metabolic byproducts. By measuring the initial acid or base consumption at different pH values, the buffering capacity associated with the functional groups in the cell wall can be determined as a function of the pH of the medium. Lipopolysaccharides (LPS), the major constituent of the outermost layer of the cell wall of Gram-negative bacteria, contribute little to the buffering capacity of the intact cell wall. Comparing the buffering capacities of the cell walls of live cells to that of cell wall components show that, while LPS may be important in regulating cell adhesion of Gram-negative bacteria, they may not provide many anionic binding sites for metal cations. The latter must therefore migrate deeper inside the cell wall. The main sources of acidity that explain the long-term base neutralizing capacity of live cells of S. putrefaciens are release of organic acids and dissolution of metabolically produced CO₂. The redox, pH and temperature conditions regulate the type and rates of excreted metabolic products since each microorganism functions at an optimum pH and temperature. The observed release of acids under basic conditions may have important implications for the activity of live microorganisms in natural environments. For instance, by releasing protons, metal reducing bacteria, such as S. putrefaciens, may compensate for the proton consumption due to metal reduction, thereby maintaining favorable conditions for metal reduction in their immediate vicinity. Combining the results of the initial part of the acid-base consumption curves in the pH stat experiments with Zn²⁺ and Cu²⁺ binding experiments provides information about the surface chemistry of the cell walls. Carboxylate and phosphate groups are identified as the major sites for metal binding to S. putrefaciens. The binding isotherms of Cu²⁺ are best explained by invoking the presence of a small percentage of high affinity carboxylate sites in the cell wall. Using a simple cell wall complexation model shows that metal binding to cell wall functional groups is sensitive to the competition with metal complexation reactions of organic acids in solution, as well as to the acid-base properties of the high affinity sites in the cell wall. Characterization of the affinity of the organic ligands, released by cellular activity, for metal ions is needed to better constrain the affinity constants of cell wall functional groups for metal ions.