Environ. Sci. Technol. 2009, 43, 5699–5704
Deposition Kinetics of Extracellular Polymeric Substances (EPS) on Silica in Monovalent and Divalent Salts P I N G T I N G Z H U , †,‡ G U O Y U L O N G , ‡ J I N R E N N I , †,‡ A N D M E I P I N G T O N G * ,†,‡ School of Environment and Urban Studies, Shenzhen Graduate School of Peking University, Shenzhen, 518055, P. R. China, and The Key Laboratory of Water and Sediment Sciences, Ministry of Education, Department of Environmental Engineering, Peking University, Beijing, 100871, P. R. China
Received February 4, 2009. Revised manuscript received June 1, 2009. Accepted June 7, 2009.
The deposition kinetics of extracellular polymeric substances (EPS) on silica surfaces were examined in both monovalent and divalent solutions under a variety of environmentally relevant ionic strength and pH conditions by employing a quartz crystal microbalance with dissipation (QCM-D). Soluble EPS (SEPS) and bound EPS (BEPS) were extracted from four bacterial strains with different characteristics. Maximum favorable deposition rates (kfa) were observed for all EPS at low ionic strengths in both NaCl and CaCl2 solutions. With the increase of ionic strength, kfa decreased due to the simultaneous occurrence of EPS aggregation in solutions. Deposition efficiency (R; the ratio of deposition rates obtained under unfavorable versus corresponding favorable conditions) for all EPS increased with increasing ionic strength in both NaCl and CaCl2 solutions, which agreed with the trends of zeta potentials and was consistent with theclassicDerjaguin-Landau-Verwey-Overbeek(DLVO)theory. Comparison of R for SEPS and BEPS extracted from the same strain showed that the trends of R did not totally agree with trends of zeta potentials, indicating the deposition kinetics of EPS on silica surfaces were not only controlled by DLVO interactions, but also non-DLVO forces. Close comparison of R for EPS extracted from different sources showed R increased with increasing proteins to polysaccharides ratio. Subsequent experiments for EPS extracted from the same strain but with different proteins to polysaccharides ratios and from activated sludge also showed that R were largest for EPS with greatest proteins to polysaccharides ratio. Additional experiments for pure protein and solutions with different pure proteins to pure saccharides ratios further corroborated that larger proteins to polysaccharides ratio resulted in greater EPS deposition.
Introduction Extracellular polymeric substances (EPS) are biopolymers accumulating on or around microbial cell surfaces that are * Corresponding author e-mail:
[email protected]; phone: (86) 10-6275-6491; fax: (86) 10-6275-6526. † School of Environment and Urban Studies. ‡ The Key Laboratory of Water and Sediment Sciences. 10.1021/es9003312 CCC: $40.75
Published on Web 07/01/2009
2009 American Chemical Society
composed of a variety of organic compounds including proteins, polysaccharides, humic substances, lipid, DNA, and so on (1-3). Deposition of EPS on mineral surfaces has great significance on mineral dissolution (4), biomineralization (5), sediment stabilization (6), contaminants (i.e., organic pollutants and heavy metal) accumulation and degradation (7-9), as well as pollutants immobilization or mobilization (10, 11). For example, EPS were able to bind lead from aqueous solutions and thus would influence its distribution in subsurface environments (8). To better understand these processes, examination of EPS deposition behavior to surfaces is therefore necessary. Recent studies also proposed that EPS have profound influence on cell transport behavior (12, 13). Meanwhile, the deposition of EPS on mineral surfaces has been assumed to facilitate the formation of biofilms (14, 15). Full investigation of EPS deposition on surfaces is therefore also necessary to better control the transport behavior of bacteria and the formation of biofilms. However, few studies have addressed the deposition kinetics of EPS on mineral surfaces. Omoike et al. (16) utilized attenuated total internal reflection infrared spectroscopy (ATR-FTIR) to examine the interaction of EPS with goethite (R-FeOOH) surface and found that nucleic acid (minor component of EPS) played an important role in the bacterial deposition to goethite. By employment of quartz crystal microbalance with dissipation (QCM-D), Kwon et al. (17) investigated the adsorption of dextran, which was chosen to represent polysaccharides in EPS, on alumina and silica. The authors found that dextran had different conformations when adsorbed on Al2O3 and SiO2 surfaces. Subsequently, using atomic force microscopy (AFM), Kwon et al. (18) compared the interactions of silica surfaces with three different homopolymers selected to represent different components of EPS. The study showed that phosphate containing polymers such as DNA and phospholipids had significant contribution to the EPS adhesion to silica surfaces. However, the deposition kinetics of real EPS (directly extracted from bacterial cell surface) to silica surfaces under environmentally relevant ionic strength and pH conditions have never been explored. The objective of this paper is to systematically examine the kinetics of EPS deposition on silica surfaces. Since composition of EPS might vary from organism to organism, soluble EPS (SEPS) and bound EPS (BEPS) (SEPS and BEPS can be classified according to their relative proximity to the bacterial cell surface) extracted from four bacterial strains with different characteristics (cell type and mobility), Escherichia coli BL21 (gram-negative, nonmotile), Pseudomonas sp. QG6 (gram-negative, motile), Rhodococcus sp. QL2 (grampositive, nonmotile), and Bacillus subtilis (gram-positive, motile), were examined. Since these four strains have different characteristics and can be widely found in the natural environment, the EPS examined in this study (extracted from these four strains and also from activated sludge) have relevance to a wide variety of EPS present in natural environment. A QCM-D was employed to determine the deposition rates for all EPS in both NaCl and CaCl2 solutions over a wide range of environmentally relevant ionic strengths and pH under both favorable and unfavorable conditions. As a well-established technique, QCM-D has been widely used as a real-time tool to quantify relatively low levels of deposition (i.e., ng/cm2) and characterize biomolecular binding events at the solid-liquid interface (19, 20). Currently, QCM-D has been proven as one of the best tools to determine the deposition kinetics of macromolecular products, including polysaccharides (17), DNA (21, 22), proteins (23), and large polymers (24). In this study, the initial deposition rates VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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of all EPS were determined according to the QCM-D experiments. The influence of solution chemistry and valence, and the components of EPS on EPS deposition kinetics were discussed. The results revealed that EPS deposition generally agreed with Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, combining effect of both van der Waals and double layer force. Close comparison of R for all EPS suggested that proteins to polysaccharides ratio played an important role on EPS deposition, which was confirmed by the subsequent experiments for EPS extracted from the same bacterial strain but with different proteins to polysaccharides ratios and from activated sludge. Additional experiments for pure protein (BSA) showed that R for BSA were larger than those for all EPS. Deposition experiments of solutions with a series of pure proteins (BSA) to pure saccharides (glucose) ratios further confirmed R increased with increasing ratio of proteins to polysaccharides. These observations indicated that the proteins to polysaccharides ratio had great influence on EPS deposition on silica surfaces.
Material and Methods EPS Extraction and Characterization. SEPS and BEPS were extracted from four bacterial strains: Escherichia coli BL21 (gram-negative, nonmotile), Pseudomonas sp. QG6 (gramnegative, motile), Rhodococcus sp. QL2 (gram-positive, nonmotile), and Bacillus subtilis (gram-positive, motile). Bacteria were grown and harvested according to protocols described in a previous publication (25) as well as in the Supporting Information. After harvest, the cell pellets were resuspended in Milli-Q water for the subsequent EPS extraction process. SEPS were separated via centrifugation at 4000 rpm for 20 min at 4 °C. Following the SEPS extraction, the pellets were resuspended in Milli-Q water and the cell suspension was then transferred to a sterilized extraction beaker to release BEPS via cation exchange resin (CER) technique (3). CER (Dowex Marathon C, 20-50 mesh, sodium form, Fluka 91973), which was soaked in Milli-Q water overnight prior to use, was added to the extraction beaker with a dosage of 2.5 g/g bacterial mass. The bacteria-CER suspension was then stirred at 600 rpm for 1.5 h at 4 °C. This was followed by a settlement of the suspension for 3 min to separate CER. The extracted BEPS were collected by centrifugation at 8000 rpm for 20 min at 4 °C. Both extracted SEPS and BEPS were filtered through sterilized 0.22 µm cellulose acetate filters to remove residuals and bacterial cells. The obtained EPS were then divided into several portions and stored in sterilized bottles at -18 °C. Aliquots of EPS solutions were taken out of the freezer and allowed to warm up to room temperature immediately before use. The chemical components of different EPS were characterized: dry weight (DW) and volatile dry weight (VDW) were determined at temperatures of 105 and 550 °C, respectively (26); TOC was measured using a TOC-meter (Tekmer Fusion, Teledyne Instruments, CA); both total nitrogen (TN) and total phosphorus (TP) were measured with the methods recommended by European Standard (European standard EN 1189, 2001); proteins and humic acids (HA) contents were determined by the modified Lowry method (27) using bovine albumin serum and humic acid as respective standards; polysaccharides content was measured according to the Dubois method (28) using glucose as standard. Electrophoretic mobility measurements of EPS were performed with EPS TOC of 70 ( 1.0 mg-L-1 in both NaCl and CaCl2 solutions at pH of 6.0 (adjusted with 0.1 M HCl) and 8.0 (adjusted with 0.1 M NaOH) at room temperature (25 °C) using a Zetasizer Nano ZS90 (Malvern Instruments, UK). Quartz Crystal Microbalance with Dissipation (QCMD). A QCM-D E1 system (Q-Sense AB, Gothenburg, Sweden) was utilized to examine the deposition kinetics of EPS on silica surfaces. QCM-D experiments were preformed with 5 5700
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MHz AT-cut quartz sensor crystals with silica-coated surface (batches 070624 and 081110). Before use, crystals were cleaned as described previously (29). The EPS deposition experiments were performed in a flow-through mode, using a peristaltic pump (ISMATEC, Switzerland) operating in clockwise mode. Specifically, the pump was connected to the sensor crystal outlet, and the studied solutions, stored in a sterilized 50 mL polypropylene conical tube (Becton Dickinson, NJ) connected to the sensor crystal inlet, were fed through the crystal sensor chamber at a flow rate of 0.1 mL-min-1. EPS deposition was examined on both bare silica surfaces (in the presence of an energy barrier) and PLL-coated silica surfaces (in the absence of an energy barrier). EPS and bare silica crystal display bulk negative, yielding an overall repulsive electric double layer energy barrier between them (30). To eliminate the repulsive energy barrier, silica crystals can be precoated with a layer of positively charged poly-Llysine (PLL) hydrobromide (molecular weight 70 000-150 000) (P-1274, Sigma-Aldrich, St. Louis, MO). Detailed protocol of silica surfaces modification with PLL can be found in the Supporting Information. Experiments in the presence of an energy barrier (unfavorable condition) were conducted in both NaCl (with seven ionic strengths ranging from 1 to 200 mM) and CaCl2 (with five ionic strengths ranging from 0.3 to 5 mM) solutions at pH 6.0 (adjusted with 0.1 M HCl) and pH 8.0 (adjusted with 0.1 M NaOH). The QCM-D system was pre-equilibrated with desired salt solution for a minimum of 30 min to establish a stable baseline (the drift of average normalized frequency was less than 0.2 Hz within 30 min). After pre-equilibration, an EPS solution at desired ionic strength and pH at the TOC of 70 ( 1.0 mg-L-1 was injected into the crystal chamber. The duration of injection ranged from 20 min to several hours. Corresponding favorable deposition experiments were conducted on PLL-coated silica surfaces in both NaCl and CaCl2 solutions at the same EPS TOC concentration as that of unfavorable conditions. Details about favorable EPS deposition experiments are provided in the Supporting Information. For all experiments, the flow rate of desired solutions through the measurement chamber was maintained constant at 0.1 mL-min-1. QCM-D Data Analysis. For the experiments performed both in the presence and absence of an energy barrier, the deposition rate can be determined from the slope of the initial (linear) portion of the change in normalized frequency ∆f3 versus time curve (21, 22), since a perfect linear curve (R2 ) 0.9987) of average number of deposited EPS on crystal versus frequency shift (f3) was observed (see detailed curve in Figure S1): kf )
d∆f3 dt
(1)
The deposition rate at different solution conditions in the presence of an energy barrier is then presented in terms of the deposition efficiency (R), which is the ratio of the deposition rate in the presence of an energy barrier (kfp) relative to the corresponding deposition rate in the absence of an energy barrier (kfa): R)
kfp kfa
(2)
Results and Discussion Characteristics of EPS. The general characteristics of EPS examined in this study are presented in Table S1. The VDW to DW ratios of all EPS were larger than 95%, indicating that the predominant components of EPS were organic compounds, which was consistent with observations of previous
studies (26, 31). The values of the TOC and C/N ratios for different EPS varied from 58.3 to 554.0 mg C g-1 EPS DW and from 0.9 to 2.7, respectively, indicating that difference was present among different sorts of EPS. Meanwhile, the values of TP varied from 23.0 to 94.1 mg P g-1of EPS DW, indicating the presence of a small amount of DNA and lipid in EPS. The biochemical components of EPS are also provided in Table S1. It can be observed that the contents of HA were relatively low, ranging from 2.8 to 22.3 mg g-1 VDW, suggesting that HA were a minor component of all examined EPS. The values of proteins and polysaccharides varied from 397.4 to 810.7 and 14.9 to 154.6 mg g-1 of EPS VDW, respectively, indicating that proteins and polysaccharides were the major components of all EPS. Further comparison of the contents of proteins and polysaccharides for all EPS yielded that proteins to polysaccharides ratios were larger than 3.0, demonstrating that proteins was the dominant component of examined EPS. Electrophoretic Mobility of EPS. The influence of solution ionic composition and ionic strength on the electrophoretic mobilities and zeta potentials of all EPS at pH 6.0 are presented in Figure S2. The electrophoretic mobilities for all EPS at pH 6.0 and pH 8.0 were nearly equivalent (data obtained at pH 8.0 are not shown). Zeta potentials of both SEPS (solid triangle) and BEPS (open triangle) were negative and became less negative with the increase of ionic strength in both NaCl and CaCl2 solutions due to compression of the electrostatic double layer. At the same ionic strength (1 and 5 mM), zeta potentials of all EPS in CaCl2 solutions (Figure S2, right) were less negative relative to those in NaCl solutions (Figure S2, left), which was possibly due to Ca2+ complexing with EPS resulting in the neutralization of surface charge (32). Close investigation of Figure S2 showed that at low ionic strengths in both NaCl and CaCl2 solutions (from 1 to 50 mM in NaCl, from 0.3 to 1 mM in CaCl2), slight difference of zeta potentials for SEPS and BEPS extracted from same strain was present, whereas, at high ionic strengths (100-200 mM NaCl, 3-5 mM CaCl2), zeta potentials for SEPS and BEPS extracted from same strain were nearly equivalent. These results suggested that the components of EPS only had slight influence on zeta potentials at low ionic strengths. EPS Deposition on PLL-Modified Silica Surface. Favorable deposition on PLL-modified silica surfaces was examined for all EPS in both NaCl and CaCl2 solutions at pH 6.0 and 8.0. Figure S3a presents representative normalized frequency shifts at the third overtone (f3) as SEPS extracted from E. coli deposited on a PLL-coated silica surface in 1, 20, and 200 mM NaCl solutions. The number of EPS deposited on the surfaces is linearly proportional to the frequency shift (Figure S1). Therefore, the increase in the magnitude of frequency shift with elapsed time indicates the number of EPS deposited with time elapses. Figure 1 illustrates favorable deposition rates (kfa) of SEPS extracted from E.coli in both NaCl and CaCl2 solutions at pH 6.0. kfa at pH 6.0 and 8.0 were equivalent (data at pH 8.0 are not shown). At low ionic strengths in both NaCl (1-10 mM) and CaCl2 (0.3-0.6 mM) solutions, maximum kfa were observed, indicating that EPS were stable to aggregation during the deposition process at these conditions. The average maximum kfa were comparable at low NaCl and CaCl2 ionic strengths, suggesting fast deposition of mostly unaggregated EPS on PLL-coated surfaces. With the increase of ionic strength (above 10 mM NaCl and 0.6 mM CaCl2), kfa slightly decreased due to the occurrence of simultaneous EPS aggregation in solutions. EPS size measurement via Zetasizer Nano ZS90 showed that the average diameter of E.coli SEPS at 200 mM NaCl and at 5 mM CaCl2 increased from ∼170 to ∼190 nm, and from ∼170 to ∼216 nm, respectively, within 30 min of introducing the premeasured amount of salt stock solutions into the stable SEPS suspension.
FIGURE 1. Favorable deposition rates of SEPS extracted from E.coli on PLL-modified silica surfaces in both NaCl (a) and CaCl2 (b) solutions at pH 6.0 (adjusted with 0.1 M HCl) as a function of solution ionic strength. Duplicate measurements were conducted over entire ionic strength range, with error bars representing standard deviations. The lines are meant to guide the eye. At the same ionic strength (1 and 5 mM), kfa in CaCl2 solutions were lower relative to those in NaCl solutions due to the enhanced aggregation in CaCl2 solutions. The average diameter of E.coli SEPS at 1 mM CaCl2 increased from ∼170 to ∼200 nm within 30 min of adding CaCl2 solution into SEPS suspension, whereas, at 1 mM NaCl, the average diameter of E.coli SEPS kept constant (∼170 nm) over the same time period. The results showed that the aggregation of EPS in CaCl2 solution was more significant than in NaCl solution, which also explained the observed faster drop of kfa with increasing ionic strength in CaCl2 solutions. The more significant aggregation that occurred in CaCl2 solutions was possibly due to the effect of “Ca2+ Bridge”, promoting the self-assembling of EPS (33). These above observations were also true for BEPS of E.coli and EPS (both SEPS and BEPS) extracted from three other bacterial strains (Figures S4 and S5). Close comparison of Figures 1, S4, and S5 showed that at the same solution conditions, kfa for EPS extracted from different sources followed the order of QG6 > E.coli > QL2 > BST. This was true over all ionic strength range in both NaCl and CaCl2 solutions. Whereas, EPS size measurement showed that hydrodynamic radius of EPS extracted from different sources followed the order of QG6 < E.coli < QL2 < BST. The observations showed that favorable deposition of EPS was governed by convectivediffusive transport of EPS to the surfaces, which highly depended on the hydrodynamic radius of the EPS. Effect of Solution Chemistry and Valence on EPS Deposition Kinetics. By normalizing the EPS deposition rates on bare silica surfaces by the favorable deposition rates at corresponding electrolyte ionic strengths, the deposition efficiencies (R) of EPS extracted from four bacterial strains in both NaCl and CaCl2 solutions over a wide range of ionic strength at pH 6.0 and pH 8.0 were derived. Figure 2 presents representative R of SEPS extracted from E.coli. In both NaCl and CaCl2 solutions, R increased with increasing ionic strength at both pH 6.0 and 8.0. This deposition behavior was consistent with the trends of zeta potentials of EPS versus ionic strength (Figure S2) and thus generally agreed with classic DLVO theory. At low ionic strengths, electrostatic repulsion occurred between the negatively charged EPS and VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Deposition efficiencies (r) of SEPS extracted from E.coli on bare silica surfaces in both NaCl (a) and CaCl2 (b) solutions at pH 6.0 (adjusted with 0.1 M HCl) and pH 8.0 (adjusted with 0.1 M NaOH) as a function of solution ionic strength. Duplicate measurements were conducted over entire ionic strength range, with error bars representing standard deviations. The lines are meant to guide the eye. silica surfaces, resulting in unfavorable deposition (R < 1). The increase of ionic strength compressed electrostatic double layer between the EPS and silica surface, resulting in higher deposition efficiencies of EPS. When the ionic strength equaled or exceeded the critical deposition ionic strength, sufficient charge screening and compression of electrostatic interaction forces between EPS and silica surfaces induced favorable deposition (R ≈1). These above observations also held true for E.coli BEPS as well as EPS extracted from three other bacterial strains (Figures S6 and S7). Further investigation of Figures 2, S6, and S7 showed that R of EPS extracted from four bacterial strains were comparable at higher ionic strengths (200 mM NaCl, 5 mM CaCl2) and were close to maximum value of 1.0, indicating that the deposition rates at these conditions were equivalent to those achieved under favorable conditions. The results showed that at higher ionic strengths, the deposition behavior of all EPS was similar, suggesting that EPS components had insignificant influence on EPS deposition under these conditions. Comparison of R at pH 6.0 (open symbols) and pH 8.0 (solid symbols) for SEPS (Figures 2 and S6a-b) and BEPS (Figure S7a-d) extracted from E.coli and QG6 as a function of NaCl and CaCl2 ionic strength revealed that the EPS deposition behavior was similar at these two pH conditions. At the same ionic strengths (1 and 5 mM), R for all EPS in CaCl2 solutions were greater than those in NaCl solutions at the same pH, which agreed with the less negative zeta potentials of EPS in CaCl2 solutions relative to those in NaCl solutions under the same conditions (Figure S2). The presence of Ca2+ also reduce the charge of the silica surface by binding to silanol groups (22). Zeta potential measurement of crushed quartz sand showed that zeta potentials in CaCl2 solutions were less negative relative to those in NaCl solutions (data not shown) at the same ionic strength. A greater reduction of electrostatic repulsion between EPS and silica surfaces in CaCl2 solutions relative to that in NaCl solutions leads to a larger deposition rate. Effect of EPS Components on EPS Deposition Kinetics. R of SEPS and BEPS extracted from the same source were compared, and it was found that at the same ionic strength during unfavorable deposition (R < 1), R of SEPS extracted from nonmotile strains E.coli and QL2 were larger than those of BEPS from the same strain in both NaCl and CaCl2 5702
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solutions, whereas R of SEPS extracted from motile strains QG6 and BST were smaller than those of BEPS (Figures 2, S6, and S7). These trends cannot be explained by zeta potentials of SEPS and BEPS at the same ionic strength (Figure S2) (for most cases, zeta potentials of BEPS were less negative than those of SEPS). The deposition efficiency apparently was not only controlled by electric double layer repulsion, but also by non DLVO processes, such as H-bonds forces and electrosteric interaction. The functional groups in EPS, such as hydroxyl, amino, and phosphate, could form hydrogen bonding with the predominant surface species neutral silanols (18). Hydrophobic groups such as nonpolar groups in proteins may allow EPS to approach silica surface closely (12). This may be followed by conformational changes in EPS allowing other functional groups to come close enough to the surface for the formation of short-range attractive polymeric interactions (34). Close inspection of different components of EPS presented in Table S1 demonstrated that the proteins to polysaccharides ratio (including hydroxyl and amino functional groups) followed the same trends of observed R for both SEPS and BEPS, that larger proteins to polysaccharides ratio corresponds to larger R. The results indicated that proteins to polysaccharides ratio might play an important role in EPS deposition on silica surface. To test the contribution of proteins to polysaccharides ratio to EPS deposition, comparison of deposition kinetics of both SEPS and BEPS extracted from four bacterial strains was also made, where the proteins to polysaccharides ratio followed the order BST-BEPS > QG6-BEPS > E.coli-SEPS > E.coli-BEPS > BST-SEPS > QG6-SEPS > QL2-SEPS > QL2BEPS (Table S1). Figures 2, S6, and S7 showed that under all ionic strengths during unfavorable deposition (R < 1), the trend of R was in agreement with the trend of proteins to polysaccharides ratio. Therefore, it is reasonable to deduce that the proteins to polysaccharides ratio plays an important role in EPS deposition. To further demonstrate that proteins to polysaccharides ratio indeed has significant contribution to EPS deposition, subsequent experiments for EPS extracted from the same bacterial strain but with different proteins to polysaccharides ratios were performed. SEPS and BEPS were extracted from four bacterial strains at three different cultivation phases. Table S2 enumerates the detailed cultivation time for each EPS and its corresponding proteins to polysaccharides ratio. It can be seen from Table S2 that proteins to polysaccharides ratio for all EPS decreased with increasing cultivation time, which agreed with previous study (35). Figure 3 presents R of both SEPS and BEPS extracted from four bacterial strains under three different cultivation periods at three representative ionic strengths (20 mM NaCl, 0.3 and 3 mM CaCl2). At the same ionic strength, R for all examined EPS decreased with increasing cultivation time (decreasing ratio of proteins to polysaccharides). Close comparison of R for these 24 different EPS (Table S2) also showed that R increased with increasing proteins to polysaccharides ratio at the same ionic strength; the result applied to all three examined ionic strengths. These observations clearly demonstrated that R increased with increasing proteins to polysaccharides ratio, indicating that the proteins to polysaccharides ratio has significant influence on EPS deposition. Deposition of BEPS extracted from an activated sludge (see preparation protocol in the Supporting Information) with proteins to polysaccharides ratio of 1.8 was examined. Figure 4 presents R of sludge BEPS in NaCl solutions with ionic strength ranging from 1 to 200 mM at pH 6.0 and 8.0. R of sludge BEPS increased with increasing solution ionic strength, which agreed with observations of EPS extracted from pure strains (Figures 2, S6, and S7). Comparison of R at pH 6.0 (open hexagon) and pH 8.0 (solid hexagon) over entire ionic strength range examined showed that BEPS
To further test that the proteins to polysaccharides ratio had great influence on EPS deposition, additional deposition experiments for pure protein (BSA) and solutions with different ratios of pure proteins (BSA) to pure saccharides (glucose) (Figure S8) were performed with the influent concentration of 70 ( 1.0 mg-L-1 TOC, which was the same as those for EPS experiments. Figure S8a presents R of BSA (open diamond) in NaCl solutions as a function of ionic strength ranging from 1 to 200 mM at pH 6.0. R of BSA increased with increasing solution ionic strength, which agreed with observations of EPS extracted from pure strains and sludge (Figures 2, 4, S6, and S7). Close comparison of R of BSA (open diamond) with those of BST-BEPS (open square) with the proteins to polysaccharides ratio of 28.1 (the largest ratio listed in Table S1) showed that R of pure protein were larger compared to those of BST-BEPS over the entire ionic strength range examined under unfavorable conditions (R < 1). The result further corroborated that larger ratio of proteins to polysaccharides resulted in greater EPS deposition. Figure S8b presents the relationship between R and pure protein BSA to pure glucose ratios in 20 mM NaCl solutions at pH 6.0. It can be clearly seen from Figure S8b that R increased with increasing ratio of BSA to glucose. The results agreed with observations for EPS deposition that R increased with increasing proteins to polysaccharides ratios. These results further confirmed that the proteins to polysaccharides ratio had significant influence on EPS deposition.
Implications
FIGURE 3. Deposition efficiencies (r) of both SEPS and BEPS extracted from E.coli (a and b), QG6 (c and d), QL2 (e and f), and BST (g and h) under different cultivation time on bare silica surfaces in the presence of 20 mM NaCl (left), 0.3 mM CaCl2 (middle), and 3 mM CaCl2 (right) at pH 6.0 (adjusted with 0.1 M HCl). Each column represents the average of duplicate measurements under the same experimental conditions, with error bars representing standard deviations.
FIGURE 4. Deposition efficiencies (r) of BEPS for activated sludge and QL2 on bare silica surfaces in NaCl solutions at pH 6.0 (adjusted with 0.1 M HCl) and pH 8.0 (adjusted with 0.1 M NaOH) as a function of solution ionic strength. Error bars represent standard deviations of duplicate measurements. The lines are meant to guide the eye. deposition behavior was similar at these two examined pH conditions, which also agreed with observations of EPS extracted from pure strains. Close comparison of R of sludgeBEPS with those of QL2-BEPS (open circle) with the proteins to polysaccharides ratio of 3.0 (the lowest value listed in Table S1) showed that R of sludge-BEPS were relatively smaller compared to QL2-BEPS over the entire ionic strength range examined. R of sludge-BEPS were also smaller than those of QL2-BEPS with proteins to polysaccharides ratio of 2.3 (after cultivation of 72 h) at the same ionic strengths. These results further confirmed that larger proteins to polysaccharides ratio resulted in greater EPS deposition.
This study showed that deposition of EPS, the ubiquitous biomacromolecules in natural environment, on silica surfaces was significant under solution ionic strengths and pH relevant to subsurface environment. The deposition behavior of EPS was sensitive to solution ionic strength and ion valence, as well as their own components. Deposition efficiencies of EPS on bare silica surfaces increased with increasing ionic strength in both monovalent and divalent solutions, which was consistent with classic DLVO theory. At the same ionic strength, deposition efficiency of EPS was much greater in divalent solutions than in monovalent solutions. EPS favorable deposition on silica surfaces occurs at ∼200 mM NaCl, which was greater than the typical monovalent cation ionic strength of freshwaters, and at 5 mM CaCl2 that was within the ionic strength range of calcium cations (36). Therefore, in freshwaters, the deposition of EPS to silica surfaces might be mainly controlled by divalent cations such as Ca2+. The significance of proteins to polysaccharides ratio on EPS deposition held true for all examined EPS (extracted from either pure strains with different characteristics or sludge). Greater proteins to polysaccharides ratio could induce faster EPS deposition in aquatic environments.
Acknowledgments This work was supported by the National Natural Science Foundation of China-Young Scientists Fund under grant 40802054. We acknowledge the editor and anonymous reviewers for their very insightful comments. We are also grateful to Prof. Bengt Kasemo from Chalmers University of Technology, Gothenburg for his useful comments.
Supporting Information Available Characteristics of EPS (Table S1); proteins to polysaccharides ratio of EPS under different cultivation phases (Table S2); relationship of the deposited EPS number versus frequency shift (f3) (Figure S1); zeta potentials of EPS (Figure S2); EPS deposition profiles (Figure S3); favorable deposition rates of EPS (Figures S4 and S5); deposition efficiencies of EPS (Figures S6 and S7); deposition efficiencies of pure proteins and solutions with different ratios of pure proteins to pure VOL. 43, NO. 15, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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saccharides (Figure S8); growth and harvest protocol for bacterial strains and sludge; crystal cleaning protocols; favorable EPS deposition experiments. This information is available free of charge via the Internet at http://pubs.acs.org.
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