Environ. Sci. Technol. 2005, 39, 9574-9582
Interaction Force Profiles between Cryptosporidium parvum Oocysts and Silica Surfaces T . L . B Y R D A N D J . Y . W A L Z * ,† Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520
The interaction force profile between single Cryptosporidium parvum oocysts and silica particles was measured in aqueous solutions using an atomic force microscope. The oocysts were immobilized during the measurements by entrapment in Millipore polycarbonate membranes with a 3 µm pore size. Experiments were performed in both NaCl and CaCl2 solutions at ionic strengths ranging from 1 to 100 mM. For both electrolytes the decay length of the repulsive force profile, obtained via the slope of a plot of the logarithm of interaction force versus separation, was found to be essentially independent of the ionic strength and always much larger than the theoretical Debye length of the system. In addition, the magnitude of the force was found to be essentially the same for both electrolytes, suggesting that the long-range repulsive forces are primarily steric in nature. Fitting the force to an expression for the steric repulsive force between two grafted brush layers yields a layer thickness of approximately 115 nm. These results support the idea that the oocysts are covered by a relatively thick layer of uncharged (or weakly charged) carbohydrates, possibly mixed with a thinner layer of charged protein.
Introduction Cryptosporidium parvum, first discovered in 1912 by E. E. Tyzzer (1), is a protozoan parasite that can be found in many water sources worldwide. The organism was initially thought to be a rare opportunistic pathogen that primarily affected livestock and other domestic animals; however in 1976 the link between Cryptosporidium parvum and diarrheal illness was established (2). It was during this time that the organism was isolated from the first human cases who were suffering from severe gastroenteritis over a 2 week period. The diarrheal illness came to be known as Cryptosporidiosis, and since that time period numerous cases of Cryptosporidiosis have been reported globally. Studies of surface water sources in the United States have shown the presence of Cryptosporidium parvum in 55% of surface water samples. In a study by Rose et al., 17% of drinking water samples have been shown to be contaminated with Cryptosporidium parvum (3). Similarly, studies by LeChevallier et al. found C. parvum contamination in 87% of raw water samples and 27% of drinking water samples (4, 5). According to these authors, the prevalence of C. parvum increases in surface waters where there is an increase in runoff * Corresponding author phone: (540)231-4213; fax: (540)231-5022; e-mail:
[email protected]. † Current address: Department of Chemical Engineering (0211), Virginia Tech, Blacksburg, VA 24060. 9574
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 24, 2005
of domestic animal wastes from the land or inadequate water treatment facilities. However, outbreaks of C. parvum have been associated with the contamination of all types of water supplies, including well waters and swimming pools (6). Of all the outbreaks of Cryptosporidium parvum that have occurred within the United States, none has had a more widespread impact or generated more attention to the dangers of C. parvum contamination than the 1993 waterborne outbreak that occurred in Milwaukee, WI. Corso et al. estimate that of the 1.61 million residents of the greater Milwaukee area, 403 000 residents contracted Cryptosporidiosis (7). This massive outbreak was contributed to an ineffective filtration process that led to inadequate removal of C. parvum oocysts in one of two municipal water treatment plants. This outbreak not only produced poor health and the loss of life, but it also resulted in a significant negative impact on the economy of the area due to medical costs and productivity losses associated with the outbreak. According to Corso et al., the total cost associated with the 1993 Milwaukee outbreak was in excess of $96 million (7). The oocyst is the predominant form of Cryptosporidium parvum found in the environment. These oocysts are spherical to slightly ovoid, range in diameter from 3.9 to 5.9 µm (8), and are composed of four inner sporozoites surrounded by a thick shell wall. This wall provides robust protection against harsh environmental conditions and is referred to as a near-inert protective covering for C. parvum at the host-oocyst-environment interface (9). The oocyst wall is between 40 and 50 nm thick and can be divided into an inner and an outer layer (10-12). The inner layer of the oocyst wall contains within it a suture, whose length is approximately half of the oocyst’s circumference. The outer layer is between 5 and 10 nm thick and is thought to be composed primarily of acidic glycoprotein. The anchored proteins of this variably electron dense layer extend outward from the oocyst surface. This extension is generated by the charge repulsion of surface ionizable groups distributed along the polypeptide backbone that leads to the formation of a charged, brushlike structure on the surface of the oocyst. These extended proteins are composed primarily of cysteine, proline, and histidine, and they tend to resemble a sparse filamentous material when viewed by means of thin-section electron microscopy (11). The oocyst wall also contains both glycolipids and phospholipids which are loosely attached or firmly embedded within the wall structure (13). These moieties have been found to play an important role in determining oocyst wall permeability, surface charge, and hydrophobicitysall of which are important factors in oocyst survival in the environment (10). Between the inner and outer layers of the oocyst cell wall lies a thin (∼5 nm) layer of complex lipid. This “central layer”, combined with the thick oocyst wall inner layer, is believed to contribute to both the rigidity and elasticity of the oocyst wall (11). The wall of the oocyst greatly hinders the effectiveness of traditional chemical disinfection techniques, such as chlorination. While techniques such as ozone disinfection (1416), mixed oxidant disinfection (17, 18), and ultraviolet light irradiation (19, 20) have shown some effectiveness, the primary control strategy used in drinking water purification is physical removal using sand-bed filters. In many parts of the world, such as Switzerland, France, and Finland, removal using riverbank filtration is also commonly practiced (21). In either case, understanding the basic interaction force profile between oocysts and mineral surfaces, specifically silica, would be of great value. Knowledge of these force profiles would also be needed for the eventual development 10.1021/es051231e CCC: $30.25
2005 American Chemical Society Published on Web 11/15/2005
of models for predicting the rate of transport of oocysts in groundwater. This paper describes a comprehensive experimental study focused on determining the force versus distance profile between single C. parvum oocysts and silica particles in aqueous solutions of NaCl and CaCl2. The measurements were made by first immobilizing the oocysts in a polycarbonate membrane filter via simple filtration. The force profile between an oocyst and a 5 µm silica particle was then measured via atomic force microscopy (AFM) using the colloid probe technique developed by Ducker et al. (22) and Butt (23).
Prior Work Considine et al. used an atomic force microscope (AFM) to measure the force of interaction between C. parvum oocysts and a model sand surface (silicate glass) in 1 mM KNO3 (24). The authors found that a repulsive force always exists and the decay length of the force, 15-16 nm, was significantly larger than the known Debye length of the solution. The authors attributed this result to the contribution of a “hairy” layer, most likely consisting of surface proteins extending into solution, which would impose a steric repulsion between the oocyst and sand surface, plus an electrostatic repulsion. When exposing the oocyst to dissolved calcium and dissolved organic acids, Considine et al. found that the hairy layer collapsed to varying extents and that the zeta potentials of the oocysts became less negative, indicating a charge neutralization effect. Dai et al. (25) studied the adhesion of C. parvum to four different materials of different surface charge density and hydrophobicity. The authors found that the adhesion was controlled primarily by surface charge, dominating any hydrophobic effects (25). In a separate study, Dai and Boll (26) examined the adhesion of C. parvum oocysts to soil particles. It was found that adhesion occurred between oocysts and soil particles of opposite charge, while no adhesion occurred between oocysts and soil particles of like charge. Hsu et al. (27) studied the influence of ionic strength and pH on the filtration of C. parvum oocysts in packed columns of glass and polystyrene beads. Their results showed that an increase in ionic strength of NaClO4 resulted in increased removal of viable C. parvum oocysts. The authors also found that an increase in the pH of their system resulted in a lower removal of the oocysts. On the basis of the findings it was believed that the removal rate of the oocysts resulted from the reduction of the electrostatic repulsion between the oocysts and the packed column constituents. Dai and Hozalski studied and compared the behavior of C. parvum oocysts with that of latex microspheres in water treatment filters. Their results showed that oocysts will effectively travel through sandy groundwater aquifers which contain low ionic strength water and significant organic matter concentrations (28). The authors also found that this transport is restricted in relatively hard groundwaters with low organic matter concentrations. Tufenkji and Elimelech performed experiments that focused on C. parvum oocyst retention in packed spherical glass bead columns as an ideal model for porous media. Their results were then compared to results predicted using classical colloidal filtration theory and were found to deviate significantly. The authors explained this deviation via use of a dual-mode deposition model for the oocysts, involving simultaneous fast and slow deposition rates (29). Recently, Kuznar and Elimelech measured the deposition of viable C. parvum oocysts onto an ultrapure quartz surface in both monovalent and divalent salts using a radial stagnation point flow system (30). The authors found no deposition of oocysts for the full range of ionic strengths in the monovalent salt, even though deposition was predicted at the higher ionic strengths using DLVO theory. Deposition
FIGURE 1. SEM image of a 5 µm silica particle glued to a cantilever tip. did occur in the presence of the divalent salt, though the degree was substantially lower than that predicted using DLVO theory. These results suggested an additional repulsive force whose presence could not be accounted for using traditional DLVO theory. It was proposed that an electrosteric repulsion existed between the oocysts and the silica surface.
Experimental Section Apparatus. A Digital Instruments NanoScope IIIa multimode atomic force microscope (Digital Instruments, CA) was used for measuring the force profiles. The colloidal probes used in these experiments were created by gluing (Norland optical adhesive 68, lot 173) a 5 µm silica particle (Bangs Laboratories) to the end of a silicon nitride cantilever tip (Veeco, model MLCT-AUNM). The colloidal probe was then exposed to ultraviolet light by means on an UV/O3 chamber for 10 min to allow the glue to cure. An SEM micrograph of the cantilever tip with a glued particle is shown in Figure 1. The cantilever probe shown in this figure is a silicon nitride cantilever probe with gold coating on the upper surface of the probe (to provide a visible reference). This particular cantilever probe is one of several probes located on the cantilever. The probe shown is a triangular probe, 220 µm in length with a spring constant of 0.03 N/m, as provided by the manufacturer. Sample Preparation. Viable oocysts used in all experiments preformed were supplied by Sterling Parasitology Laboratory (SPL) from the University of Arizona. According to the supplier, all oocysts were shed from the same calf that was originally infected with an Iowa isolate and obtained from Dr. Harley Moon at the National Animal Disease Center in Ames, IA. These oocysts were purified using discontinuous sucrose and cesium chloride centrifugation gradients. The oocysts were stored in an antibiotic solution containing 0.01% Tween 20 (to prevent aggregation), 100 U of penicillin, and 100 ug of gentamicin per mL (to prevent bacterial growth from occurring within the sample). Prior to performing experiments, the 1 mL sample of 1 × 106 oocysts that was purchased from SPL was centrifuged, according to supplier’s specifications, at 12 000 rpm (13 278 rcf) for 1 min using an Eppendorf 5415D microfuge with a fixed angle rotor. After centrifugation, the antibiotic solution supernatant was removed and replaced with deionized water (Barnstead). This procedure was repeated two more times to ensure the removal of all antibiotic solution initially present. After all solution was removed, the oocysts were resuspended by means of a vortex mixer and were stored at 4 °C. No further treatments were performed on the oocysts. It was important during experiments that the oocysts remain immobilized for the duration of the experiment. In the experiments of Considine et al. (24), the oocysts were VOL. 39, NO. 24, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
9575
FIGURE 2. AFM image of a trapped oocyst and the surrounding membrane surface. The image was obtained with a 5 µm silica particle glued to the tip of a cantilever, which is the primary reason for the lack of detail. glued to a silicon wafer surface. We felt, however, that gluing the oocysts in this manner could possibly alter the structure of their outer protein layers, thus we instead sought to immobilize them via physical means. Our method involved trapping the oocysts in the pores of polycarbonate membranes with a pore size (3 µm) that was slightly below the typical oocyst diameter (3.9-5.9 µm). To prepare samples for analysis, 40 µL of 1 × 106 organisms/mL stock oocyst solution was combined with 960 µL of 1.0 mM ionic strength solution to create a 40 000 organism solution. This solution was then placed in a syringe and was injected through a 13 mm diameter Millipore polycarbonate membrane by means of a syringe filter holder. Applying a positive pressure resulted in the oocysts being pushed into the membrane pores. Even though the oocysts were immobile, a significant portion remained above the surface where it could be accessed by the colloidal probe on the AFM cantilever. Once the oocysts were mechanically trapped within the membrane pore, the membrane was immediately removed from the syringe filter holder and placed on top of an AFM sample puck, which was then transferred to the top of the AFM piezo scanner. The sample was raised until it came into contact with the AFM fluid cell apparatus, which sealed the fluid cell. A 1.0 mM ionic strength aqueous solution was then injected into the fluid cell. While different ionic strengths were used in the measurements, the sequence was to work from lowest (1.0 mM) to highest ionic strength. Care was taken during this entire process to ensure that a liquid film was always present on the membrane surface to avoid exposure of the oocyst to air. Solution Preparation. Aqueous sodium chloride solutions of concentration 1.0, 3.16, 10.0, 31.6, and 100.0 mM were prepared using ACS-grade NaCl (Mallinckrodt AR, lot no. 7581 A13634) and deionized water. The solutions were not buffered, and the pH of the solution ranged between 5.5 and 5.7. Similarly, aqueous calcium chloride solutions of concentration 0.333, 1.05, 3.33, 10.5, and 33.3 mM were made using ACS-grade CaCl2 (Aldrich, lot no. 07516JU) and 9576
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 24, 2005
deionized water (these concentrations of CaCl2 produced equivalent ionic strengths to those of the NaCl solutions mentioned above). The pH of these solutions, again not buffered, ranged between 5.6 and 6.9. All solution concentrations were verified by means of pH and conductivity measurements. Positioning the Oocysts. During the force measurements, it was important that the silica particle probe be positioned directly over the top of the oocyst, otherwise a lateral component to the force could arise. This was done by imaging a large scan area until an oocyst was located. The scanned image (see Figure 2) was then analyzed in 3-D imaging mode using the NanoScope software to verify that an actual oocyst was present. This was done by comparing the relative heights and shape of the oocyst to that of the surrounding membrane (see Figure 3). The oocyst was then centered in the center of the AFM scan. The scan size was then incrementally reduced, with the oocyst repeatedly centered within the image after each scan. This process was repeated until a scan size of 5 µm × 5 µm was obtained. AFM Force Measurements. Once an oocyst was determined to be directly under the colloidal probe, the AFM was switched from imaging mode to force mode. A minimum of 50 force measurements were then obtained in force mode using a scan rate of 0.996 Hz and a ramp size of 1 µm. Each force curve generated by the AFM Nanoscope software was an average of five individual force measurements. Once force curves had been obtained for the lowest strength solution (1 mM NaCl), the colloidal probe was disengaged from the oocyst surface. The fluid cell was then drained and flushed with the next highest ionic strength solution. Positioning and force measurements were then repeated for each ionic strength. In the case where both a monovalent and a divalent salt solution were used with the same oocyst surface, the fluid cell was first drained and flushed with the lowest ionic strength monovalent salt (1 mM NaCl), then drained and flushed again with the lowest ionic strength solution of the divalent salt (0.333 mM CaCl2). Upon collecting the force
FIGURE 3. Side view of an oocyst trapped in a membrane. As in Figure 2, this image was obtained with a 5 µm silica particle glued to the tip of an AFM cantilever. measurements using the Nanoscope software, the force curve files were then exported to Mathematica for further analysis.
Results Silica Particle/Polycarbonate Membrane Force Profiles. To validate the experimental system, we first performed a series of experiments in which the interaction force profile between a 5 µm silica particle and the surface of the polycarbonate membrane was measured. To avoid problems with the membrane pores, a relatively smooth section of the membrane was located. Experiments were performed using a single particle at the same membrane location in aqueous NaCl. The results of these measurements are presented in Figure 4 and Table 1. Figure 4 shows a semilog plot of the measured force (determined from the cantilever deflection) versus particle/membrane separation distance, h. The force for each ionic strength has been normalized by the contact value, F0, to better illustrate the change in slope. The results are clearly linear, indicating that the force decays exponentially as expected from a simple screened electrostatic repulsion of the form
F ) B exp(-κh)
(1)
where B is a pre-exponential constant and κ is the Debye parameter (decay constant). This equation has been found to be valid for the electrostatic interaction between large particles and surfaces for the conditions when (i) the particle size is much larger than the decay length (meaning that the Derjaguin approximation can be applied) and (ii) the separation distance is larger than the Debye length, allowing the superposition of electric potentials. Under these conditions, B is a function of the electric potentials on each surface, ψ01 and ψ02, and is given by (31)
B ) 64πr0
( )
( ) ( )
zeψ01 zeψ02 kT 2 κR tanh tanh ze 4kT 4kT
(2)
Here, r is the dielectric constant of the solution, 0 is the
FIGURE 4. Force profiles measured between a 5 µm silica particle and a polycarbonate membrane surface at varying ionic strengths of an aqueous NaCl solution. The forces have been normalized by the value at contact, F0, to better illustrate the change in slope. permittivity of free space, z is the valence of the symmetrical electrolyte, e is the charge of a proton, and R is the oocyst radius. Table 1 compares the decay length of these force profiles (obtained from the slope of the lines in Figure 4) with the expected Debye length of the solutions, calculated as
κ-1 )
(
0rkT
2000e2NA
)
I-1/2
(3)
where NA is Avogadro’s constant and I is the solution ionic VOL. 39, NO. 24, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
9577
TABLE 1. Comparison between Predicted and Measured Decay Lengths for the Measured Interaction between a 5 µm Silica Particle and a Polycarbonate Membrane in Aqueous NaCl Solutions ionic strength mM
predicted Debye length nm
measured decay length nm
1.0 3.16 10.0 31.6
9.84 5.53 3.11 1.75
9.40 5.20 3.0 1.70
strength. The agreement was found to be quite good, with an average deviation between the measured and predicted decay length of 4.2%. Force Profiles between C. parvum Oocysts and Silica Particles in NaCl and CaCl2. Typical force profiles measured between a silica particle and a single C. parvum oocyst in aqueous NaCl solutions are shown in Figure 5. Compared to the silica/membrane results shown in Figure 4, these force profiles show much lower sensitivity to ionic strength. Specifically, while the absolute magnitude of the force at a given separation distance does change slightly (this point is discussed in greater detail below), the slope of the lines seem to be quite similar, even for an increase in the salt concentration of 2 orders of magnitude. This point can be seen more clearly in Figure 6, which gives a log-log plot of the average decay length of the repulsive force versus ionic strength for three different experiments in NaCl solutions. These decay lengths were determined by the slope of the linear component of the force profiles shown in Figure 5 (for example, in the range of 2060 nm). It should be mentioned that while Figure 5 shows only one force profile for each salt concentration, each data point in Figure 6 actually represents an average over many such profiles. According to eqs 1 and 3, for a purely electrostatic repulsion, the decay length should scale with ionic strength to the -1/2 power, which should be the value of the slopes of the log-log plots in Figure 6. As seen, however, the decay lengths obtained for the interaction between the silica particle and oocyst display a much weaker power-law dependence, ranging between -0.07 and -0.09.
FIGURE 6. Log-log plot showing the variation of the decay length of the measured force profiles with ionic strength. The decay lengths were obtained by taking the slope of a plot of the natural log of the measured force vs separation distance. Each data point represents an average of many individual force profiles, and the error bars correspond to the 95% confidence limits. For purely electrostatic interactions, the slope of each line should be -1/2.
FIGURE 7. Force vs separation distance for a 5 µm silica particle interacting with a trapped oocyst at various ionic strengths of an aqueous CaCl2 solution. The legend provides the ionic strength of each of the CaCl2 solutions.
FIGURE 5. Force vs separation distance for a 5 µm silica particle interacting with a trapped oocyst at various ionic strengths of an aqueous NaCl solution. 9578
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 24, 2005
Similar behavior was observed for experiments conducted in the aqueous CaCl2 solutions, which are presented in Figure 7. Again, the force profiles show little dependence on the solution ionic strength, and the scaling exponent between the decay length of the repulsive force and the solution ionic strength is much less than the expected value of -1/2 (presented in Figure 6). While the force profiles in Figures 5 and 7 are certainly quite similar, there are, nonetheless, small differences that should be mentioned. In addition to the slight change in decay length with ionic strength, it was found that the magnitude of the force at contact was also slightly dependent
FIGURE 8. Variation in the magnitude of the repulsive force at contact with ionic strength in aqueous (a) NaCl and (b) CaCl2 solutions. Each data point represents an average over many force profiles, and the error bars provide the 95% confidence limits. on ionic strength. This can be seen in Figure 8, which shows the value of the force at contact versus ionic strength for both the NaCl and CaCl2 solutions, respectively. Although there is significant scatter (indicated by the size of the 95% confidence limits), the trend is quite clear. [The primary cause of this scatter is the well-known difficulty in determining the exact point of contact (termed the region of constant compliance) in the AFM experiments. This problem is made more difficult with cantilevers of very low spring constants, such as those used here. It is also clear that the magnitude of the scatter is greater in the CaCl2 solutions; however, the cause of this difference is currently not known.] The force profiles presented above for NaCl and CaCl2 each involved separate experiments with different oocysts, meaning that it is difficult to directly compare results obtained in the two different electrolyte solutions. (It should be mentioned, however, that the oocysts were all from the same lot.) Because it was felt that such a comparison could be meaningful, an additional set of experiments was performed in which force measurements on a single oocyst were taken with both NaCl and CaCl2 solutions flushed into the sample cell. Force profiles obtained at ionic strengths of 1.0 and 31.6 mM are shown in Figure 9. As seen, the force profiles at each ionic strength are essentially equal, indicating that the electrolyte valence has little effect. A point should be made here concerning the values of separation distance in the force profiles measured with the oocysts. In an AFM experiment, the point of zero separation is determined as the point where the movement of the piezo (which moves the sample holder containing the oocyst) and the cantilever (with the attached particle) become equal, termed the point of constant compliance. With terminally anchored polymers or proteins on the surface of the oocyst, which is believed to be the case here, this point of compliance will actually correspond to the point where the adsorbed layer can no longer be compressed, meaning that the true distance between the silica particle and the oocyst surface will be slightly larger. This offset is not a significant factor here, however, because (1) we are simply comparing the effect of added
FIGURE 9. Two graphs comparing the effect of electrolyte on the measured force profile. The same silica particle and oocyst were used for all four force curves. As seen, at both 1 mM (a) and 31.6 mM (b) ionic strengths, switching from NaCl to CaCl2 produces no significant change in the measured force.
TABLE 2. Comparison between Predicted and Measured Decay Lengths for the Measured Interaction between Two 5 µm Silica Particles, One Attached to the End of the AFM Cantilever and the Other Trapped in a Polycarbonate Membranea ionic strength mM
predicted Debye length nm
measured decay length nm
1.0 3.16 10.0 31.6 100.0
9.84 5.53 3.11 1.75 0.984
9.79 6.42 3.17 1.79 1.04
a
Measurements were made in aqueous NaCl solutions.
electrolyte on the nature of the force profiles, and (2) an offset in the separation distance will not affect the slope of a plot of the log of force versus separation distance (e.g., Figures 5 and 7), which is how the decay lengths are determined. As a final test of the accuracy of the experimental approach used here, an additional validation experiment was performed in which 5 µm diameter silica particles were trapped in the membrane and the force profile between a single trapped particle and the silica particle attached to the end of the AFM cantilever was measured. This measurement was performed to test whether the elasticity of the membrane trapping the oocyst was allowing the oocyst to actually move during the force measurement. If this were happening, then the measured force profiles and decay lengths would reflect properties of the membrane rather than the oocyst. The decay lengths obtained by these measurements, along with the expected Debye length of the solution, are given in Table 2. The values were found to be in excellent agreement, with an average deviation of 5.2%. This agreement clearly indicates that the membrane is not the cause of the unusual decay lengths discussed above for trapped C. parvum oocysts, since any such contribution from the membrane would have been evident in the results with the trapped silica particle. VOL. 39, NO. 24, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
9579
Discussion The results above clearly indicate that while the addition of salt does reduce the magnitude of the repulsive interaction between a silica particle and a C. parvum oocyst, the effect is far smaller than that predicted using traditional DLVO theory between two charged particles. Nonetheless, electrophoresis measurements performed by numerous researchers clearly indicate that these oocysts are negatively charged (24, 26, 30, 34-40) at near-neutral values of pH, such as those used in these experiments. Thus the observed insensitivity to ionic strength is quite unexpected. One possible structure for the outermost layers of the oocyst that would explain this observed behavior is that of a thin, charged inner layer, such as that produced by charged surface proteins, covered by a larger layer of uncharged material. Specifically, the repulsive interaction between a negatively charged silica particle and an oocyst with this type of surface structure would consist of both a large, longrange steric repulsion coupled with a weaker electrostatic repulsion. The primary effect of increasing the ionic strength of the solution would be to weaken the electrostatic component of the force (any possible effect of ionic strength on the steric component is expected to be relatively small). Experimental evidence of such a structure was provided by the work of Nanduri et al. (12). These authors first stained oocysts using ruthenium red and then used a phenol mixture to dissociate the surface protein group into an aqueous phase. Chromatographic analysis indicated that 90% of the dissociated material had a molecular weight larger than 106 Da. Compositional analysis indicated that 82% of the dissociated material was carbohydrates, with glucose and galactose as the predominant sugars. In addition, transmission electron micrographs of the stained oocysts indicated that the outermost layer of the oocyst consisted of dense, uniformly distributed aggregates that were 20-30 nm thick. The authors conclude that this carbohydrate matrix could play an important role in oocyst adhesion. As mentioned above, Kuznar and Elimelech used a radial stagnation point flow system to study the deposition kinetics of viable oocysts onto ultrapure quartz surfaces in both KCl and CaCl2 aqueous solutions (30). In KCl solutions, deposition remained insignificant up to concentrations greater than 100 mM. While deposition in CaCl2 solutions was significantly higher (this point is discussed in greater detail below), even at 100 mM CaCl2 the attachment efficiency, characterizing the probability that an oocyst will deposit on contacting the surface, remained less than 0.4. (By comparison, DLVO theory predicts an attachment efficiency of essentially 1.0 at 100 mM CaCl2.) To further explore the nature of the repulsive forces presented in Figures 5 and 7, we fit these force profiles to the Alexander-de Gennes equation for the steric interaction between two surfaces covered with grafted polymer layers in a good solvent (41, 42). Specifically, for two flat plates covered with polymer of thickness L, the repulsive force per unit area, F/A, can be predicted using
( )[( ) ( ) ]
F(D) kT ) 3 A s
2L D
9/4
-
D 2L
3/4
for h < 2L (4)
Here, s is the distance between the chains on the surface and D is the separation distance between the plates. This equation is based on a balance between increasing osmotic pressure and decreasing elastic restoring forces which result when two parallel plates with polymer brush surfaces are brought within a distance less than the thickness of their polymer layers. This particular equation assumes that the polymer is grafted to the interface and that the polymer chains are linear, flexible, neutral chains in a good solvent. 9580
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 24, 2005
FIGURE 10. Two graphs showing the fitting of force profiles measured in 10.0 mM NaCl solution with the Alexander-de Gennes theory of eq 6. In each graph, the symbols are the measured results while the line is the fit. For (a), the s and L values obtained were 50.1 and 113 nm, respectively, while for (b), the s and L values obtained were 50.0 and 113 nm, respectively. Applying this equation to our particular system required two modifications. First, since only one of our surfaces is covered with polymer, the 2L was replaced with L in both the equation itself and the upper separation limit. Second, to account for the effect of curvature, the Derjaguin approximation was utilized. Specifically, the force of interaction between two spherical particles of radii R1 and R2 separated by distance h can be approximated as
F(h) ) 2π
(
R1R2 R1 + R2
)∫
∞
h
F(D) dD A
(5)
This approximation assumes that the particle radii are much larger than the characteristic range of the force profile, which is valid here. Substituting eq 4 into this expression and changing the upper limit of integration to L instead of ∞ (needed because the force expression only applies for h < L) yields
F(h) )
(
2π
)( ){
R1R2 kT R1 + R2 s3
4L L 5 h
[( )
5/4
]
-1 +
]}
4L h 7/4 -1 7 L for h < L (6)
[( )
Shown in Figure 10 are fits of this equation to force profiles measured in 10.0 mM NaCl. Since the above equation is valid for purely steric interactions, only the data above a separation of 15 nm was used in each fitting. At this distance from the interface the electrostatic forces that may be present in the system will be negligible for this ionic strength solution. It is clear that this equation can provide a fairly good fit to the measured force profiles. The values of s and L obtained in each fitting was essentially the same for both curves, 50 nm for s and 113 nm for L. The value of L provides an estimate of the thickness of the uncharged carbohydrate layer covering the oocysts. Repeating this fitting procedure using force profiles obtained in the different ionic strength solutions (same oocyst) produced similar values of the layer thickness,
FIGURE 11. Example of the adhesion force measured in 1.0 mM CaCl2. The adhesive force causes the sudden upward jump from a negative (or attractive) force at approximately 100 nm separation. as did a similar analysis performed in CaCl2 solutions on a different oocyst. For example, in 10 mM CaCl2, the values of s and L obtained were 55.1 and 115 nm, which compare favorably with the values obtained in 10 mM NaCl with a different oocyst. This finding suggests that these parameters are somewhat representative of the structure of these oocysts. (Note that due to the slight offset in determining the absolute separation distance between the particle and oocyst surface discussed above, it is likely that the true thickness of the carbohydrate layer is slightly larger than the values mentioned here.) Explanation of Oocyst Deposition Behavior. One final question that needs to be addressed is the impact of electrolyte type on oocyst deposition. As mentioned above, stagnation point flow experiments by Kuznar and Elimelech found significantly higher attachment efficiencies for oocysts depositing onto quartz surfaces in CaCl2 solutions than in NaCl solutions at equivalent ionic strengths (30). The trend is difficult to understand in light of the nearly identical force curves for NaCl and CaCl2 solutions shown in Figure 9. One possible explanation for this result is the observation that the force profiles measured in CaCl2 solutions frequently displayed a significant adhesion force. An example of this force can be seen in Figure 11, which shows the force profile made in 1.0 mM CaCl2. Unlike the earlier force profiles, this force curve was taken upon retraction of the oocyst from the silica particle (i.e., as the two surfaces were being pulled apart after contact). The adhesion force produces the sudden jump in the force profile from a negative (meaning attractive) value to a positive or zero value. Using the maximum negative value achieved before the jump, we can estimate the magnitude of this adhesive force to be approximately 115 pN. It should be mentioned that this adhesion was not always observed in the CaCl2 solutions and was sometimes also observed in the NaCl solutions. However, the frequency at which an adhesion force was observed was substantially greater in the CaCl2 solutions. Equally interesting is the fact that this adhesive force occurs not at contact, but at significantly larger separations, such as 100 nm for the force curve shown in Figure 11. Recall that this distance is approximately the same value as the thickness of the steric polymer layers obtained from fitting the force profiles in Figure 10. These results suggest that the calcium ions are facilitating adhesion between the outermost
region of the uncharged carbohydrate layer and the surface of the silica particle. One possible mechanism would be binding of the calcium ions to both the carbohydrate and the silica particle, meaning that the calcium would serve as a bridge between the two surfaces. It is well-known that calcium ions can adsorb to the surface of oppositely charged surfaces, such as silica, while various authors have studied the interaction of calcium ions with carbohydrates (see Lu et al. (43), Hamazaki (44), and Sharom and Grant (45)). However, the repulsive force experienced by the actual compression of the carbohydrate layer, such as shown in Figures 5 and 7, would not be altered by the ionic strength or ion valence. In conclusion, the force profile measurements presented here provide some insights into the nature of the outermost layer of C. parvum oocysts. The structure that provides the best match to the measured force profiles, which is also consistent with independent experimental work on the nature of the oocyst surface layer, is that of a relatively thick (approximately 115 nm) layer of uncharged carbohydrates molecules, primarily glucose, possibly mixed with a thinner layer of charged protein molecules. While such a structure could lead to a negative electrophoretic mobility, the magnitude of this mobility would be weakened by the displacement of the plane of shear by the carbohydrate layer. One question that remains unanswered by these results, however, is the mechanism governing the capture of these oocysts in natural aquifers. Specifically, river bank filtration, in which the oocysts are removed by natural aquifers, is known to be an effective mechanism for purification. One possibility is that the carbohydrate layer surrounding the oocyst is not completely neutral but does, in fact, contain a small number of negative groups. A weak charge might also explain the weak dependence between decay length and ionic strength shown in Figure 6. If so, then it is possible that capture occurs on local patches that are highly positively charged in the heterogeneous subsurface aquifer. To better explore this possibility, we plan to repeat these experiments using positively charged particles, such as alumina at neutral pH values.
Acknowledgments This work was supported by the United States Department of Agriculture, Award No. 2002-35102-12600, and by the National Science Foundation, Division of Chemical and Transport Systems, Award CTS-0350630. The authors also gratefully acknowledge the benefit of conversations with M. Elimelech and Z. A. Kuznar in the chemical engineering department at Yale.
Literature Cited (1) Tyzzer, E. E. Cryptosporidium parvum (sp. nov.), a coccidium found in the small intestine of the common mouse. Arch. Protistenk. 1912, 26, 394-412. (2) Meisel, J. L.; Perera, D. R.; Meligro, C.; Rubin, C. E. Overwhelming diarrhea associated with Cryptosporidium in an immunosuppressed patient. Gastroenterology 1976, 1156-1160. (3) Rose, J. B.; Gerba, C. P.; Jakubowski, W. Survey of potable watersupplies for Cryptosporidium and Giardia. Environ. Sci. Technol. 1991, 25, 1393-1400. (4) Lechevallier, M. W.; Norton, W. D.; Lee, R. G. Giardia and Cryptosporidium spp. in filtered drinking-water supplies. Appl. Environ. Microbiol. 1991, 57, 2617-2621. (5) Lechevallier, M. W.; Norton, W. D.; Lee, R. G. Occurrence of Giardia and Cryptosporidium spp. in surface-water supplies. Appl. Environ. Microbiol. 1991, 57, 2610-2616. (6) Ramirez, N. E.; Ward, L. A.; Sreevatsan, S. A review of the biology and epidemiology of cryptosporidiosis in humans and animals. Microbes Infect. 2004, 6, 773-785. (7) Corso, P. S.; Kramer, M. H.; Blair, K. A.; Addiss, D. G.; Davis, J. P.; Haddix, A. C. Cost of illness in the 1993 waterborne Cryptosporidium outbreak, Milwaukee, Wisconsin. Emerging Infect. Dis. 2003, 9, 426-431. VOL. 39, NO. 24, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
9581
(8) Medema, G. J.; Schets, F. M.; Teunis, P. F. M.; Havelaar, A. H. Sedimentation of free and attached Cryptosporidium oocysts and Giardia cysts in water. Appl. Environ. Microbiol. 1998, 64, 4460-4466. (9) Smith, M.; Thompson, K. C. CryptosporidiumsThe Analytical Challenge; Royal Society of Chemistry, 2001. (10) Reduker, D. W.; Speer, C. A.; Blixt, J. A. Ultrastructure of Cryptosporidium-parvum oocysts and excysting sporozoites as revealed by high-resolution scanning electron-microscopy. J. Protozool. 1985, 32, 708-711. (11) Harris, J. R.; Petry, F. Cryptosporidium parvum: Structural components of the oocyst wall. J. Parasitol. 1999, 85, 839-849. (12) Nanduri, J.; Williams, S.; Aji, T.; Flanigan, T. P. Characterization of an immunogenic glycocalyx on the surfaces of Cryptosporidium parvum oocysts and sporozoites. Infect. Immun. 1999, 67, 2022-2024. (13) Robertson, L. J.; Campbell, A. T.; Smith, H. V. Induction of folds or sutures on the walls of Cryptosporidium-parvum oocysts and their importance as a diagnostic feature. Appl. Environ. Microbiol. 1993, 59, 2638-2641. (14) Li, L. J.; Haas, C. N. Inactivation of Cryptosporidium parvum with ozone in treated drinking water. J. Water Supply Res. Technol.sAqua 2004, 53, 287-297. (15) Corona-Vasquez, B.; Samuelson, A.; Rennecker, J. L.; Marinas, B. J. Inactivation of Cryptosporidium parvum oocysts with ozone and free chlorine. Water Res. 2002, 36, 4053-4063. (16) Parker, J. F. W.; Greaves, G. F.; Smith, H. V. The effect of ozone on the viability of Cryptosporidium-parvum oocysts and a comparison of experimental methods. Water Sci. Technol. 1993, 27, 93-96. (17) Biswas, K.; Craik, S.; Smith, D. W.; Belosevic, M. Synergistic inactivation of Cryptosporidium parvum using ozone followed by monochloramine in two natural waters. Water Res. 2005, 39, 3167-3176. (18) Son, H.; Cho, M.; Chung, H.; Choi, S.; Yoon, J. Bactericidal activity of mixed oxidants: Comparison with free chlorine. J. Ind. Eng. Chem. 2004, 10, 705-709. (19) Morita, S.; Namikoshi, A.; Hirata, T.; Oguma, K.; Katayama, H.; Ohgaki, S.; Motoyama, N.; Fujiwara, M. Efficacy of UV irradiation in inactivating Cryptosporidium parvum oocysts. Appl. Environ. Microbiol. 2002, 68, 5387-5393. (20) Belosevic, M.; Craik, S. A.; Stafford, J. L.; Neumann, N. F.; Kruithof, J.; Smith, D. W. Studies on the resistance/reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts exposed to medium-pressure ultraviolet radiation. FEMS Microbiol. Lett. 2001, 204, 197-203. (21) Tufenkji, N.; Ryan, J. N.; Elimelech, M. The promise of bank filtration. Environ. Sci. Technol. 2002, 36, 422a-428a. (22) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Direct measurement of colloidal forces using an atomic force microscope. Nature 1991, 353, 239-241. (23) Butt, H. J. Measuring electrostatic, van der Waals, and hydration forces in electrolyte-solutions with an atomic force microscope. Biophys. J. 1991, 60, 1438-1444. (24) Considine, R. F.; Dixon, D. R.; Drummond, C. J. Oocysts of Cryptosporidium parvum and model sand surfaces in aqueous solutions: an atomic force microscope (AFM) study. Water Res. 2002, 36, 3421-3428. (25) Dai, X.; Boll, J.; Hayes, M. E.; Aston, D. E. Adhesion of Cryptosporidium parvum and Giardia lamblia to solid surfaces: the role of surface charge and hydrophobicity. Colloids Surf., B 2004, 34, 259-263. (26) Dai, X.; Boll, J. Evaluation of attachment of Cryptosporidium parvum and Giardia lamblia to soil particles. J. Environ. Qual. 2003, 32, 296-304.
9582
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 24, 2005
(27) Hsu, B. M.; Huang, C. P.; Pan, J. R. Filtration behaviors of Giardia and CryptosporidiumsIonic strength and pH effects. Water Res. 2001, 35, 3777-3782. (28) Dai, X. J.; Hozalski, R. M. Evaluation of microspheres as surrogates for Cryptosporidium parvum oocysts in filtration experiments. Environ. Sci. Technol. 2003, 37, 1037-1042. (29) Tufenkji, N.; Elimelech, M. Spatial distributions of Cryptosporidium oocysts in porous media: Evidence for dual mode deposition. Environ. Sci. Technol. 2005, 39, 3620-3629. (30) Kuznar, Z. A.; Elimelech, M. Adhesion kinetics of viable Cryptosporidium parvum oocysts to quartz surfaces. Environ. Sci. Technol. 2004, 38, 6839-6845. (31) Verwey, E. J. W.; Overbeek, J. T. G. Theory of the Stability of Lyophobic Colloids; Elsevier: Amsterdam, 1948. (32) Fayer, R. Cryptosporidium: a water-borne zoonotic parasite. Vet. Parasitol. 2004, 126, 37-56. (33) Fujino, T.; Matsui, T.; Kobayashi, F.; Haruki, K.; Yoshino, Y.; Kajima, J.; Tsuji, M. The effect of heating against Cryptosporidium oocysts. J. Vet. Med. Sci. 2002, 64, 199-200. (34) Considine, R. F.; Dixon, D. R.; Drummond, C. J. Laterally resolved force microscopy of biological microspheres-oocysts of Cryptosporidium parvum. Langmuir 2000, 16, 1323-1330. (35) Considine, R. F.; Drummond, C. J.; Dixon, D. R. Force of interaction between a biocolloid and an inorganic oxide: Complexity of surface deformation, roughness, and brushlike behavior. Langmuir 2001, 17, 6325-6335. (36) Drozd, C.; Schwartzbrod, J. Hydrophobic and electrostatic cell surface properties of Cryptosporidium parvum. Appl. Environ. Microbiol. 1996, 62, 1227-1232. (37) Karaman, M. E.; Pashley, R. M.; Bustamante, H.; Shanker, S. R. Microelectrophoresis of Cryptosporidium parvum oocysts in aqueous solutions of inorganic and surfactant cations. Colloids Surf., A 1999, 146, 217-225. (38) Kuznar, Z. A.; Elimelech, M. Role of surface proteins in the deposition kinetics of Cryptosporidium parvum oocysts. Langmuir 2005, 21, 710-716. (39) Searcy, K. E.; Packman, A. I.; Atwill, E. R.; Harter, T. Association of Cryptosporidium parvum with suspended particles: Impact on oocyst sedimentation. Appl. Environ. Microbiol. 2005, 71, 1072-1078. (40) Thomas, F.; Bard, E.; Rouillier, M. C.; Prelot, B.; Mathieu, L. Filtration-elution of Cryptosporidium oocysts assisted by electrostatic interactions. Colloids Surf., A 2001, 195, 135-142. (41) de Gennes, P. G. Polymers at an interface; a simplified view. Adv. Colloid Interface Sci. 1987, 27, 189-209. (42) de Gennes, P. G. C. R. Acad. Sci. 1985, 300, 839-843. (43) Lu, Y.; Deng, G. C.; Miao, F. M.; Li, Z. M. Sugar complexation with calcium ion. Crystal structure and FT-IR study of a hydrated calcium chloride complex of D-ribose. J. Inorg. Biochem. 2003, 96, 487-492. (44) Hamazaki, H. Ca2+-dependent binding of human serum amyloid-P component to Alzheimers β-amyloid peptide. J. Biol. Chem. 1995, 270, 10392-10394. (45) Sharom, F. J.; Grant, C. W. M. Glycosphingolipid spin labels Ca2+ effects on sphingolipid distribution in bilayers containing phosphatidyl serine. Biochem. Biophys. Res. Commun. 1975, 67, 1501-1506.
Received for review June 27, 2005. Revised manuscript received October 6, 2005. Accepted October 7, 2005. ES051231E
