Influence Of Extracellular Polymeric Substances Research Articles

Effects of recirculation dredging on density, strength, settling and oxygen concentration of fluid mud in the port of Emden

Abstract Purpose Recirculation dredging is a port maintenance concept developed in the Port of Emden, Germany to create a navigable fluid mud layer. This study investigates the effects of recirculation on key sediment properties, including density, yield stress, and oxygen concentration. Methods Six field monitoring surveys were carried out at two locations at different times of the year to assess changes before and after recirculation. Bathymetry, bulk density, yield stress, and oxygen concentration profiles were measured in situ. The settling properties and oxygen concentration levels on collected fluid mud samples were analyzed in the laboratory. Results The investigation reveals minimal changes in the density of recirculated fluid mud. However, the post-recirculation measurements showed a decrease in yield stress, ranging from 18 to 51% at Große Seeschleuse (GS) and 36% to 52% at Industriehafen (IH). The yield stress and density vary depending on the frequency of dredging. After structural density (1166 kg m−3 in GS and 1173 kg m−3 in IH), the yield stress of fluid mud increased exponentially. Therefore, monitoring of the yield stress is important for recirculation. A slight increase in oxygen concentration was observed post-recirculation, especially during winter. Yet, the rapid decline in oxygen levels post-mixing in the laboratory showed that sustaining long-term elevated oxygenation levels is not feasible by recirculation dredging alone. Conclusions The findings highlight the effectiveness of the recirculation on the yield stress, density, and oxygen concentration of fluid mud and illustrate the importance of considering both density and yield stress in sediment management practices. Future research should address the temporal evolution of density, yield stress, and oxygen levels following a dredging intervention and the influence of extracellular polymeric substances (EPS) and organic matter decay on sediment behavior.

Influence of extracellular polymeric substances on arsenic bioaccumulation and biotransformation in biofilms

It is well recognized that biofilms can biosorb and biotransform heavy metals in aquatic environments. However, the effects of extracellular polymeric substance (EPS) on inorganic arsenic (As) bioaccumulation and biotransformation in biofilms are still unrevealed and need to be investigated. In order to explore the above scientific issues, the As accumulation and speciation in EPS-containing or EPS-free biofilms and growth medium under As(V)/As(III) exposure conditions were measured. After the removal of EPS, the amount of As uptake (Asup) and As adsorption (Asad) in biofilms were significantly reduced, no matter whether exposed to As(V) or As(III). FTIR analysis further suggested that the interaction between these functional groups with As was limited after the removal of EPS. In the EPS-containing biofilms, the Asad was mainly As(V) with low toxicity. However, after the removal of EPS, the Asad was mainly As(III) with high fluidity, and no methylated As was found. Moreover, the removal of EPS inhibited As(III) oxidation and methylation by biofilms, resulting in the decrease of As(V) and methylated As in the growth medium. The findings of this study emphasized the essential impact of EPS on the biosorption and biotransformation of As in biofilms. This study provides a unique understanding of the role of biofilms in As biogeochemical cycle, and water quality purification function in water environments.

Extracellular polymeric substances altered the physicochemical properties of molybdenum disulfide nanomaterials to mitigate its toxicity to Chlorella vulgaris

Although the toxic effects of two-dimensional nanomaterials (2D-NMs) have been widely reported, the influence of extracellular polymeric substances (EPS) on the environmental fate and risk of 2D-NMs in aquatic environments is largely unknown, and the processes and mechanisms involved remain to be revealed. Herein, we investigated the impact of EPS secreted by microalgae (Chlorella vulgaris (C. vulgaris)) on the environmental transformation and risk of molybdenum disulfide (MoS2). We found that the attachment of EPS increased the thickness of MoS2 (from 2 nm to 5 nm), changed it from a monolayer sheet to a fuzzy multilayer structure, and promoted the formation of defects on MoS2. The blue-shift of the peak associated with the plasmon resonances in the 1 T phase and the generation of electron-hole pairs suggested that EPS altered the surface electronic structure of MoS2. EPS interacted mainly with the S atoms on the 1 T phase, and the attachment of EPS promoted the oxidation of MoS2. The reduction in hydrodynamic diameter (Dh) and the decrease in zeta potential indicated that EPS inhibited the agglomeration behavior of MoS2 and enhanced its dispersion and stability in aqueous media. Notably, EPS reduced the generation of free radicals (superoxide anion (•O2−), singlet oxygen (1O2), and hydroxyl radicals (•OH−)). Furthermore, EPS mitigated the toxicity of MoS2 to C. vulgaris, such as attenuated reduction in biomass and chlorophyll content. Compared to pristine MoS2, MoS2 + BG11 + EPS exhibited weaker oxidative stress, membrane damage and lipid peroxidation. The adsorption of EPS on MoS2 surface reduced the attachment sites of MoS2, making MoS2 less likely to be enriched on the cell surface. The findings have significant contribution for understanding the interactions between EPS and MoS2 in aquatic ecosystems, providing scientific guidance for risk assessment of 2D-NMs.

Stability and mobility of zinc oxide nanoparticles in aquatic environment: Influence of extracellular polymeric substances from cyanobacteria and microalgae

The stability and mobility of engineered nanoparticles in the natural water environment are influenced by various environmental factors, such as electrolyte conditions and the presence of natural organic matters. Cyanobacterial and microalgal extracellular polymeric substances (EPSs) are complex organic mixtures ubiquitous in eutrophic waters. In this study, bound EPSs (B-EPSs) and dissolved EPSs (D-EPSs) of Microcystis sp. and Chlorella vulgaris were extracted as the representative natural organic matters in the aquatic environment. Time-resolved dynamic light scattering and quartz crystal microbalance with dissipation monitoring were applied to investigate the influence of the extracted EPSs on zinc oxide nanoparticle (ZnO NP) aggregation and deposition behaviors. Data showed that EPSs with higher protein fractions were more efficient in hindering the aggregation of ZnO NPs in NaCl solutions and low CaCl2 concentration solutions. In higher CaCl2 concentration solutions (>50 mM), the presence of D-EPSs promoted aggregation by forming intermolecular bridging. By accelerating the Brownian movement of NPs and providing electrostatic and hydrophilic attraction between NPs and silica surfaces, B-EPSs facilitated ZnO NP deposition. On the contrary, D-EPSs hindered the deposition through steric repulsion. Moreover, B-EPS-coated ZnO NPs formed a more rigid layer, while the D-EPS-coated ZnO NPs formed a softer layer.

Influence of extracellular polymeric substances on electrochemical behaviours of stainless steels in circulating cooling water

Stainless steel alloys, often used in circulating cooling systems of power plants using reclaimed water as coolant, can be affected by biocorrosion induced by biofilm formation. Extracellular polymeric substances (EPS) produced by bacteria play an important role in the different stages of biofilm formation, maturation and maintenance. The influence of loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS), isolated from Citrobacter freundii, on the adsorption properties and electrochemical behaviours of stainless steels 304, 316 L and 317 (SS304, SS316 L and SS317) in circulating cooling water, were investigated by evaluating the adhesion forces of SSs (AFM), surface analysis (SEM, XPS) and the effect of EPS on SSs (electrochemical measurements, ICP-MS). The results show that the LB-EPS and TB-EPS of the solutions influence the surface adsorption of stainless steels (SSs). The adhesion forces of SSs in TB-EPS are higher than those of LB-EPS. A statistical difference in the adhesion forces of SSs was detected, with higher adhesion force of SS317, followed by SS316L and finally by SS304. The adhesion forces to SSs are influenced by both the electrostatic force and metal surface hydrophobicity. LB-EPS and TB-EPS can inhibit the dissolution of metal elements on SS surfaces in circulating cooling water. The dissolution rate of each metal element on the surface of SS304, SS316 L and SS317 is different, and their dissolution trends are basically the same: Fe > Ni > Cr > Mn. In the presence of TB-EPS or LB-EPS, the dissolution rate of Fe on SS304, SS316L and SS317 decreases by 30.61%, 24.20% and 27.43% or 77.95%, 86.70% and 83.96%, respectively. Electrochemistry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) indicate that the formation of a protective film composed of organic complexes such as C-(Fe, Ni), C–O-(Fe, Ni), N-(Fe, Ni), N–O-(Ni, Fe) and FeOOH on the SS surfaces can slow down the corrosion rate of stainless steel to some extent. Moreover, EPS films formed on SS317 and SS316 L have higher resistance and lower conductivity than those formed on SS304.

Surface biomineralization of uranium onto Shewanella putrefaciens with or without extracellular polymeric substances

The influence of extracellular polymeric substances (EPS) on the interaction between uranium [U(VI)] and Shewanella putrefaciens (S. putrefaciens), especially the U(VI) biomineralization process occurring on whole cells and cell components of S. putrefaciens was investigated in this study. The removal efficiency of U(VI) by S. putrefaciens was decreased by 22% after extraction of EPS. Proteins were identified as the main components of EPS by EEM analysis and were determined to play a major role in the biosorption of uranium. SEM-EDS results showed that U(VI) was distributed around the whole cell as 500-nanometer schistose structures, which consisted primarily of U and P. However, similar uranium lamellar crystal were wrapped only on the surface of EPS-free S. putrefaciens cells. FTIR and XPS analysis indicated that phosphorus- and nitrogen-containing groups played important roles in complexing U (VI). XRD and U LIII-edge EXAFS analyses demonstrated that the schistose structure consisted of hydrogen uranyl phosphate [H2(UO2)2(PO4)2•8H2O]. Our study provides new insight into the mechanisms of induced uranium crystallization by EPS and cell wall membranes of living bacterial cells under aerobic conditions.

The Role of Extracellular Polymeric Substances in the Toxicity Response of Anaerobic Granule Sludge to Different Metal Oxide Nanoparticles.

Wastewater treatment plants (WWTP) are regarded as the last barriers for the release of incompletely separated and recycled nanoparticles (NPs) into the environment. Despite the importance and ubiquity of microbial extracellular polymeric substances (EPSs) in the complex wastewater matrix, the interaction between NPs and EPSs of anaerobic microflora involved in wastewater treatment and the resultant impact on the biomass metabolomics are unclear. Thus, the impacts of different metal oxide (TiO2, ZnO, and CuO) NPs on functional bacteria in anaerobic granular sludge (AGS) and the possible toxicity mechanisms were investigated. In particular, the binding quality, enhanced resistance mechanism, and chemical fractional contribution of EPSs from AGS against the nanotoxicity of different NPs was assessed. The results showed that CuO NPs caused the most severe inhibition towards acetoclastic and hydrogenotrophic methanogens, followed by ZnO NPs, whereas TiO2 NPs caused no inhibition to methanogenesis. Excessive EPS production, especially the protein-like substances, was an effective strategy for reducing certain NPs’ toxicity by immobilizing NPs away from AGS cells, whereas the metabolism restriction on inner microorganisms of AGS induced by CuO NPs can deteriorate the protective role of EPS, indicating that the roles of EPS may not be amenable to generalizations. Further investigations with lactate dehydrogenase (LDH) and reactive oxygen species (ROS) assays indicated that there are greatly essential differences between the toxicity mechanisms of metal NPs to AGS, which varied depending on the NPs’ type and dosage. In addition, dynamic changes in the responses of EPS content to different NPs can result in a significant shift in methanogenic and acidogenic microbial communities. Thus, the production and composition of EPSs will be a key factor in determining the fate and potential effect of NPs in the complex biological matrix. In conclusion, this study broadens the understanding of the inhibition mechanisms of metal oxide NPs on the AGS process, and the influence of EPSs on the fate, behavior, and toxicity of NPs.

Production of Hydroxyl Radicals from Oxygenation of Simulated Acid Mine Drainage in Presence of Extracellular Polymers Substances.

The reaction mechanisms Fe(II) abiotic oxidation produce ·OH by CaCO3-induced in acid mine drainage (AMD) are well-documented, but little is known about the influence of extracellular polymeric substances (EPS) secreted by microorganisms on Fe(II) oxidation in AMD. In this study, ·OH production was experimently measured from oxygenation of simulated AMD in the presence of EPS. The cumulative ·OH increased from 56.75 to 158.70 µM within 24h at pH 3 with the increase in EPS concentration from 0 to 12mg/L. An appropriate pH (about 6) and EPS (6mg/L) concentration were required for the moderate rate of Fe(II) oxidation. Besides, the yield of ·OH increased remarkably with the addition of Fe3+. In the presence of EPS, ·OH production is attributed mainly the complexation of Fe(II) with EPS, of which is rich of carboxyl and hydroxyl groups. The findings provide fundamental supplement of ·OH production from Fe(II) oxidation by microorganisms in natural AMD.

Interaction of temperature, salinity and extracellular polymeric substances controls trace element incorporation into tufa calcite

AbstractThe influence of extracellular polymeric substances on carbonate mineral growth in natural settings remains one of the most poorly understood contributors to the growth of non‐marine carbonate sediments. The influences of these materials are complicated by their association with living cells creating local microenvironments via metabolism and enzyme production, and by our uncertainty about the extracellular polymeric substances materials themselves. Different mixtures of extracellular polymeric substance molecules may behave in different ways, and differences in the local physical environment may alter how the mixtures influence mineral formation, and even result in different patterns of polymerization. Here, the influence of extracellular polymeric substances on calcite precipitation rate and Mg/Cacalcite in the absence of cells is investigated using extracts of extracellular polymeric substances from temperate fluvial tufa biofilm. The influence is complex, with the concentration of extracellular polymeric substances in solution altering deposition rate and trace element incorporation. Moreover, the results show interaction of the presence/absence of extracellular polymeric substances and both temperature and salinity. However, despite extracting extracellular polymeric substances from the same parent sample, a uniform influence was not found in these experiments, implying that the mixture is sufficiently variable within a sample for microenvironments within the biofilm to either promote or inhibit mineralization. As sedimentologists, we can no longer take the view that extracellular polymeric substances are a bystander material, or that they have a single set of coherent and predictable or intuitive influences. Rather, the emphasis must be on investigating the specific mixtures present in nature, and their complex and dynamic interaction with both mineral surfaces and hydrochemical conditions.

Influence of extracellular polymeric substances on the heteroaggregation between CeO2 nanoparticles and soil mineral particles

Interaction with soil mineral particles (SMPs) and organic matters can significantly determine the fate of nanoparticles (NPs) in the environment such as waters, sediments, and soils. In this study, the heteroaggregation of CeO2 NPs with different soil minerals (kaolinite, montmorillonite, goethite and hematite) and the influence of extracellular polymeric substance (EPS) were studied. The obvious heteroaggregation between CeO2 NPs with different SMPs were demonstrated via co-settling and aggregation kinetics experiments. The variety in the heteroaggregation between CeO2 NPs with different SMPs is mainly induced by the difference in their surface properties, such as surface charge, specific surface areas and surface complexation. The presence of EPS can result in great inhibition on the heteroaggregation between CeO2 NPs with the positive charged goethite by enhancing the electrostatic repulsion between NPs and mineral colloids. However, the influence of EPS on the interaction between CeO2 NPs with negative charged SMPs is more dependent on the steric stabilization. The presence of EPS may promote the migration of CeO2 NPs in environment and then increase their risks to human health and ecosystems. These findings contribute to better understanding interactions between NPs and SMPs and have important implications on predicting the behaviors and risks of NPs in the natural environment.

Extracellular Polymeric Substances Drive Symbiotic Interactions in Bacterial‒Microalgal Consortia

The importance of several factors that drive the symbiotic interactions between bacteria and microalgae in consortia has been well realised. However, the implication of extracellular polymeric substances (EPS) released by the partners remains unclear. Therefore, the present study focused on the influence of EPS in developing consortia of a bacterium, Variovorax paradoxus IS1, with a microalga, Tetradesmus obliquus IS2 or Coelastrella sp. IS3, all isolated from poultry slaughterhouse wastewater. The bacterium increased the specific growth rates of microalgal species significantly in the consortia by enhancing the uptake of nitrate (88‒99%) and phosphate (92‒95%) besides accumulating higher amounts of carbohydrates and proteins. The EPS obtained from exudates, collected from the bacterial or microalgal cultures, contained numerous phytohormones, vitamins, polysaccharides and amino acids that are likely involved in interspecies interactions. The addition of EPS obtained from V. paradoxus IS1 to the culture medium doubled the growth of both the microalgal strains. The EPS collected from T. obliquus IS2 significantly increased the growth of V. paradoxus IS1, but there was no apparent change in bacterial growth when it was cultured in the presence of EPS from Coelastrella sp. IS3. These observations indicate that the interaction between V. paradoxus IS1 and T. obliquus IS2 was mutualism, while commensalism was the interaction between the bacterial strain and Coelastrella sp. IS3. Our present findings thus, for the first time, unveil the EPS-induced symbiotic interactions among the partners involved in bacterial‒microalgal consortia.

Extracellular polymeric substances of acidophilic microorganisms play a crucial role in heavy metal ions adsorption

The influences of extracellular polymeric substances (EPS) on heavy metal ions biosorption by acidophilic microorganisms have not been systematically reported previously. In this study, three acidophilic strains were chosen as model microbes to evaluate the effect of EPS on the adsorption of heavy metal ions (Cu2+, Ni2+, Cd2+, Zn2+) from aqueous solutions. The results indicated that buffering capacities and protonation/deprotonation abilities of these acidophiles were enhanced in the presence of EPS. Acid–base titration and FTIR analysis revealed the carboxyl, phosphoryl, amino and hydroxyl groups have provided EPS binding sites for heavy metal ions via deprotonation. The extreme thermophilic archaea had the highest total site concentration of 7.93 ± 0.52 × 10−4 mol/g on the cell surface. Meanwhile, cells with EPS had better performance in adsorption when compared with cells without EPS. The biosorption abilities of the extreme thermophilic archaea with EPS toward Cu2+, Ni2+, Cd2+ and Zn2+ were 48 μmol/g, 37.5 μmol/g, 32.5 μmol/g and 28 μmol/g, respectively. This study could provide theoretical support for the treatment of heavy metal ions in acid mine drainage by acidophiles.

Influence of extracellular polymeric substances from activated sludge on the aggregation kinetics of silver and silver sulfide nanoparticles

Influence of extracellular polymeric substances from activated sludge on the aggregation kinetics of silver and silver sulfide nanoparticles

Can ultrafiltration singly treat the iron- and manganese-containing groundwater?

The presence of Fe2+ and Mn2+ would cause severe ultrafiltration (UF) membrane fouling and limited its extensive application in treating the groundwater. A pilot-scale gravity-driven membrane (GDM) process which coupled the dual roles of biocake layer and UF membrane was introduced to treat the groundwater under high Mn2+concentrations and low temperature conditions. The results indicated that flux stabilization was observed during long-term GDM filtration with average stabilized fluxes of 3.6–5.7 L m−2 h−1. GDM process conferred efficient removals of Fe2+ and Mn2+ with both average removals > 95%. Pre-adding manganese oxides (MnOx) could effectively shorten the ripening period of manganese removal from 50 to 30 days, and simultaneously contribute to the Mn2+ removal and flux improvements. The presence of Mn2+ facilitated the formation of heterogeneous structures of biocake layer to primarily determine the flux stabilization of GDM, while the influence of extracellular polymeric substances (EPS) concentrations was nearly negligible. Besides, the Mn2+ removal was primarily attributed to the biocake layer other than UF membrane itself, and the chemically auto-catalytic oxidation by MnOx particles played the pivotal role. Therefore, these findings provide relevance for establishing new strategies in treating the iron-and manganese-containing groundwater.

Digestion Properties of Intracellular Polymers and Extracellular Polymeric Substances and Influences of Extracellular Polymeric Substances on Anaerobic Digestion of Sludge

AbstractAnaerobic digestion is a method adopted to treat sludge, but its effectiveness is limited by the existence of extracellular polymeric substances (EPSs). For the first time, this study separ...

Comparison and optimization of extraction methods of extracellular polymeric substances in anammox granules: From maintaining protein secondary structure perspective

Extracellular proteins are crucial for the formation and structural maintenance of anammox granules. However, the influence of extracellular polymeric substances (EPS) extraction methods on the secondary structure of extracellular proteins has not been studied fully. In this work, ten EPS extraction methods were applied to anammox granules and compared systematically. The EPS extraction methods were then applied to bovine serum albumin (BSA), a representative of extracellular proteins, to investigate their influence on protein secondary structure by circular dichroism (CD) spectroscopy. The ten EPS extraction methods yielded EPS concentration varying from 5.2 ± 0.1 to 84.8 ± 2.0 mg/g volatile suspended solid (VSS). Tween-20 treatment resulted in the most similar CD spectrum and protein secondary structure to the control. Treatment 0.5% Tween-20 for 4 h was found to be the best condition to preserve protein secondary structure while achieving a high EPS yield (44.4 ± 1.4 mg/g VSS) and limited cell lysis for anammox granules. This study provides an optimized protocol to extract granular EPS when the role of the secondary structure of extracellular proteins in regulating aggregation, adsorption and biofilm formation of microbes is to be elucidated.

The influence of extracellular polymeric substances on the coagulation process of cyanobacteria

The hydrophobicity and flocculation properties of cyanobacterial cells are closely related to their extracellular polymeric substances (EPS). During the treatment of drinking water, the coagulation and removal of EPS-wrapped cyanobacterial particles from natural water sources is very difficult. In this work, a series of surface characteristics of cyanobacterial cells with different EPS fractions were analyzed to evaluate their influences on the coagulation process. With the removal of EPS, the coagulation efficiency of cyanobacteria was gradually improved. The intracellular microcystin release showed that the cyanobacterial cells in each EPS removal phase were almost intact with few broken cells. The surface of cyanobacterial cells had higher hydrophobicity and lower zeta potentials with each step of the EPS extraction, which improved the ratio of particles that were in an unstable state. Furthermore, the deeper the EPS extraction phase, the larger the decreased in size of cyanobacterial particles, thus increasing their specific surface area for adsorption with coagulant. It was concluded that the coagulation mechanism of EPS-wrapped cyanobacterial particles was: the cyanobacterial cells were first peeled off through attraction by opposite charges from the coagulant, and then they were adsorbed before settling down. This study provides a scientific basis for the removal of cyanobacteria by enhancing coagulation.

Interactive influence of extracellular polymeric substances (EPS) and electrolytes on the colloidal stability of silver nanoparticles

The colloidal stability of silver nanoparticles (AgNPs) was evaluated using time-resolved dynamic light scattering, electrophoretic mobility and dissolved Ag concentration in the presence of electrolytes and extracellular polymeric substances (EPS).

Biorecovery of cobalt and nickel using biomass-free culture supernatants from Aspergillus niger

In this research, the capabilities of culture supernatants generated by the oxalate-producing fungus Aspergillus niger for the bioprecipitation and biorecovery of cobalt and nickel were investigated, as was the influence of extracellular polymeric substances (EPS) on these processes. The removal of cobalt from solution was >90% for all tested Co concentrations: maximal nickel recovery was >80%. Energy-dispersive X-ray analysis (EDXA) and X-ray diffraction (XRD) confirmed the formation of cobalt and nickel oxalate. In a mixture of cobalt and nickel, cobalt oxalate appeared to predominate precipitation and was dependent on the mixture ratios of the two metals. The presence of EPS together with oxalate in solution decreased the recovery of nickel but did not influence the recovery of cobalt. Concentrations of extracellular protein showed a significant decrease after precipitation while no significant difference was found for extracellular polysaccharide concentrations before and after oxalate precipitation. These results showed that extracellular protein rather than extracellular polysaccharide played a more important role in influencing the biorecovery of metal oxalates from solution. Excitation–emission matrix (EEM) fluorescence spectroscopy showed that aromatic protein-like and hydrophobic acid-like substances from the EPS complexed with cobalt but did not for nickel. The humic acid-like substances from the EPS showed a higher affinity for cobalt than for nickel.

Influence of Extracellular Polymeric Substances on the Adsorption of Cadmium onto Three Bacterial Species

In this study, we measured the effect of EPS on Cd and proton adsorption behaviors by measuring the extent of adsorption onto biomass with and without the EPS removed via a cation exchange resin. We conducted both Cd adsorption experiments and potentiometric titrations of biomass using three common bacterial species: one Gram-positive (Bacillus subtilis) and two Gram-negative (Shewanella oneidensis, Pseudomonas putida) species. The Cd adsorption experiments were conducted as a function of metal loading in order to probe whether environmentally-low metal loadings lead to different adsorption mechanisms and roles for EPS than the higher metal loadings of most previous adsorption studies. We suspended each biomass sample in a solution of dissolved Cd in 0.01 M NaClO4 at metal loadings of 1, 2, 5, and 74 μmol/g. Surface complexation modeling (SCM) was used to determine stability constants for the important Cd-bacteria complexes, and the effect of metal loading on the resulting calculated stability constant values was determined.In general, the measured bulk Cd adsorption behavior is unaffected by EPS removal. However, our potentiometric titration results suggest that EPS removal does alter the distribution of site types, but not the mass-normalized total site concentration within the biomass. SCM suggests that high affinity sulfhydryl sites control Cd binding under low metal loading conditions for B. subtilis and P. putida, and that sulfhydryl sites are present both on the cells and within the EPS for these species. Conversely, the SCM results suggest that Cd-sulfhydryl binding is un-important on the EPS of S. oneidensis.