Characterization of Flocs and Floc Size Distributions Using Image Analysis.

A nonintrusive digital imaging process was developed to study particle size distributions created through flocculation and sedimentation. Quantification of particle size distributions under different operating conditions can be of use in the understanding of aggregation mechanisms. This process was calibrated by measuring standardized polystyrene particles of known size and was utilized to count and measure individual kaolin clay particles as well as aggregates formed by coagulation with polyaluminum chloride and flocculation. Identification of out-of-focus flocs was automated with LabVIEW and used to remove them from the database that was analyzed. The particle diameter of the test suspension of kaolinite clay was measured to be 7.7 ± 3.8 µm and a linear relationship was obtained between turbidity and the concentration of clay particles determined by imaging. The analysis technique was applied to characterize flocs and floc particle size distribution as a function of coagulant dose. Removal of flocs by sedimentation was characterized by imaging, and the negative logarithm of the fraction of turbidity remaining after settling had a linear relationship with the logarithm of aluminum dose. The maximum floc size observed in the settled water was less than 120 µm, which was in accordance with the value predicted by a model for the capture velocity of the experimental tube settler of 0.21 mm/s.

Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1.

Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.

Correlation equation for predicting attachment efficiency (α) of organic matter-colloid complexes in unsaturated porous media.

Naturally occurring polymers such as organic matter have been known to inhibit aggregation and promote mobility of suspensions in soil environments by imparting steric stability. This increase in mobility can significantly reduce the water filtering capacity of soils, thus jeopardizing a primary function of the vadose zone. Improvements to classic filtration theory have been made to account for the known decrease in attachment efficiency of electrostatically stabilized particles, and more recently, of sterically stabilized particles traveling through simple and saturated porous media. In the absence of an established unsaturated transport expression, and in the absence of applicable theoretical approaches for suspensions with asymmetric and nonindifferent electrolytes, this study presents an empirical correlation to predict attachment efficiency (α) for electrosterically stabilized suspensions in unsaturated systems in the presence of nonideal electrolytes. We show that existing models fall short in estimating polymer-coated colloid deposition in unsaturated media. This deficiency is expected given that the models were developed for saturated conditions where the mechanisms controlling colloid deposition are significantly different. A new correlation is derived from unsaturated transport data and direct characterization of microspheres coated with natural organic matter over a range of pH and CaCl(2) concentrations. The improvements to existing transport models include the following: adjustment for a restricted liquid-phase in the medium, development of a quantitative term to account for unsaturated transport phenomena, and adjustments in the relative contribution of steric stability parameters based on direct measurements of the adsorbed polymer layer characteristics. Differences in model formulation for correlations designed for saturated systems and the newly proposed correlation for unsaturated systems are discussed, and the performance of the new model against a comprehensive set of experimental observations is evaluated.

Fluid shear influences on the performance of hydraulic flocculation systems.

Gravity driven hydraulic flocculators that operate in the absence of reliable electric power are better suited to meet the water treatment needs of green communities, resource-poor communities, and developing countries than conventional mechanical flocculators. However, current understanding regarding the proper design and operation of hydraulic flocculation systems is insufficient. Of particular interest is the optimal fluid shear level needed to produce low turbidity water. A hydraulic tube flocculator was used to study how fluid shear levels affect the settling properties of a flocculated alum-kaolin suspension. A Flocculation Residual Turbidity Analyzer (FReTA) was used to quantitatively compare the sedimentation velocity distributions and the post-sedimentation residual turbidities of the flocculated suspensions to see how they were affected by varying fluid shear, G, and hydraulic residence time, θ, while holding collision potential, Gθ, constant. Results show that floc breakup occurred at all velocity gradients evaluated. High floc settling velocities were correlated with low residual turbidities, both of which were optimized at low fluid shear levels and long fluid residence times. This study shows that, for hydraulic flocculation systems under the conditions described in this paper, low turbidity water is produced when fluid shear is kept at a minimum. Use of the product Gθ for design of laminar flow tube flocculators is insufficient if residual turbidity is used as the metric for performance. At any Gθ within the range tested in this study, best performance is obtained when G is small and θ is long.

Method for quantitative analysis of flocculation performance.

The sedimentation rate and the post-sedimentation residual turbidity of flocculated suspensions are properties central to the design and operation of unit processes following flocculation in a water treatment plant. A method for comparing flocculation performance based on these two properties is described. The flocculation residual turbidity analyzer (FReTA) records the turbidity of flocculent suspensions undergoing quiescent settling. The fixed distance across which flocs must travel to clear the measurement volume allows sedimentation velocity distributions of the flocculent suspension to be calculated from the raw turbidity data. By fitting the transformed turbidity data with a modified gamma distribution, the mean and variance of sedimentation velocity can be obtained along with the residual turbidity after a period of settling. This new analysis method can be used to quantitatively compare how differences in flocculator operating conditions affect the sedimentation velocity distribution of flocs as well as the post-sedimentation residual turbidity.

Impact of dissolved organic matter on colloid transport in the vadose zone: deterministic approximation of transport deposition coefficients from polymeric coating characteristics.

Although numerous studies have been conducted to discern colloid transport and stability processes, the mechanistic understanding of how dissolved organic matter (DOM) affects colloid fate in unsaturated soils (i.e., the vadose zone) remains unclear. This study aims to bridge the gap between the physicochemical responses of colloid complexes and porous media interfaces to solution chemistry, and the effect these changes have on colloid transport and fate. Measurements of adsorbed layer thickness, density, and charge of DOM-colloid complexes and transport experiments with tandem internal process visualization were conducted for key constituents of DOM, humic (HA) and fulvic acids (FA), at acidic, neutral and basic pH and two CaCl(2) concentrations. Polymeric characteristics reveal that, of the two tested DOM constituents, only HA electrosterically stabilizes colloids. This stabilization is highly dependent on solution pH which controls DOM polymer adsorption affinity, and on the presence of Ca(+2) which promotes charge neutralization and inter-particle bridging. Transport experiments indicate that HA improved colloid transport significantly, while FA only marginally affected transport despite having a large effect on particle charge. A transport model with deposition and pore-exclusion parameters fit experimental breakthrough curves well. Trends in deposition coefficients are correlated to the changes in colloid surface potential for bare colloids, but must include adsorbed layer thickness and density for sterically stabilized colloids. Additionally, internal process observations with bright field microscopy reveal that, under optimal conditions for retention, experiments with FA or no DOM promoted colloid retention at solid-water interfaces, while experiments with HA enhanced colloid retention at air-water interfaces, presumably due to partitioning of HA at the air-water interface and/or increased hydrophobic characteristics of HA-colloid complexes.

An uncertain role for Cu(II) in stimulating Mn(II) oxidation by Leptothrix discophora SS-1.

In an effort to improve understanding of the role of Cu(II) in bacterial Mn(II) oxidation, a model Mn(II)-oxidizing bacterium, Leptothrix discophora SS-1, was grown in presence of toxic and non-toxic concentrations of Cu(II), Cd(II) and Mn(II). Mn(II)-oxidizing activity increased by 40% when cells were grown in the presence of 0.05 microM of Cu(II) and increased twofold at 0.18 microM Cu(II). Toxic levels of Cd(II) did not stimulate Mn(II) oxidizing activity, indicating that Mn(II) oxidation is not a response to metal toxicity. Stimulation by Cu(II) confirms the specific role of Cu(II) in Mn(II) oxidation. Comparison of transcript levels of the multicopper oxidase mofA gene in the presence and absence of added Cu(II) do not indicate a statistically significant change in mofA transcript levels in cultures supplemented with Cu(II). Thus, the exact role of Cu(II) in Mn(II) oxidation and its affect on mofA gene expression remain uncertain.

Curli produced by Escherichia coli PHL628 provide protection from Hg(II).

The role of curli, amyloid extracellular fibers, in the tolerance of Escherichia coli PHL628 to Hg(II) was examined. Our findings indicate that by sorbing Hg(II) extracellularly, curli protect the cells. To our knowledge, this is the first time a protective role of curli against toxic metals has been demonstrated.

Flocculation model and collision potential for reactors with flows characterized by high Peclet numbers.

A mechanistically-based model is developed to characterize flocculation in the context of flow regimes with high Peclet numbers such as would occur in serpentine flow reactors. These flow conditions are obtained in gravity-driven hydraulic flocculators without mechanical agitation that are an important component of sustainable water treatment systems where energy efficiency and cost are receiving increasing emphasis. The model incorporates a fractal description of flocs, estimates of floc separation distances, estimates of relative velocities of floc particles dependent on the relevant flow regime, and provides an overall prediction of the required reaction time for formation of settleable flocs based on influent turbidity, alum dose, and energy dissipation rate. Viscosity is shown to be significant for the early stage of flocculation and turbulent eddies are shown to be significant for the final stage of flocculation. The collision potential defined as the product of the hydraulic residence time (θ) and the cube root of the energy dissipation rate (ɛ), i.e., ɛ(1/3)θ, is shown to be a better predictor of flocculator performance than the conventional product of θ and the velocity gradient (G), i.e., Gθ.

Mass transfer model of nanoparticle-facilitated contaminant transport in saturated porous media.

A one-dimensional model has been evaluated for transport of hydrophobic contaminants, such as polycyclic aromatic hydrocarbon (PAH) compounds, facilitated by synthetic amphiphilic polyurethane (APU) nanoparticles in porous media. APU particles synthesized from poly(ethylene glycol)-modified urethane acrylate (PMUA) precursor chains have been shown to enhance the desorption rate and mobility of phenanthrene (PHEN) in soil. A reversible process governed by attachment and detachment rates was considered to describe the PMUA binding in soil in addition to PMUA transport through advection and dispersion. Ultimately, an irreversible second-order PMUA attachment rate in which the fractional soil saturation capacity with PMUA was a rate control was found to be adequate to describe the retention of PMUA particles. A gamma-distributed site model (GS) was used to describe the spectrum of physical/chemical constraints for PHEN transfer from solid to aqueous phases. Instantaneous equilibrium was assumed for PMUA-PHEN interactions. The coupled model for PMUA and PHEN behavior successfully described the enhanced elution profile of PHEN by PMUA. Sensitivity analysis was performed to analyze the significance of model parameters on model predictions. The adjustable parameter alpha in the gamma-distribution shapes the contaminant desorption distribution profile as well as elution and breakthrough curves. Model simulations show the use of PMUA can be also expected to improve the release rate of PHEN in soils with higher organic carbon content. The percentage removal of PHEN mass over time is shown to be influenced by the concentration of PMUA added and this information can be used to optimize cost and time require to accomplish a desired remediation goal.

Functional tomographic fluorescence imaging of pH microenvironments in microbial biofilms by use of silica nanoparticle sensors.

Attached bacterial communities can generate three-dimensional (3D) physicochemical gradients that create microenvironments where local conditions are substantially different from those in the surrounding solution. Given their ubiquity in nature and their impacts on issues ranging from water quality to human health, better tools for understanding biofilms and the gradients they create are needed. Here we demonstrate the use of functional tomographic imaging via confocal fluorescence microscopy of ratiometric core-shell silica nanoparticle sensors (C dot sensors) to study the morphology and temporal evolution of pH microenvironments in axenic Escherichia coli PHL628 and mixed-culture wastewater biofilms. Testing of 70-, 30-, and 10-nm-diameter sensor particles reveals a critical size for homogeneous biofilm staining, with only the 10-nm-diameter particles capable of successfully generating high-resolution maps of biofilm pH and distinct local heterogeneities. Our measurements revealed pH values that ranged from 5 to >7, confirming the heterogeneity of the pH profiles within these biofilms. pH was also analyzed following glucose addition to both suspended and attached cultures. In both cases, the pH became more acidic, likely due to glucose metabolism causing the release of tricarboxylic acid cycle acids and CO(2). These studies demonstrate that the combination of 3D functional fluorescence imaging with well-designed nanoparticle sensors provides a powerful tool for in situ characterization of chemical microenvironments in complex biofilms.

Iron requirement for Mn(II) oxidation by Leptothrix discophora SS-1.

A common form of biocatalysis of Mn(II) oxidation results in the formation of biogenic Mn(III, IV) oxides and is a key reaction in the geochemical cycling of Mn. In this study, we grew the model Mn(II)-oxidizing bacterium Leptothrix discophora SS-1 in media with limited iron (0.1 microM iron/5.8 mM pyruvate) and sufficient iron (0.2 microM iron/5.8 mM pyruvate). The influence of iron on the rate of extracellular Mn(II) oxidation was evaluated. Cultures in which cell growth was limited by iron exhibited reduced abilities to oxidize Mn(II) compared to cultures in medium with sufficient iron. While the extracellular Mn(II)-oxidizing factor (MOF) is thought to be a putative multicopper oxidase, Mn(II) oxidation in the presence of zero added Cu(II) was detected and the decrease in the observed Mn(II) oxidation rate in iron-limited cultures was not relieved when the medium was supplemented with Cu(II). The decline of Mn(II) oxidation under iron-limited conditions was not accompanied by siderophore production and is unlikely to be an artifact of siderophore complex formation with Mn(III). The temporal variations in mofA gene transcript levels under conditions of limited and abundant iron were similar, indicating that iron limitation did not interfere with the transcription of the mofA gene. Our quantitative PCR results provide a step forward in understanding the regulation of Mn(II) oxidation. The mechanistic role of iron in Mn(II) oxidation is uncertain; the data are consistent with a direct requirement for iron as a component of the MOF or an indirect effect of iron resulting from the limitation of one of many cellular functions requiring iron.

Spatial distributions of copper in microbial biofilms by scanning electrochemical microscopy.

The spatial distribution of Cu was determined in Escherichia coli PHL628 biofilms using a scanning electrochemical microscope (SECM) consisting of a microelectrode in conjunction with a piezoelectric micropositioning system. Aqueous labile copper species were determined using voltametric stripping after reductive deposition of Cu for 4 min on the microelectrode at -0.7 V (vs Ag/AgCl). The position of the bulk solution-biofilm interface was determined from the change in current produced by 0.4 mM hydroxymethyl ferrocene that was added as a redox indicator. After a 2 h exposure to 0.2 mM copper, Cu was located in the upper region of the biofilm with a penetration depth less than 150 microm. A one-dimensional diffusive transport model adequately described the spatial distribution of copper in the biofilm, but the Cu retardation factor in the biofilm was more than 6-fold larger than that calculated from the isotherm for Cu binding to suspensions of E. coli PHL628 cells. There are several possible reasons for this difference, including an increase in the amount of extracellular polymer per cell within the biofilm and/or tortuosity that might hinder Cu transport into biofilms. The SECM technique in combination with model calculations provides direct evidence in support of the concept that formation of a biofilm may confer resistance to transient spikes in the bulk solution concentration of toxic metal species by retarding metal diffusion and reducing the metal exposure of cells within the biofilm.

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy.

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 microm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 microm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 microm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).

Engineered polymeric nanoparticles for bioremediation of hydrophobic contaminants.

Sorption of hydrophobic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs), to soil has been shown to limit their solubilization rate and mobility. In addition, sequestration of contaminants by sorption to soil and by partitioning in nonaqueous phase liquids (NAPLs) reduces their bioavailability. Polymeric nano-network particles have been demonstrated to increase the "effective" solubility of a representative hydrophobic organic contaminant, phenanthrene (PHEN) and to enhance the release of PHEN from contaminated aquifer material. In this study, we investigate the usefulness of nanoparticles made from a poly(ethylene) glycol modified urethane acrylate (PMUA) precursor chain, in enhancing the bioavailability of PHEN. PMUA nanoparticles are shown to increase the mineralization rate of PHEN crystal in water, PHEN sorbed on aquifer material, and PHEN dissolved in a model NAPL (hexadecane) in the presence of aquifer media. These results show that PMUA particles not only enhance the release of sorbed and NAPL-sequestered PHEN but also increase its mineralization rate. The accessibility of contaminants in PMUA particles to bacteria also suggests that particle application may be an effective means to enhance the in-situ biodegradation rate in remediation through natural attenuation of contaminants. In pump-and-treat or soil washing remediation schemes, bioreactors could be used to recycle extracted nanoparticles. The properties of PMUA nanoparticles are shown to be stable in the presence of a heterogeneous active bacterial population, enabling them to be reused after PHEN bound to the particles has been degraded by bacteria.

Behavior of lead in a model microbial predator-prey system.

Predation at the microbial level can affect the fate of toxic trace metals. Metals associated with bacterial prey can be released into the dissolved phase following digestion by a predator, and/or metals can remain in the predator and be transferred potentially to the next level of the food chain. Toxic metal ions in the aqueous phase also are expected to modify the growth and predation rate of a microbial predator. A model predator-prey system was developed to test the effects of Pb on cells and to help elucidate the fate of Pb in this type of interaction. Established methods that have been shown to be suitable for distinguishing dissolved, prey-bound, predator-bound, and ingested Pb were used to establish the pathway of Pb over time. Growth parameters were measured using batch reactors for the protozoan predator Tetrahymena thermophila and the bacterial prey Pseudomonas putida without Pb and at several concentrations of Pb. The effect of prey density on predation and Pb phase distribution also was investigated. Results demonstrate that some kinetic parameters related to prey consumption and growth of T. thermophila are altered by Pb. Upon addition of predator to prey cells in equilibrium with dissolved Pb, dissolved and prey-bound Pb become associated with the predator through ingestion and adsorption. Ingested Pb is excreted later as a bound metal associated with T. thermophila waste matter. A preliminary mathematical model was developed to describe predator-prey dynamics and their influence on the behavior and fate of Pb. Growth data were used to obtain model parameters, and model simulations for Pb fractionation are compared to experimental observations.

Predicting the rate and extent of cadmium and copper desorption from soils in the presence of bacterial extracellular polymer.

The movement of cationic transition metals through the subsurface is strongly retarded by sorption to the porous media. However, dissolved organic ligands can compete with soil surfaces by providing binding sites for metals in solution. An extracellular polymer produced by a bacterium isolated from soil was used in this study to observe and model the influence of a naturally occurring ligand on the release of adsorbed metals from two test soils. Experimental results show that the presence of dissolved extracellular polymer enhanced the rate and extent of desorptive release of soil-bound cadmium and copper. A kinetic model that uses a gamma distribution of rate constants to account for the physical and chemical heterogeneity of the soil matrix was employed to describe the release of cadmium and copper in batch experiments. Model parameters describing soil, metal and extracellular polymer interactions were obtained through separate experiments. With these parameters the model successfully predicted the influence of dissolved polymer on the rate and extent of release of cadmium and copper from soil in independent batch experiments. These results suggest that the presence of natural metal-binding ligands such as bacterial extracellular polymers can act to increase the driving force for desorption by lowering the aqueous concentration of free unbound metals in solution.

Engineered polymeric nanoparticles for soil remediation.

Hydrophobic organic groundwater contaminants, such as polynuclear aromatic hydrocarbons (PAHs), sorb strongly to soils and are difficult to remove. We report here on the synthesis of amphiphilic polyurethane (APU) nanoparticles for use in remediation of soil contaminated with PAHs. The particles are made of polyurethane acrylate anionomer (UAA) or poly(ethylene glycol)-modified urethane acrylate (PMUA) precursor chains that can be emulsified and cross-linked in water. The resulting particles are of colloidal size (17-97 nm as measured by dynamic light scattering). APU particles have the ability to enhance PAH desorption and transport in a manner comparable to that of surfactant micelles, but unlike the surface-active components of micelles, the individual cross-linked precursor chains in APU particles are not free to sorb to the soil surface. Thus, the APU particles are stable independent of their concentration in the aqueous phase. In this paper we show that APU particles can be engineered to achieve desired properties. Our experimental results show that the APU particles can be designed to have hydrophobic interior regions that confer a high affinity for phenanthrene (PHEN) and hydrophilic surfaces that promote particle mobility in soil. The affinity of APU particles for contaminants such as PHEN can be controlled by changing the size of the hydrophobic segment used in the chain synthesis. The mobility of colloidal APU suspensions in soil is controlled by the charge density or the size of the pendent water-soluble chains that reside on the particle surface. Exemplary results are provided illustrating the influence of alternative APU particle formulations with respect to their efficacy for contaminant removal. The ability to control particle properties offers the potential to produce different nanoparticles optimized for varying contaminant types and soil conditions.

Mobilization of adsorbed copper and lead from naturally aged soil by bacterial extracellular polymers.

Sorption of pollutants is a dominant phase transfer process affecting the fate and transport of metals through the subsurface. The movement of contaminants is retarded by sorption to the stationary subsurface porous media and can seriously hinder remediation efforts. Research has shown that the binding of adsorbed metals becomes more pronounced the longer the contaminant is in the subsurface and the release rates of aged metal contaminants have not received the research attention given to freshly added metals in laboratory studies. Metal release rates are also influenced by the presence of dissolved ligands that compete with mineral soil surfaces by providing binding sites. Dissolved organic matter such as bacterial extracellular polymers are common in natural soil solutions and the metal binding properties of bacterial polymers are well established. Therefore, binding of metals to dissolved biopolymers may result in mobilization of an adsorbed metal. This is important for cases where the metals are assumed to be relatively immobile such as in the case of land applied biosolids. In addition, naturally occurring adherent bacteria commonly produce extracellular polymers and thus may modify the bioavailability of meal contaminants at the point of their attachment. In this study samples from three sites, one a land applied sludge test site, were used to investigate the ability of bacterial extracellular polymers to release metals from soils with long-term exposures. The presence of ?200mg/L bacterial extracellular polymer was found to increase the short-term (less than 350h) release of Cu and Pb by a factor of 2-4-fold.

Development of a model microbial predator-prey system suitable for studies of the behavior of toxic trace metals.

Interactions between microbial predators and their prey can significantly influence the behavior of toxic trace metals. Ingested bacterial prey-bound metals can either accumulate within a predator or be excreted and potentially reintroduced into the dissolved phase. A defined predator-prey system suitable for developing a more fundamental understanding of metal behavior in simple microbial food chains was designed and tested by using lead (Pb) as a representative cationic transition metal. Desired features of this system were the ability to define the chemical speciation of dissolved metals as well as to distinguish between prey- and predator-bound metals. Pseudomonas putida and the ciliate protozoan Tetrahymena thermophila were selected as representative bacterial prey and predator species, respectively. In addition, the use of fluorescent microspheres was evaluated as an experimental surrogate for bacterial prey. Filtration techniques for size-selective separation were developed so that the distribution of Pb between cells of T. thermophila, cells of P. putida or microspheres, and the dissolved phase could be assessed. Filtration units were selected based on their ability to perform separations with minimal metal loss at circumneutral pH. Five-micron polycarbonate filter membranes successfully separated T. thermophila from P. putida with good cell retention and low metal loss. Centrifuge filters successfully separated dissolved and particle-bound metal (<5,000 nominal molecular wt limit). Exemplary experimental results are presented and show that predation on Pb-exposed cells of P. putida or microspheres increases uptake of Pb by T. thermophila.