Bacterial surface layers, such as extracellular polymeric substances (EPS), are known to play an important role in metal sorption and biomineralization; however, there have been very few studies investigating how environmentally induced changes in EPS production affect the cell's surface chemistry and reactivity. Acidbase titrations, cadmium adsorption assays, and Fourier transform infrared spectroscopy (FT-IR) were used to characterize the surface reactivities of Hymenobacter aerophilus cells with intact EPS (WC) or stripped of EPS (SC) and purified EPS alone. Linear programming modeling of titration data showed SC to possess functional groups corresponding to phosphoryl (pK a ϳ6.5), phosphoryl/amine (pK a ϳ7.9), and amine/hydroxyl (pK a ϳ9.9). EPS and WC both possess carboxyl groups (pK a ϳ5.1 to 5.8) in addition to phosphoryl and amine groups. FT-IR confirmed the presence of polysaccharides and protein in purified EPS that can account for the additional carboxyl groups. An increased ligand density was observed for WC relative to that for SC, leading to an increase in the amount of Cd adsorbed (0.53 to 1.73 mmol/liter per g [dry weight] and 0.53 to 0.59 mmol/liter per g [dry weight], respectively). Overall, the presence of EPS corresponds to an increase in the number and type of functional groups on the surface of H. aerophilus that is reflected by increased metal adsorption relative to that for EPS-free cells.Acid-base titrations are frequently used to characterize microbial cell surface reactivity, in particular, the ability of the cell to adsorb and desorb protons (19,21,47). This ability is conferred by the presence of proton-reactive surface functional groups that are also responsible for the surface adsorption of other cations, including dissolved metals. Thus, a microbe's ability to immobilize metals and influence metal transport is largely dependent on the nature of the reactive sites found at the cell-water interface, namely, their concentrations and chemical affinities (in terms of equilibrium surface stability constants) for cations such as protons and metals.Both Gram-negative and Gram-positive bacteria have been characterized extensively using acid-base titration to determine their reactivity with respect to geochemical processes (18,22,33). To date, most work has focused on mesophilic and strictly heterotrophic model organisms; however, some work has also been done with cyanobacteria (29, 44) and thermophiles (19,47). While proton sorption assays provide information on surface site densities and acidity constants, a more direct assessment of a microbe's ability to interact with aqueous metals is the metal adsorption assay, where a cell's ability to adsorb metal ions from solution is measured over a range of pH values. Metal adsorption assays have been used to characterize microbes from a wide variety of environments to determine their potential for bioremediation of heavy metal contamination (21, 26), their influence on geochemical cycling (5, 16), and their ability to serve as nucleation sites f...