Environ. Sci. Technol. 1999, 33, 3609-3614
Adsorption of Bacteriophages on Clay Minerals S A N D I P C H A T T O P A D H Y A Y * ,† A N D ROBERT W. PULS‡ National Research Council, U.S. Environmental Protection Agency, National Risk Management Research Laboratory, Subsurface Protection and Remediation Division, 919 Kerr Research Drive, Ada, Oklahoma 74820
The ability to predict the fate of microorganisms in soil is dependent on an understanding of the process of their sorption on soil and subsurface materials. Presently, we have focused on studying the thermodynamics of sorption of bacteriophages (T-2, MS-2, and φX-174) on clays (hectorite, saponite, kaolinite, and clay fraction of samples collected from a landfill site). The thermodynamic study not only determines the feasibility of the process but also provides information on the relative magnitudes of the different forces under a particular set of conditions. The total free energy of interaction during sorption of bacteriophages on clays (∆G) has been assumed to be the summation of ∆GH (∆G due to hydrophobic interactions) and ∆GEL (∆G due to electrostatic interactions). The magnitude of ∆GH was determined from the different interfacial tensions (γ) present in the system, while ∆GEL was calculated from ζ-potentials of the colloidal particles. Calculated results show that surface hydrophobicities of the selected sorbents and sorbates dictate sorption. Among the selected bacteriophages, maximum sorption was observed with T-2, while hectorite has the maximum sorption capacity. Experimental results obtained from the batch adsorption studies also corroborated those obtained from the theoretical study.
Introduction Although water is abundant on earth in all forms, inland freshwater is paradoxically globally scarce. This has led to our dependence on groundwater. The demand for groundwater is increasing, and by the year 2000, the Nation’s groundwater supply is expected to be 33% of the total water used in the United States (1). Artificial recharging of present groundwater sources is becoming important as the usage of groundwater increases, and recharging with wastewater is an attractive alternative. Therefore, knowledge of the fate of microorganisms in soil and aquatic environments is necessary to effectively assess the possibility of contamination of groundwater from wastewater. Such a knowledge is also required to assess the necessity of disinfecting the groundwater and to determine the efficiency of different treatment methods used for the attenuation of microbial content. Processes leading to the colonization of microorganisms on any solid-water interfaces are adsorption, desorption, growth, and erosion. Any model predicting the fate of microorganisms should be based on mechanistic studies of * Corresponding author present address: ManTech Environmental Research Services Corporation, P.O. Box 1198, Ada, OK 74821-1198; telephone: (580)436-8580; fax: (580)436-8501; e-mail: Chattopadhyay.
[email protected]. † National Research Council. ‡ U.S. Environmental Protection Agency. 10.1021/es9811492 CCC: $18.00 Published on Web 09/11/1999
1999 American Chemical Society
these processes. The present focus is on understanding the thermodynamics of sorption of bacteriophages on clays and soils. Bacteriophages were selected as surrogate human pathogenic viruses. Clays have been selected as model sorbates, as clays affect the hydraulic permeability and the movement of microorganisms in soil system. The thermodynamic study involves determination of ∆G, whose sign and magnitude allow a prediction of the favored directionality of the process. The ∆G also represents the total forces participating during the sorption process. As bacteria and viruses are ionogenic, their interactions with sorbent surfaces can be categorized as hydrophobic and electrostatic depending on the nature of sorbent and sorbate at a particular set of conditions. When Zerda et al. (2) studied the sorption of different viruses on silica, whose surface charge was artificially modified, they found that, at pH values above and below the isoelectric points (IEP) of the viruses, electrostatic forces dictated sorption. However, when the viruses in nearisoelectric states were studied, they adsorbed on all types of silica, irrespective of the charges, indicating sorption by nonelectrostatic forces. Therefore, the total free energy of interaction, represented by ∆G, can be considered to be the summation of electrostatic (∆GEL) and hydrophobic (∆GH) interactions (3, 4). Similar conclusions were obtained by Chattopadhyay and Traina (5) for sorption of ionic organic compounds on clays. Though the occurrence and relative importance of hydrophobic interactions in the soil-virus system have been recognized by other researchers (4, 6, 7), limited information is available in this regard. To calculate the magnitude of ∆GH, determination of surface hydrophobicities of sorbates and sorbents were necessary. Among the several methods available to study the surface hydrophobicities, we have used interfacial tension (γ) measurements and have also developed suitable thermodynamic equations to predict the magnitude of ∆GH from the different γ values present in the system. The magnitudes of ∆GEL for sorption of bacteriophages on clays were determined with three models, which solve the linearized Poisson-Boltzmann equation by assuming different shapes of the sorbate and the sorbent (8). Detailed discussions on these models are provided later. We have calculated the values of ∆GEL with all the models and have selected the best plausible model that describes the presently studied system. Finally, the calculated values of ∆G were justified with the results obtained from the sorption experiments.
Materials and Methods Three bacteriophages were selected for the study based on their availability, stability, and ease of assaying. These bacteriophages were (i) T-2, whose host bacterium is Escherichia coli ATTC 11303-B2; (ii) MS-2, whose host bacterium is E. coli ATTC 15597-B1; and (iii) φX-174, whose host bacterium is E. coli ATTC 13706-B1. The above bacteriophages and their host bacteria were supplied by Dr. G. Peter Breidenbach, ManTech Environmental Research Services Corporation, Ada, OK. The bacteriophages were grown on lawns of host bacteria by the agar-overlay method and assayed by the procedures described by Adams (9). The T-2 (head dimensions: 65 nm × 95 nm) is a tailed, doublestranded DNA bacteriophage with 48% nucleic acid content. The MS-2 is a single-stranded RNA bacteriophage with 31% nucleic acid content, and its diameter ranges from 24 to 26 nm. The φX-174 is a ssDNA bacteriophage with 26% nucleic acid content, and its diameter ranges from 25 to 27 nm. The reported IEPs of these bacteriophages are as follows: 4.2 for T-2 (10), 3.9 for MS-2 (11), and 6.6 for φX-174 (12). VOL. 33, NO. 20, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Water Contact Angle (θw) for the Selected Sorbents, Sorbates, and Substrates sorbents
θw (deg)
bacteriophages
θw (deg)
substrate
θw (deg)
hectorite saponite kaolinite Norman clay
57 ( 2.6 26 ( 1.4 27 ( 5.3 28 ( 5.3
T2 MS2 φX-174
96 ( 8.6 50 ( 9.5 42 ( 6.4
glass microslide Teflon chamber slide
39 ( 5.6 105 ( 5.5 59 ( 3.1
Well-characterized phyllosilicates and soil samples obtained from a Norman, OK, landfill site were used as sorbents for the present study. The phyllosilicates were obtained from the Source and Special Clays Repository of the Clay Minerals Society (MO). The selected phyllosilicates were kaolinite (KGa-2), hectorite (SHCa-1), and saponite (SapCa-1). Size fractionation of the Norman soil samples was done to collect the clay fraction; henceforth, these samples will be referred as the Norman clay fraction. This clay fraction, which contained an array of phyllosilicates and organic matter, is expected to demonstrate the effects of heterogeneity in the sorbent composition on sorption. The total organic carbon content, as determined by combustion method, was found to be 0.18 ( 0.06%. X-ray diffraction (XRD) measurements of Norman clay fraction indicated the presence of smectites, illite, kaolinite, mica, and quartz. Among the phyllosilicates, the cation-exchange capacities (CECs) of hectorite and saponite (89.2 and 80.4 cmol kg-1, respectively) (13) were considerably higher than the CEC of kaolinite (3.3 cmol kg-1) (14). Hectorite and saponite were also found to have a higher BET surface area (93.4 and 34.6 m2 g-1, respectively) (5) than that of kaolinite (14.11 m2 g-1) (15). The CEC and the BET surface area of the Norman clay fraction were determined as 7.6 cmol kg-1 and 92.3 m2 g-1, respectively. The hectorite and saponite samples were Na-saturated, and the procedure followed for preparing clay suspensions was as outlined by Chattopadhyay and Traina (5). All clay samples were suspended in Millipore water, and a background electrolyte concentration of 0.01 M NaCl was maintained for all the samples. Batch adsorption experiments were performed in 15-mL polypropylene round-bottom centrifuge tubes (Falcon, Becton Dickinson). In each tube approximately 1 mL of virus stock solution was added to 10 mL of clay suspension so that the amount of bacteriophages added to each clay suspension was 106 PFU (plaque-forming units). The clay concentrations ranged between 6 and 10 mg mL-1 of suspension. The control tubes received only virus solution in background electrolyte. The clay-virus suspensions were vortexed and then placed in an orbit shaker (Lab-Line Instruments, Inc.) at 50 rpm for 3 h at 7 ( 1 °C. Subsamples were withdrawn at regular intervals and centrifuged at 1500g for 30 min in a RC-5-B centrifuge (Ivan Sorvall, Inc.). Measurements were made over a period of 5 days at an interval of 24 h to allow sufficient time for the bacteriophage-clay systems to reach equilibrium. The amount of bacteriophages sorbed on the selected clay samples were determined from the difference in the concentrations of viruses in the control liquid phase (considered as the initial concentration) and the supernatants of samples withdrawn (16). All experiments were performed in triplicate. Contact angles (θ) made by water droplets on monolayers of bacteriophages and clay samples were measured by using the Axisymmetric Drop Shape Analysis-Contact Diameter (ADSA-CD) technique (17, 18). In the ADSA-CD method, the average contact diameter of a sessile drop on a surface, as viewed from above, was measured to determine the θ that the drop makes with the surface. Sessile drops were formed with 1 µL of double, distilled water. Contact diameters of 30-40 sessile drops of water were measured in two directions (perpendicular to each other) about 30-60 s after delivery 3610
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using a microscope. These contact diameters were then used as inputs to a FORTRAN program (17) that calculated the values of θ. Other inputs to this program were as follows: difference in density between water and vapor phase (0.997 g cm-3), volume of water droplets, surface tension of water (γwv) at 20 °C (72.75 erg cm-2), and acceleration due to gravity (980.665 cm s-2). Monolayers of bacteriophages and clay samples were prepared by pouring concentrated suspensions on Lab-TekII chamber slides (Nalge Nunc International, IL). Wettability characteristics (as determined by the magnitudes of water contact angles) of these chamber slides were compared to that of standard glass slides. The chamber slides were found to be more hydrophobic than the glass slides (Table 1), and this might be the reason for observing a higher degree of adherence of the phages to the chamber slides. To remove the excess water from the suspensions on the slides, the clay samples were dried at room temperature in a P2O5 desiccator, while the phage samples were dried at a temperature of 4 °C to avoid damage and death to virus particles. The θ on Teflon slides was measured to serve as a reference point and to ensure the accuracy of the measurement technique. The electrophoretic mobilities (EM) and the corresponding pH values were determined for all bacteriophages and clay suspensions. The EM was determined with ZetaPlus (Brookhaven Instruments Corporation), which measured the velocity of the charged, colloidal particles in liquids. The instrument ZetaPlus also calculated the ζ-potential from the solution conditions and the particle mobility. The ζ-potential is the potential at the slipping plane that separates the particle and its bound water molecules from the freely moving water molecules in the solution. The instrument was calibrated with a standard latex dispersion before use. The disposable square plastic cell was rinsed twice with the sample solution. The electrodes were cleaned by rinsing with Millipore water, sonicating for few seconds in water, and then rinsing with the solution of interest. All experiments were conducted according to the instruction manual of ZetaPlus.
Data Analysis Studies (19-21) have indicated that adhesion of sorbates on sorbents is dependent on various forces, such as attractive London dispersion forces, repulsive Lennard-Jones forces, van der Waals forces, hydrogen bridges, steric or orientation effects, hydration effects, and hydrophobic effects. On summarizing, the total force leading to adhesion of sorbates on sorbents were divided into three major groups (20, 21): (i) electrostatic (EL) interactions; (ii) Lifshitz-van der Waals electrodynamic forces (LW), which is comprised of van der Waals-Keesom or orientation forces, van der Waals-Debye or induction forces, and van der Waals-London or dispersion forces; and (iii) polar forces or acid-base interactions (AB). Therefore, the overall ∆G is
∆G ) ∆GEL + ∆GLW + ∆GAB
(1)
This is in contrast to the classical DLVO theory, which predicts that the total force is the summation of repulsive electrostatic forces and attractive van der Waals forces. Both ∆GLW and ∆GAB are dependent on interfacial tensions, as ∆GLW is a function of γLW and ∆GAB is a function of γAB, where γLW and
FIGURE 1. Schematic representation of the components of hydrophobic forces. γAB are the apolar and polar components of interfacial tension, respectively. Also, γ (21) is the summation of γLW and γAB. Based on the above considerations, we have grouped ∆GLW and ∆GAB as ∆GH, where ∆GH is a function of γ. The applicability of the nomenclature comes from the fact that ∆GH is the fraction of the total ∆G that is due to surface hydrophobicities. Therefore, eq 1 can be rewritten as
∆G ) ∆GEL + ∆GH
(2)
A similar type of distinction was also made by Hato (22), who indicated that the long-ranged attractive forces acting between two macroscopic bodies in an aqueous medium can be divided into two distinct components: electrostatic and hydrophobic. In a suspension, the interactions between bacteriophages and clay particles can be categorized as between two clay particles (c-c), between two bacteriophages (b-b), and between clay and bacteriophage (c-b). Therefore, eq 2 can be expanded as H
EL
H
EL
H
∆G ) (∆G + ∆G )c-c + (∆G + ∆G )b-b + (∆G + ∆GEL)c-b (3) Figure 1 illustrates the development of the thermodynamic equations required for the calculations of the different ∆GH values. The ∆G for any process can be obtained from the difference between the forces present before and after the occurrence of the process. In the cases of b-b and c-c interactions:
(∆GH)b-b ) -2γbw and (∆GH)c-c ) -2γcw
(4)
where γbw is the γ between bacteriophage and surrounding water, and γcw is the γ between clay and water. When the bacteriophages adhere to the clay particles, γbw and γcw are replaced with γbc (γ between bacteriophage and clay) at the point of contact. Therefore, the work done is
(∆GH)c-b ) γbc - γcw - γbw
(5)
The interfacial tensions indicated in eqs 4 and 5 can only be obtained from empirical equations as direct measurements of these values are not possible. We have used the empirical equations predicted by Neumann et al. (23) to calculate the magnitudes of γbc, γbw, and γcw from the experimentally determined values of γwv and θ made by water on the monolayers of clays and bacteriophages (θc and θb, respectively). The advantage of this method is that only one liquid can be used to obtain the necessary data. The values of θc and θb in conjunction with γwv were used to determine γcv (γ between clay and vapor) and γbv (γ between bacteriophage and vapor) by using the following equations:
cos θc )
(0.015γcv - 2.0)xγcvγwv + γwv , and γwv[0.015xγcvγwv - 1] cos θb )
(0.015γbv - 2.0)xγbvγwv + γwv γwv[0.015xγbvγwv - 1]
(6)
With the calculated value of γcv and γbv, γcw and γbw were determined from
γcw )
[(γcv)1/2 - (γwv)1/2]2 1 - 0.015(γcvγwv)1/2
γbw )
[(γbv)1/2 - (γwv)1/2]2 1 - 0.015(γbvγwv)1/2 (7)
The FORTRAN program written by Neumann et al. (23) was used to calculate the above values. Finally, the magnitude of γbc was determined from
γbc )
[(γbv)1/2 - (γcv)1/2]2
(8)
1 - 0.015(γcvγbv)1/2
The ∆GH was obtained from the summation of (∆GH)c-c, (∆GH)b-b, and (∆GH)c-b for each sorbate-sorbent combination. The ∆GEL was calculated from the ζ values of the suspensions of clays and bacteriophages. As indicated earlier, three models were used to determine the magnitudes of ∆GEL. The equations used for these models are discussed briefly below. The “plate-plate” (pp) model assumes that clay particles and bacteriophages behave as parallel plates with respect to each other. Verwey and Overbeek (24) used the linear supposition approximation to derive the following expression for ∆GEL between two infinite parallel plates separated by distance “h” in a 1:1 electrolyte solution:
( )
kT 8 ∆GEL ) (4πκ) π e
2
tanh
( ) ( )
eψ01 eψ02 -κh tanh e (9) 4kT 4kT
where is the permittivity of the liquid medium and is equal to the product of the dielectric constant of water (80.2 at 20 °C) and permittivity of vacuum (8.854 × 10-12 Fm-1), κ is the inverse Debye length (the Debye length is the thickness of the double layer), k is the Boltzmann’s constant (1.381 × 10-23 J K-1), T is the absolute temperature in K, e is the charge of the electron (1.602 × 10-19 C), and ψ01 and ψ02 are the surface potentials on each plate. The κ values for 1:1 electrolyte solution were obtained from van Oss (25). The surface potentials (ψ01 and ψ02) were determined from the respective ζ potentials, as
(
ψ0 ) ζ 1 +
z κz e R
)
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where z is the distance between the surface of the charged particle and the slipping plane (z ≈ 3-5 Å), and R is the Stokes radius of the particles (19). The above equation is applicable for relatively small ζ values (values < 60 mV) (19). A second equation for the “plate-plate” model, proposed by Hogg et al. (26), has also been used to determine ∆GEL:
(
1-
∆GEL ) 2κψ01ψ02 exp(-κh)
ψ201
ψ202
TABLE 2. Change in pH and EM with the Addition of Bacteriophages to Sorbents Before Combining Sorbent and Bacteriophage Suspensions sorbent pH EMa ψ0 (mV)
)
hectorite saponite kaolinite Norman clay T-2 MS-2 φX-174
+ exp(-κh) 2ψ01ψ02
1 - exp(-2κh)
(11)
In the “sphere-sphere” (ss) model all the interacting particles in the system are considered as spheres. The equation for this model was derived by Hogg et al. (26) to obtain the ∆GEL, as
∆GEL )
a1
{(ψ01 + ψ02)2 ln[1 + e-κh] +
4a2(a1 + a2)
(ψ02 - ψ01)2 ln[1 - e-κh]} (12)
where a1 and ψ01 are the radius and the surface potential of the larger sphere, and a2 and ψ02 are the radius and the surface potential of the smaller sphere. The “plate-sphere” (ps) model assumes that one of the interacting particles behaves as a sphere while the other behaves as a flat plate. Suresh and Waltz (27) have provided the expression for the interaction energy between a single sphere of radius R interacting with a flat plate in a 1:1 electrolyte solution as
∆GEL )
( )
16 kT R e
2
tanh
( ) ( )
eψ01 eψ02 -κh tanh e 4kT 4kT
(13)
All the above models were used to determine the ∆GEL for the c-b systems. The interaction energies for the b-b systems were calculated by using the “sphere-sphere” model only as the selected bacteriophages were, in general, spherical. The interaction energies for the c-c systems were determined by using both the “plate-plate” and “sphere-sphere” models.
Results and Discussion Sorption in the selected bacteriophage-clay suspensions has been quantified as the percentage decrease in the freely suspended bacteriophage over a period of 5 days. There was extensive sorption of the bacteriophages on the selected sorbents, ranging from 97% for T-2 on hectorite to 84% for φX-174 on saponite and kaolinite (Table 2). Similar observations were also made by other researchers for different bacteriophage-sorbent systems, such as sorption of bacteriophage T-4 on coal (28), MS-2 on different Indian soils (29), and φX-174 on soil (30). Among the selected clays, maximum sorption was observed on hectorite, followed by the Norman clay fraction. The high sorption on hectorite was due to its high surface area. When Lipson and Stotzky (31) compared sorption of reoviruses on kaolinite and montmorillonite, they observed that more reoviruses sorbed on montmorillonite (>95%) than on kaolinite (85%). The high sorption of bacteriophages on the Norman clay was probably due to its high BET surface area and its surface hydrophobicity (will be discussed later). The similarity in the sorption capacities of saponite and kaolinite despite their differences in CECs was probably due to the presence of positive charges on the edges of kaolinite particles. Among the selected bacteriophages, more T-2 sorbed on the selected clays, followed by MS-2 and φX-174. The lower sorption of φX-174 as compared to that of T-2 or MS-2 can be due to its higher IEP (12) as desorption rate 3612
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-0.86 -2.68 -1.94 -0.13 -1.90 -2.33 -2.22
6.4 7.2 6.14 5.8 6.7 6.6 6.8
-13.6 -34.6 -29.3 -1.94 -29.47 -36.50 -34.96
After Combining Sorbent and Bacteriophage Suspensions T-2 MS-2 OX-174 sorbent hectorite saponite kaolinite Norman clay a
amount amount amount pH sorbed (%) pH sorbed (%) pH sorbed (%) 8.3 7.6 6.2 6.9
97 ( 3 88 ( 2 92 ( 4 93 ( 3
8.5 7.2 6.2 6.7
94 ( 5 89 ( 5 85 ( 2 92 ( 3
8.4 7.7 6.3 6.9
93 ( 3 84 ( 6 84 ( 2 88 ( 5
The unit of EM is m s-1 V-1 cm.
increases with increase in IEP, and equilibrium sorption is a balance between adsorption and desorption. The high degree of sorption of T-2 can be explained by its size, shape, and surface hydrophobicity. As the surface area of T-2 is approximately 6 times more than either MS-2 or φX-174, the overall number of sorption sites available for interaction is also high. Therefore, the increase in sorption was due to an increase in the number of available surface charge sites and not due to an increase in overall charge strength. Furthermore, as indicated by Dowd et al. (12), the probability of a virus particle coming under the influence of attractive forces (such as van der Waals forces) is directly proportional to its surface area. Besides having a larger diameter, the tail of T-2 has 6 short spikes and 6 long fibers. It is possible that the presence of the fibers was responsible for a higher degree of sorption of T-2. Stenstro¨m and Kjelleberg (32) have observed that the presence of fimbriae on Salmonella typhimurium resulted in a higher degree of adhesion of these cells to clays as compared to adhesion of nonfimbriated cells. The role of tail fibers on the adsorption of phages to surfaces has also been reported by Kellenberger et al. (33). Sorption is also dependent on surface hydrophobicity, which is proportional to the respective water contact angles (θc and θb). Experimental results showed that θc for hectorite is the highest (57 ( 2.6°), with θc for hectorite > Norman clay fraction > kaolinite > saponite (Table 1). No statistically significant difference existed between Norman clay, kaolinite, and saponite. Such similar hydrophobicities along with higher surface areas of the Norman clay particles are responsible for their higher sorption capacity as compared to that of saponite and kaolinite. Among the bacteriophages selected for this study, T-2 was found to be the most hydrophobic, with a θb value of 96 ( 8.6°. The surface hydrophobicity of MS-2 was found to be less than that of T-2 and higher than that φX-174. Shields (4) had also found that MS-2 is more hydrophobic than φX-174 on comparing their relative hydrophobicities with hydrophobic interaction chromatography (HIC) using octyl-Sepharose and hydrocarbon partitioning. It is possible to relate the surface hydrophobicities of the bacteriophages to their nucleic acid content, as θb values have increased with an increase in the nucleic acid content. The pH and EM of clay and bacteriophage suspensions were monitored before and after mixing (Table 2). On reviewing the initial pH values of all the suspensions, we found that these values were greater than or equal to the
TABLE 3. Summary of Hydrophobic Interactionsa phage T2
MS2
φX-174
a
associated clay
γcw (mJ m-2)
γcv (mJ m-2)
γbw (mJ m-2)
γbv (mJ m-2)
γbc (mJ m-2)
∆GHc-b (mJ m-2)
∆GHc-c (mJ m-2)
∆GHb-b (mJ m-2)
∆GH (mJ m-2)
hectorite saponite kaolinite Norman clay hectorite saponite kaolinite Norman clay hectorite saponite kaolinite Norman clay
9.4 ( 0.47 0.7 ( 0.15 0.9 ( 0.80 1.1 ( 0.79 9.4 ( 0.47 0.7 ( 0.15 0.9 ( 0.80 1.1 ( 0.79 9.4 ( 0.47 0.7 ( 0.15 0.9 ( 0.80 1.1 ( 0.79
49.1 ( 1.45 66.1 ( 0.66 65.7 ( 2.63 64.9 ( 2.62 49.1 ( 1.45 66.1 ( 0.66 65.7 ( 2.63 64.9 ( 2.62 49.1 ( 1.45 66.1 ( 0.66 65.7 ( 2.63 64.9 ( 2.62
37.0 ( 24.88 37.0 ( 24.88 37.0 ( 24.88 37.0 ( 24.88 8.1 ( 6.71 8.1 ( 6.71 8.1 ( 6.71 8.1 ( 6.71 4.1 ( 1.85 4.1 ( 1.85 4.1 ( 1.85 4.1 ( 1.85
34.7 ( 24.69 34.7 ( 24.69 34.7 ( 24.69 34.7 ( 24.69 53.8 ( 4.46 53.8 ( 4.46 53.8 ( 4.46 53.8 ( 4.46 57.5 ( 3.57 57.5 ( 3.57 57.5 ( 3.57 57.5 ( 3.57
3.3 17.8 17.3 16.2 0.5 6.0 5.5 4.5 1.6 4.0 3.5 2.7
-43.2 -20.0 -20.7 -21.9 -17.1 -2.9 -3.5 -4.7 -11.9 -0.9 -1.5 -2.6
-18.9 -1.5 -1.8 -2.3 -18.9 -1.5 -1.8 -2.3 -18.9 -1.5 -1.8 -2.3
-74.1 -74.1 -74.1 -74.1 -16.2 -16.2 -16.2 -16.2 -8.2 -8.2 -8.2 -8.2
-136.2 -95.6 -96.6 -98.3 -52.2 -20.6 -21.6 -23.2 -39.0 -10.6 -11.5 -13.1
γlv ) 72.75 ( 0.3 mJ m-2.
TABLE 4. Summary of Electrostatic Interactions
phage T2
MS2
φX-174
associated clay hectorite saponite kaolinite Norman clay hectorite saponite kaolinite Norman clay hectorite saponite kaolinite Norman clay
∆GELc-b values obtained with different methods pp - VO pp - HHF ps ss (mJ m-2) (mJ m-2) (mJ m-2) (mJ m-2) 0.172 0.424 0.364 0.025 0.210 0.518 0.444 0.030 0.202 0.498 0.427 0.029
-0.476 0.173 0.197 -1.694 -1.069 0.279 0.128 -2.675 -0.920 0.275 0.162 -2.441
0.008 0.020 0.017 0.001 0.025 0.061 0.052 0.004 0.023 0.056 0.048 0.003
IEPs of the respective clays and bacteriophages. This indicates that the particles in suspensions were mostly negatively charged. After the bacteriophage suspensions were combined with the clay suspensions, a maximum of 2.1 units of pH increase was observed. Maximum increase was obtained when bacteriophages interacted with hectorite, followed by Norman clay, saponite, and kaolinite. Correspondingly, the highest amount of bacteriophages was sorbed on hectorite. The EM of all bacteriophages and clays indicated that they were negatively charged before sorption (Table 2). On comparing the EM of the clay minerals, we found that Norman clay was the least negative and saponite was the most negative. It might be possible that the presence of organic matter in the Norman clay fraction has masked the negative charges on the clay particles and/or there might be a predominance of clays with low negative charge. Among the 2:1 type clay minerals, hectorite was found to be less negative than saponite, and this is because the entire permanent negative charge of hectorite is located on its octahedral layer unlike that of saponite (13). The most negative among the bacteriophages was T-2, which was also the most hydrophobic, followed by φX-174 and MS-2. The values of interfacial tensions required to determine ∆GH were calculated from the θ values. Table 3 indicates the calculated values of γcv, γcw, γbv, γbw, and γbc as determined with the empirical equations proposed by Neumann et al. (23). Additional corroboration of the earlier-mentioned order of surface hydrophobicities of the sorbents and the sorbates can also be obtained by comparing the values of γcv and γcw. As an example, hectorite, which was found to be the most hydrophobic among all the sorbent samples from Table 1, has the lowest γcv and the highest γcw. Similar observation was also made with T-2. Additionally, the ease of sorption
0.002 0.014 0.012 -0.008 0.001 0.045 0.037 -0.034 0.002 0.042 0.034 -0.030
(∆GEL/∆G)cb (%)
∆GELc-c (mJ m-2)
∆GELb-b (mJ m-2)
∆GEL (mJ m-2)
∆G (mJ m-2)
0.02 0.10 0.08 0.01 0.14 2.13 1.49 0.07 0.19 6.81 3.32 0.13
0.08 0.49 0.36 0.00 0.08 0.49 0.36 0.00 0.08 0.49 0.36 0.00
0.006 0.006 0.006 0.006 0.024 0.024 0.024 0.024 0.021 0.021 0.021 0.021
0.10 0.52 0.39 0.01 0.13 0.58 0.44 0.03 0.13 0.57 0.43 0.03
-136.08 -95.04 -96.18 -98.28 -52.04 -20.04 -21.16 -23.19 -38.87 -10.01 -11.11 -13.04
of a bacteriophage on a clay sample can be deduced from the magnitudes of γbc. However, caution should be used before drawing definite conclusions as the values of γbc are only partially responsible for dictating the sign and magnitude of the total ∆G. All ∆GH values for sorption of bacteriophages on clays are negative (Table 3), indicating that the hydrophobic interactions favor sorption of bacteriophages on the selected sorbents. On comparing the ∆GH values for different interacting systems, it was found that the magnitudes of (∆GH)b-b were considerably higher than (∆GH)b-c and (∆GH)c-c and significantly affected the values of ∆GH. As was seen earlier, the surface hydrophobicity of T-2 was highest, and correspondingly that has led to high (∆GH)b-b. This implies that maximum aggregation should be observed between T-2, followed by MS-2 and φX-174. The results obtained with particle size analysis (data not presented in this paper) corroborated this inference. Similarly, maximum aggregation should be between hectorite particles, which was followed by Norman clay fraction, kaolinite, and saponite. On comparing the (∆GH)c-b for a particular clay, we found the magnitudes of (-∆GH)c-b of T-2 > MS-2 > φX-174. Correspondingly, for any bacteriophage, the (-∆GH)c-b for hectorite > Norman clay > kaolinite > saponite. Though considerable differences exist between hectorite and the other clay particles, the values of (-∆GH)c-b for Norman clay, kaolinite, and saponite are similar. Overall, maximum (-∆GH)c-b was observed when T-2 sorbed on hectorite, and minimum (-∆GH)c-b was observed when φX-174 sorbed on saponite. As discussed earlier, we have used three configurations of the sorbent-sorbate system to determine the magnitudes of (∆GEL)c-b (Table 4). Irrespective of the models used, the VOL. 33, NO. 20, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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calculated value of (∆GEL)c-b is considerably less than that of (∆GH)c-b, indicating that (∆GH)c-b dictates the process of sorption. Among the three models and four equations selected to determine (∆GEL)c-b, the plate-sphere configuration provided the most probable values. The physical shapes of the bacteriophages considered were spherical, while in general, the clay particles are usually considered to be shaped as platelets. Furthermore, on comparing the mean particle sizes of the clays and the bacteriophages (results not presented here), we found that the clay particles were, on an average, 29 times larger than the bacteriophages. This implies that a bacteriophage would view the clay particles as flat plates. Also, the values of (∆GEL)c-b obtained with the “platesphere” model are between the two extreme values obtained by the models based on the “sphere-sphere” and “plateplate” configurations. Therefore, we have used the values obtained with the “plate-sphere” model to determine the total ∆G. The (∆GEL)b-b were calculated by considering that these phages are shaped as spheres (eq 12), and (∆GEL)c-c was obtained with eqs 9, 11, and 12. The equation proposed by Verwey and Overbeek (24) was preferred over other equations used for determining (∆GEL)c-c as this equation produced a higher value of (∆GEL)c-c. Calculated results showed that maximum repulsive electrostatic forces were present between φX-174, followed by MS-2 and T-2. Correspondingly, the magnitude of the repulsive electrostatic forces was highest with saponite, which was > kaolinite > hectorite > Norman clay. The low repulsive electrostatic force for Norman clay was expected as the negative charge on the Norman clay particles was the least among the selected sorbents (Table 2). On comparing the (∆GEL)c-b for any particular clay, we found that the maximum repulsive force was obtained with φX-174, followed by MS-2 and T-2. Correspondingly, for any particular bacteriophage, the maximum repulsive force was obtained with saponite as the sorbent, followed by kaolinite, hectorite, and the Norman clay fraction. The magnitude of total (-∆G) ranged between approximately 136.08 mJ m-2 for sorption of T-2 on hectorite and 10.01 mJ m-2 for sorption of φX-174 on saponite. For a particular bacteriophage, the order of (-∆G) is hectorite > Norman clay > kaolinite > saponite, and for a particular clay, the order of (-∆G) is T-2 > MS-2 > φX-174. These distributions compare well with the amount of bacteriophages sorbed on clays at equilibrium conditions, as indicated in Table 2. Calculations based on the plate-sphere configuration have also shown that electrostatic forces form a small fraction of the total change in free energy, with a maximum of 6.81% for the φX-174-saponite system. Therefore, hydrophobic interactions dominate during sorption of the selected bacteriophages on the selected clays. The present study was intended to provide a thermodynamic explanation of the sorption of bacteriophages on clay surfaces. The study shows that determination of surface hydrophobicities and surface potentials is essential in the development of any model that predicts the sorption of such microorganisms on soil systems.
Acknowledgments Although the research described in this article has been funded wholly or in part by the U.S. EPA, it has not been
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subjected to the Agency’s peer and administrative review and therefore may not necessarily reflect the views of the Agency, and no official endorsement may be inferred.
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Received for review November 6, 1998. Revised manuscript received March 12, 1999. Accepted July 28, 1999. ES9811492
