Rapid Characterization and Modeling of Natural and Undefined Charge-Regulated Surfaces in Aqueous Systems.

The surfaces of most materials in aqueous systems are charged due to the ionization of surface functional groups. When these surfaces interact, the surface charge, electrostatic potential, and pH will vary as a function of separation distance, and this process is termed the charge-regulation effect. Charge regulation is a controlling factor in the adhesion and transport of colloids and microorganisms in aqueous systems, and its modeling requires representation of the pH-charge response of the surfaces, typically provided as the equilibrium constants (K) and site densities (N) of the dominant surface functional groups. Existing methods for obtaining these parameters demonstrate shortcomings when applied to many natural and man-made materials, such as weathered materials, materials with undefined or complex surface structures, and permeable materials, and for materials that do not provide the requisite high surface area in suspension due to small sample sizes. This hinders inclusion of the charge-regulation effect in colloid and microbial transport studies, and most studies of colloidal and microbial surface interactions use simplifying assumptions; a key example is the routine use of the constant potential assumption in DLVO modeling. Here we present a robust method that overcomes these issues and provides a rapid means to characterize charge-regulated surfaces using zeta potential data, without requiring a priori knowledge of the material composition. Applying a combined charge-regulation and Gouy-Chapman model, K and N values are obtained that accurately represent the electrostatic response of a charge-regulated surface. This method is demonstrated using activated carbon, aluminum oxide, iron (hydr)oxide, feldspar, and silica sand. The resulting K and N values are then used to show the variations in surface charge, electrostatic potential, and pH that can occur as these charge-regulated surfaces interact. This method provides a readily applied experimental approach for characterizing charge-regulated surfaces, with the overall goal to promote the inclusion of charge-regulated interactions into adhesion and transport studies with natural and undefined materials.

Variation in bacterial ATP concentration during rapid changes in extracellular pH and implications for the activity of attached bacteria.

In this study we investigated the relationship between a rapid change in extracellular pH and the alteration of bacterial ATP concentration. This relationship is a key component of a hypothesis indicating that bacterial bioenergetics - the creation of ATP from ADP via a proton gradient across the cytoplasmic membrane - can be altered by the physiochemical charge-regulation effect, which results in a pH shift at the bacteria's surface upon adhesion to another surface. The bacterial ATP concentration was measured during a rapid change in extracellular pH from a baseline pH of 7.2 to pH values between 3.5 and 10.5. Experiments were conducted with four neutrophilic bacterial strains, including the Gram-negative Escherichia coli and Pseudomonas putida and the Gram-positive Bacillus subtilis and Staphylococcus epidermidis. A change in bulk pH produced an immediate response in bacterial ATP, demonstrating a direct link between changes in extracellular pH and cellular bioenergetics. In general, the shifts in ATP were similar across the four bacterial strains, with results following an exponential relationship between the extracellular pH and cellular ATP concentration. One exception occurred with S. epidermidis, where there was no variation in cellular ATP at acidic pH values, and this finding is consistent with this species' ability to thrive under acidic conditions. These results provide insight into obtaining a desired bioenergetic response in bacteria through (i) the application of chemical treatments to vary the local pH and (ii) the selection and design of surfaces resulting in local pH modification of attached bacteria via the charge-regulation effect.

Partitioning of phenanthrene into surfactant hemi-micelles on the bacterial cell surface and implications for surfactant-enhanced biodegradation.

Recent studies have suggested that the ability of a surfactant to enhance the bioavailability of hydrophobic organic compounds (HOC) requires the formation of surfactant hemi-micelles on the bacterial cell surface and subsequent partitioning of HOC into the hemi-micelles. However, the studies did not provide direct evidence of HOC partitioning into surfactant hemi-micelles on the bacterial cell surface. In this study, direct evidence is provided to demonstrate that the nonionic surfactant Brij 30 forms hemi-micelles on the bacterial cell surface and that phenanthrene sorption at the bacterial surface is enhanced by the surfactant. These results are in agreement with the current theory describing surfactant-enhanced HOC bioavailability. This enhanced bioavailability is put into context with microbial kinetics and system partitioning processes, and it is demonstrated that the addition of surfactant can enhance, have no effect, or inhibit HOC biodegradation depending upon surfactant concentration and microbial growth rate. Understanding these non-linear relationships between surfactant-enhanced HOC bioavailability, biodegradation kinetics, and system partitioning will assist in the design and implementation of surfactant-enhanced bioremediation programs.

Alteration of bacterial surface electrostatic potential and pH upon adhesion to a solid surface and impacts to cellular bioenergetics.

In our previous study [Hong Y, Brown DG (2009) Appl Environ Microbiol 75(8):2346-2353], the adenosine triphosphate (ATP) level of adhered bacteria was observed to be 2-5 times higher than that of planktonic bacteria. Consequently, the proton motive force (Delta p) of adhered bacteria was approximately 15% greater than that of planktonic bacteria. It was hypothesized that the cell surface pH changes upon adhesion due to the charge-regulated nature of the bacterial cell surface and that this change in surface pH can propagate to the cytoplasmic membrane and alter Delta p. In the current study, we developed and applied a charge regulation model to bacterial adhesion and demonstrated that the charge nature of the adhering surface can have a significant effect on the cell surface pH and ultimately the affect the ATP levels of adhered bacteria. The results indicated that the negatively charged glass surface can result in a two-unit drop in cell surface pH, whereas adhesion to a positively charged amine surface can result in a two-unit rise in pH. The working hypothesis indicates that the negatively charged surface should enhance Delta p and increase cellular ATP, while the positively charged surface should decrease Delta p and decrease ATP, and these results of the hypothesis are directly supported by prior experimental results with both negatively and positively charged surfaces. Overall, these results suggest that the nature of charge on the solid surface can have an impact on the proton motive force and cellular ATP levels.

Variation in bacterial ATP level and proton motive force due to adhesion to a solid surface.

Bacterial adhesion to natural and man-made surfaces can be beneficial or detrimental, depending on the system at hand. Of vital importance is how the process of adhesion affects the bacterial metabolic activity. If activity is enhanced, this may help the cells colonize the surface, whereas if activity is reduced, it may inhibit colonization. Here, we report a study demonstrating that adhesion of both Escherichia coli and Bacillus brevis onto a glass surface resulted in enhanced metabolic activity, assessed through ATP measurements. Specifically, ATP levels were found to increase two to five times upon adhesion compared to ATP levels in corresponding planktonic cells. To explain this effect on ATP levels, we propose the hypothesis that bacteria can take advantage of a link between cellular bioenergetics (proton motive force and ATP formation) and the physiochemical charge regulation effect, which occurs as a surface containing ionizable functional groups (e.g., the bacterial cell surface) approaches another surface. As the bacterium approaches the surface, the charge regulation effect causes the charge and pH at the cell surface to vary as a function of separation distance. With negatively charged surfaces, this results in a decrease in pH at the cell surface, which enhances the proton motive force and ATP concentration. Calculations demonstrated that a change in pH across the cell membrane of only 0.2 to 0.5 units is sufficient to achieve the observed ATP increases. Similarly, the hypothesis indicates that positively charged surfaces will decrease metabolic activity, and results from studies of positively charged surfaces support this finding.

Effects of linear alkylbenzene sulfonate on the sorption of Brij 30 and Brij 35 onto aquifer sand.

Surfactant sorption is of considerable importance to environmental applications, including surfactant flushing to mobilize hydrophobic contaminants; effects of surfactants on the transport of dissolved contaminants, microorganisms, and colloids through porous media; and bioremediation of hydrophobic organic compounds, as well as understanding the fate and transport of surfactants as environmental contaminants themselves. Although most sorption studies consider pure surfactants, commercial detergent formulations typically consist of mixtures of nonionic and anionic surfactants. In this study, the effects of varying concentrations of the anionic surfactant linear alkylbenzene sulfonate (LAS) on micelle formation and sorption behavior of the two commonly used nonionic surfactants Brij 30 and Brij 35 onto aquifer sand were examined. A strong linear relationship was observed between the critical micelle concentration (CMC) of the Brij surfactants and the concentration of LAS in the mixture, with the CMC decreasing with increasing concentration of LAS. The relative change in CMC as a function of the LAS concentration was identical forthe two Brij surfactants, indicating that LAS interacted with their common alkyl chains. Sorption isotherms were developed for Brij 30 and Brij 35 present as single surfactants in an aqueous solution as well as when present with LAS. Although LAS had minor effects on the maximum sorption plateaus of the Brij surfactants, Brij sorption at was significantly enhanced as a function of the LAS concentration for Brij aqueous concentrations below the CMC. Application of a multi-interaction isotherm model indicated that the formation of surface aggregates (e.g., hemimicelles) decreased with increasing LAS concentration. Overall, these results provide insight into the complex sorption behavior of surfactant mixtures.

Electrostatic behavior of the charge-regulated bacterial cell surface.

The electrostatic behavior of the charge-regulated surfaces of Gram-negative Escherichia coli and Gram-positive Bacillus brevis was studied using numerical modeling in conjunction with potentiometric titration and electrophoretic mobility data as a function of solution pH and electrolyte composition. Assuming a polyelectrolytic polymeric bacterial cell surface, these experimental and numerical analyses were used to determine the effective site numbers of cell surface acid-base functional groups and Ca(2+) sorption coefficients. Using effective site concentrations determined from 1:1 electrolyte (NaCl) experimental data, the charge-regulation model was able to replicate the effects of 2:1 electrolyte (CaCl(2)), both alone and as a mixture with NaCl, on the measured zeta potential using a single Ca(2+) surface binding constant for each of the bacterial species. This knowledge is vital for understanding how cells respond to changes in solution pH and electrolyte composition as well as how they interact with other surfaces. The latter is especially important due to the widespread use of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory in the interpretation of bacterial adhesion. As surface charge and surface potential both vary on a charge-regulated surface, accurate modeling of bacterial interactions with surfaces ultimately requires use of an electrostatic model that accounts for the charge-regulated nature of the cell surface.

Influence of solution ionic strength on the collision efficiency distribution and predicted transport distance of a Sphingomonas sp. flowing through porous media.

The effects of solution ionic strength on the collision efficiency (alpha) distribution of a Sphingomonas sp. were investigated using multiple sand columns of varying lengths and analyzing the bacteria clean-bed breakthrough concentrations using a distributed colloid filtration theory (D-CFT). Five different probability density functions (PDFs) were investigated and all accurately replicated the lab-scale experimental data, whereas a single alpha value could not. The alpha distribution shifted toward smaller values with decreasing ionic strength and the PDF parameters were strongly correlated to the Debye length, indicating that electrostatic interactions had a direct impact on the alpha distribution. The results indicate that while ionic strength has a large impact on bacterial transport distances for a concentration reduction of a few orders of magnitude, as occurs at the laboratory scale, due to the distributed nature of the collision efficiency, it has a minor effect on predicted transport distances required to achieve concentration reductions on the order of 10(6), which occurs at the field scale. Because of this, bacterial inactivation (e.g., death), rather than physically removing the bacteria from solution via filtration, is likely the key process impacting the transport of viable bacteria at the field scale. Overall, for systems with a distributed alpha, the results indicate that ionic strength has a strong influence on the transport of bacteria at the lab-scale (centimeters to one meter), both ionic strength and bacterial inactivation are important at the meso-scale (tens of meters), and inactivation becomes the dominant mechanism for reducing the transport of viable bacteria at the field scale (hundreds of meters).

Relationship between micellar and hemi-micellar processes and the bioavailability of surfactant-solubilized hydrophobic organic compounds.

In a landmark study on surfactant-enhanced biodegradation of hydrophobic organic compounds (HOCs), Guha and Jaffé demonstrated that a fraction (f) of micellar-phase HOC is directly bioavailable to bacterial cells. They developed a theoretical description of fwhich provided an excellent model of the experimental results. However, a mass transfer term describing the transport of the HOC through the cell wall (m(c)) was found to vary over an order of magnitude for the different surfactants examined, and a theoretical description of it remained elusive. This elusivity also resulted in the model not being able to describe a priori why fwas zero for the non-ionic surfactant C12E23. Here, the results of a recent study on surfactant sorption are used to develop an alternative mechanism describing m(c), where hemi-micellar formation on the cell surface is incorporated into the pathway describing micellar HOC bioavailability. The revised model is validated against HOC bioavailability data for five different C12E(y) surfactants, and it is shown that a single value for the mass transfer coefficient describing transfer of the hemi-micellar HOC into the bacterial cell is able to replicate the complete C12E(y) dataset, including that of C12E23 which eluded the original model. Overall, the results indicate that surfactant sorption and hemi-micelle formation are important parameters governing surfactant-enhanced bioavailability.

Cell surface acid-base properties of Escherichia coli and Bacillus brevis and variation as a function of growth phase, nitrogen source and C:N ratio.

Potentiometric titration has been conducted to systematically examine the acid-base properties of the cell surfaces of Escherichia coli K-12 and Bacillus brevis as a function of growth phase, nitrogen source (ammonium or nitrate), and carbon to nitrogen (C:N) ratio of the growth substrate. The two bacterial species revealed four distinct proton binding sites, with pK(a) values in the range of 3.08-4.05 (pK(1)), 4.62-5.57 (pK(2)), 6.47-7.30 (pK(3)), and 9.68-10.89 (pK(4)) corresponding to phosphoric/carboxylic, carboxylic, phosphoric, and hydroxyl/amine groups, respectively. Two general observations in the data are that for B. brevis the first site concentration (N(1)), corresponding to phosphoric/carboxylic groups (pK(1)), varied as a function of nitrogen source, while for E. coli the fourth site concentration (N(4)), corresponding to hydroxyl/amine groups (pK(4)), varied as a function of C:N ratio. Correspondingly, it was found that N(1) was the highest of the four site concentrations for B. brevis and N(4) was the highest for E. coli. The concentrations of the remaining sites showed little variation. Finally, comparison between the titration data and a number of cell surface compositional studies in the literature indicates one distinct difference between the two bacteria is that pK(4) of the Gram-negative E. coli can be attributed to hydroxyl groups while that of the Gram-positive B. brevis can be attributed to amine groups.

Comparative assessment of coal tars obtained from 10 former manufactured gas plant sites in the eastern United States.

A comparative analysis was performed on eleven coal tars obtained from former manufactured gas plant sites in the eastern United States. Bulk properties analyzed included percent ash, Karl Fisher water content, viscosity and average molecular weight. Chemical properties included monocyclic- and polycyclic-aromatic hydrocarbon (PAH) concentrations, alkylated aromatic concentrations, and concentrations of aliphatic and aromatic fractions. It was found that there was at least an order-of-magnitude variation in all properties measured between the eleven coal tars. Additionally, two coal tars obtained from the same manufactured gas plant site had very different properties, highlighting that there can be wide variations in coal tar properties from different samples obtained from the same site. Similarities were also observed between the coal tars. The relative chemical distributions were similar for all coal tars, and the coal tars predominantly consisted of PAHs, with naphthalene being the single-most prevalent compound. The C(9-22) aromatic fraction, an indicator of all PAHs up to a molecular weight of approximately 276 gmole(-1), showed a strong power-law relationship with the coal tar average molecular weight (MW (ct)). And the concentrations of individual PAHs decreased linearly as MW (ct) increased up to ca. 1000 gmole(-1), above which they remained low and variable. Implications of these properties and their variation with MW (ct) on groundwater quality are discussed. Ultimately, while these similarities do allow generalities to be made about coal tars, the wide range of coal tar bulk and chemical properties reported here highlights the complex nature of coal tars.

Collision efficiency distribution of a bacterial suspension flowing through porous media and implications for field-scale transport.

The collision efficiency (alpha) distribution of a bacterial population was determined using multiple packed-bed columns of varying lengths and analyzing the bacteria clean-bed breakthrough concentrations using a distributed colloid filtration theory. This technique allows the alpha distribution to be determined independently from other effects that can cause non-exponential deposition, including detachment and blocking. It was found that multiple probability density functions (PDF's) could accurately replicate the experimental data. Regardless of which PDF was used, a distributed alpha resulted in significantly greater predicted field-scale transport than when using a single alpha. However, there were wide variations in the predicted field-scale transport between the different distributions, suggesting that lab-scale experiments may not be readily utilized to determine the specific PDF that best represents alpha at the field scale. Finally, blocking was observed in the column effluent curves, underscoring the fact that if non-clean-bed processes occur then an approach such as that utilized in the current study may be used to separate the non-clean-bed and clean-bed processes when determining the collision efficiency distribution.

Effects of nonionic surfactants on the cell surface hydrophobicity and apparent hamaker constant of a Sphingomonas sp.

Nonionic surfactants of the form CxEy were studied for their ability to alter the cell surface hydrophobicity and apparent Hamaker constants of a Sphingomonas sp. Through contact angle measurements on hydrated and dried bacterial lawns, it was found that the cell surface hydrophobicity changed systematically with both the alkyl (x) and polyoxyethylene (y) chain lengths. While differences in contact angles were observed between hydrated and dried lawns, they could not be attributed to the mere presence or absence of water, suggesting that reorientation of cell surface components may occur during drying. All surfactants examined reduced the hydrophilicity of the bacterial cell surface, with one surfactant (C18E10) making the cells hydrophobic. Effective bacterial Hamaker constants for bacteria interacting across a vacuum (Abb) and water (Abwb) and bacteria interacting in water with quartz sand (Abws) were calculated from the contact angles. It was found that the surfactants have the potential to reduce the Hamaker constants, but that the overall effects differed between dried and hydrated lawns, indicating that lawn preparation method can have a significant impact in interpretation of cell surface properties. The results also indicate that the Abws value of 10-20 J, which is often assumed in bacterial attachment and transport studies, may be an order of magnitude higher than the actual value. Finally, the results suggest that alteration of bacterial adhesion due to the presence of surfactants cannot be attributed to a single cell surface property but is rather due to multiple interactions.

Nonionic surfactant sorption onto the bacterial cell surface: a multi-interaction isotherm.

The adsorption of linear polyoxyethylene (POE) alcohol surfactants of the form CxEy onto the surface of a Sphingomonas sp. has been examined. For this study, the alkyl chain length (x) was fixed at 12 and the POE chain length (y) was varied, with y = 4, 7, 9, 10, and 23 ethylene oxide units. Langmuirian isotherms were observed for C12E4 and C12E23, and more complex isotherms were observed for the three intermediate POE chain length surfactants, with C12E7 and C12E9 exhibiting strong S-shaped isotherms. All isotherms showed plateaus near the critical micelle concentration (CMC) with the plateau decreasing with increasing POE chain length. A simple multi-interaction isotherm is proposed that models the sorption isotherm as the sum of two interactions. The first interaction describes monolayer adsorption, whereas the second interaction describes lateral interactions between sorbed surfactant molecules and the formation of surface aggregates. Varying ratios of these two interactions as a function of POE chain length gives rise to the variety of observed isotherm shapes. Results of the isotherm analysis suggest that lateral interactions dominate for surfactants with low POE chain lengths, and the lateral interactions decrease as the POE chain length is increased.

Raoult's law-based method for determination of coal tar average molecular weight.

A Raoult's law-based method for determining the number average molecular weight of coal tars is presented. The method requires data from two-phase coal tar/water equilibrium experiments, which readily are performed in environmental laboratories. An advantage of this method for environmental samples is that it is not impacted by the small amount of inert debris often present in coal tar samples obtained from contaminated sites. Results are presented for 10 coal tars from nine former manufactured gas plants located in the eastern United States. Vapor pressure osmometry (VPO) analysis provided similar average molecular weights to those determined with the Raoult's law-based method, except for one highly viscous coal tar sample. Use of the VPO-based average molecular weight for this coal tar resulted in underprediction of the coal tar constituents' aqueous concentrations. Additionally, one other coal tar was not completely soluble in solvents used for VPO analysis. The results indicate that the Raoult's law-based method is able to provide an average molecular weight that is consistent with the intended application of the data (e.g., modeling the dissolution of coal tar constituents into surrounding waters), and this method can be applied to coal tars that may be incompatible with other commonly used methods for determining average molecular weight, such as vapor pressure osmometry.

Simultaneous utilization of acetate and hydrogen by Geobacter sulfurreducens and implications for use of hydrogen as an indicator of redox conditions.

Dissolved hydrogen concentrations, in conjunction with other geochemical indicators, are becoming an accepted means to determine terminal electron acceptor processes (TEAPs) in groundwater aquifers. Aqueous hydrogen concentrations have been found to fall within specific ranges under methanogenic, sulfate-reducing, iron-reducing, and denitrification conditions. Although hydrogen is gaining in acceptance for determining subsurface TEAPs, there is a dearth of data with regards to the kinetic coefficients for hydrogen utilization in the presence or absence of an additional electron donor under different TEAPs. This study expands the kinetic data for hydrogen utilization through a series of batch experiments, which were conducted to study the utilization of acetate and hydrogen by Geobacter sulfurreducens under iron-reducing conditions. The results of these experiments indicate that the kinetic coefficients (cell yield and first-order degradation rate) describing the rate of hydrogen utilization by G. sulfurreducens under iron-reducing conditions correlate energetically with the coefficients found in previous experiments under methanogenic and sulfate-reducing conditions. In addition, with acetate and hydrogen as simultaneous electron donors, there is slight inhibition between the two electron donors for G. sulfurreducens, and this can be modeled through competitive inhibition terms in the classic Monod formulation. Finally, a key result of this study is that the TEAP-dependent hydrogen concentration in aquifers is not related solely to the microbial kinetics of the hydrogen-consuming organisms as previously suggested but is affected by the multi-substrate kinetics of hydrogen being consumed simultaneously with other electron donors as well as the availability of the electron acceptor.

Effects of porous media preparation on bacteria transport through laboratory columns.

Bacterial and colloid transport experiments related to environmental systems are typically performed in the laboratory, with sand often used as the porous media. In order to prepare the sand, mechanical sieving is frequently used to tighten the sand grain size distribution. However, mechanical sieving has been reported to provide insufficient repeatability between identical colloidal transport experiments. This work examined the deficiencies of mechanical sieving with respect to bacterial transport through sand columns. It was found that sieving with standard brass sieves (1) contaminates the sand with copper and zinc as a linear function of sieving time and (2) inefficiently sizes sand grains below 300 microm (the largest size examined in this study) due to rapid clogging of the sieves. A procedure was developed that allows utilization of brass sieves for sizing the sand grains and removes the metal contamination introduced from the sieves. Bacterial transport experiments utilizing this column preparation procedure gave repeatable breakthrough curves. Further examination of the effects of these treatments on bacterial transport showed interesting results. First, it was found that the metal contamination did not affect the clean-bed bacterial transport. Second. it was found that variations of the column flushing procedure did not alter the clean-bed breakthrough of the bacteria, but did alter the inter-particle blocking. Finally, it was found that the shape of the sand grains (oblong vs. rounded) significantly alters the bacterial transport. with the transport being dominated by the smallest dimension of the oblong grains.