Impact of the Charge-Regulation Effect on Bacterial Growth and Bioavailability of Sorbed Growth Substrates

Bacteria are typically found attached to surfaces. Their growth on surfaces can be beneficial, such as with trickling filters, bio-filters and bio-scrubbers, where the attached bacteria are used to biodegrade aqueous organic components. There are also plenty of systems where bacterial surface growth is not desirable, such as with biofilm formation in water distribution pipelines and pathogenic biofilm infections on medical implants and equipment. One consideration in all these systems is the interaction between the bacteria and the adhering surface, and specifically, how the physiochemical properties of the surface can influence bacterial growth. Previous studies demonstrated that the metabolic activity of bacteria could be altered upon adhesion onto solid surfaces. The underlying mechanism remained unclear until the theory linking the charge-regulation effect to cellular bioenergetics was developed by Hong and Brown. Surfaces in the aqueous environment are typically charged due to ionization of acidic (negatively-charged) and basic (positively-charged) functional groups at the surface. These "charge-regulated" surfaces exhibit surface charge and electrostatic potential variation as a function of pH. When charge-regulated surfaces approach each other, such as when a bacterium approaches a solid surface, the surface charge and electrostatic potential will vary as a function of separation distance. This in turn causes the local pH to vary, following the Boltzmann distribution, and this will alter the surface functional group ionization, which affects the surface charge. The end results of these complex interactions is that the surface charge, electrostatic potential and pH will all vary as a function of separation distance between the surfaces. This is the so-called charge-regulation effect and it can be modeled as a function of the dissociation constants and site density of the surface functional groups. This study is based on the hypothesis linking cellular bioenergetics, where energy is stored as proton gradient across the bacterial cytoplasmic membrane, and the charge-regulation effect, where the cell surface proton concentration is altered during adhesion. Previous studies have shown that bioenergetics is affected by the charge-regulation process and that the effect is a direct function of the acidic and basic functional groups on the adhering surface. These studies were performed under non-growth conditions, and demonstrate that activity increases for acidic surfaces and decreases for basic surface, in agreement with the hypothesis. The purpose of this study was to build upon the prior studies and examine how charge-regulated surfaces can affect the growth of bacteria. First, we developed and demonstrated a simple approach for characterizing charge-regulated surfaces using zeta potential – pH titration data. The purpose was to model the surface electrostatic potential and charge density as a function of pH. This allowed modeling of the materials used in this study and interpretation of the experimental results. Having knowledge of surface properties, preliminary biodegradation experiments were conducted using regular sand (acidic functionality) and iron-coated sand (basic functionality). The results demonstrated that attached bacterial growth was directly affected by the charge-regulation effect. The iron-coated sand carrying more positive charge at the surface exhibited antimicrobial properties as compared to the uncoated sand, which is in agreement with our working hypothesis for charge-regulated surfaces.We then focused on three different granular activated carbon (GAC) materials, all provided from Evoqua Water Technologies, LLC. These included an untreated GAC, a GAC that was modified to contain a surface-bound weak base, and a GAC that was modified to contain a surface-bound strong. Surface analysis demonstrated that the untreated GAC was negatively-charged, whereas the treated GACs exhibited increased positive charge from the weak-base to the strong-base. Enhanced nitrogen content due to the applied bases on the treated GAC surfaces was confirmed with SEM/XRD. Using the three GACs, we performed biodegradation (respirometry) experiments using benzoic acid as the growth substrate. At higher concentrations of benzoic acid, bacterial growth was reduced in the presence of the treated GACS, with the effect more pronounced with the strong-base GAC, as expected following the hypothesis. At a low concentration of benzoic acid, however, we saw the exact opposite results, and let to a new and unexpected research finding, related to how the charge-regulation effect can alter the sorption – and thus bioavailability – of ionizable growth substrates. We explored this using three different growth substrates – benzoic acid (weak acid), benzylamine (weak base), and ethylbenzene (neutral). Using these growth substrates, we examined their sorption and bioavailability with each GAC. Substrate sorption isotherm on the GACs was strongly affected by surface pH, and this strongly affected the bioavailability of the different growth substrates via charge-regulated variations in local pH. For example, the modeling results indicated an increase in local pH upon bacterial adhesion onto strong-base GAC, which carried more positive charges. The sorption of benzoic acid decreases with increasing pH, and this resulted in release of sorbed benzoic acid and an increase in the aqueous concentration available for bacterial growth. This study has demonstrated that the charge-regulation effect can impact both bacterial activity (via changes in cellular bioenergetics, which is dependent on a pH gradient across the cell membrane) and bioavailability of sorbed, ionizable growth substrates (via changes in the substrate ionization). The overall relationship between surface electrostatic properties and cellular bioenergetics provides were demonstrated through experimentation and modeling using the charge-regulation model. Understanding of this effect can allow more effective selection and specific design of surfaces given the goals of the specific system.