Environ. Sci. Technol. 1991, 2 5 , 2088-2095
Bacteriophage Adsorption during Transport through Porous Media: Chemical Perturbations and Reversibility Roger C. Bales," Stephen R. Hinkle,+ Thomas W. Kroeger,t and Kristen Stocking§ Department of Hydrology and Water Resources, University of Arizona, Tucson, Arizona 8572 1
Charles P. Gerba Department of Microbiology and Immunology, University of Arizona, Tucson, Arizona 8572 1
In a series of seven column experiments, attachment of the bacteriophage PRD-1 and MS-2 to silica beads at pH's 5.0-5.5 was at least partially reversible; however, release of attached phage was slow and breakthrough curves exhibited significant tailing. Rate coefficients for attachment and detachment were on the order of lo4 and 10-6-10-4 s-l, respectively. Corresponding time scales were hours for attachment and days for detachment. The sticking efficiency ( a )for phage attachment was near 0.01. The rate of phage release was enhanced by raising pH and introducing surface-active chemical species, illustrating the importance of chemical perturbations in promoting biocolloid transport. In a series of batch experiments, MS-2 adsorbed strongly to a hydrophobic surface, octadecyltrichlorosilane-bonded silica, a t both pH's 5 and 7. Adsorption to the unbonded silica at pH 5 was linear, but was 2.5 (with Ca2+)to 0.25% (without Ca2+)of that to the bonded surface. Neither MS-2 nor PRD-1 adsorbed to unbonded silica at pH 7. Hydrophobic effects appear to be important for adsorption of even relatively hydrophilic biocolloids. Introduction The fate of viruses in groundwater is governed by attachment to immobile substrates, generally referred to as adsorption, and by inactivation (1). In a study of over 100 groundwater samples, Yates et al. (2) found temperature to be the only measured water characteristic significantly correlated with viral inactivation. Gerba (3) cited extensive evidence to the effect that sorbed viruses are generally protected from inactivation relative to free viruses. Several factors contribute to the adhesion of viruses and other colloids to soil particles, including electrostatic attraction and repulsion, van der Waals forces, covalent-ionic interactions, hydrogen bonding, and hydrophobic effects. Murray and Parks ( 4 ) showed that free energies for adsorption of poliovirus to a variety of metal oxides corresponded well with potentials predicted by electrostatic theory. Both chemical and electrostatic interactions could be important in observations that divalent cations were more effective than monovalent cations in promoting adsorption of poliovirus to membrane filters (5). Reported virus adsorption and transport experiments have generally not involved well-characterized surfaces, but have focused on specific soils or groundwater media (6-11). These results, while quantitative, have failed to give general insight into how chemical properties of the virus, collector, and aqueous solution control attachment and release, In addition, the reversibility of virus adsorption has been studied for only a few cases ( 4 ) . While several t Present address: USGS, Water Resources Division, 10615 SE Cherry Blossom Dr., Portland, OR 97216. *Present address: STS Consultants, Ltd., 11425 W. Lake Park Dr., Milwaukee, WI 53224. $Present address: Stearns & Wheler, One Remington Park Dr., Cazenovia. IVY 13035.
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Environ. Sci. Technol., Vol. 25, No. 12, 1991
mathematical models are available for describing transport of viruses and other colloids in soil and groundwater ( l ) , all lack data for validation. The research described in this paper is part of our ongoing studies of virus attachment and transport in natural waters. Our first objective was to determine the effect of pH on the attachment of MS-2 and PRD-1 to well-characterized silica and hydrophobic surfaces. We chose MS-2, PRD-1, and silica as their surface chemical properties are well-known, offering the potential to determine the factors controlling the degree of adsorption. We examined the importance of Ca2+concentration and temperature in influencing MS-2 adsorption on silica at one pH. A second objective was to demonstrate the reversibility of bacteriophage adsorption and to determine the effect of chemical perturbations on the rates of desorption. Our third objective was to test equilibrium, first-order, and two-site colloid transport models using the quantitative data and parameter estimates developed for the chemical conditions studied. Materials and Methods B a t c h E x p e r i m e n t P r o c e d u r e s . A set of pH 5 and pH 7 batch experiments using silica and surface-modified silica was undertaken to determine the role of pH and hydrophobicity in MS-2 adsorption. Adsorption isotherms were run a t 4 "C in order to minimize viral inactivation. A set of pH 5 batch experiments was also done a t 24 "C to investigate the effect of temperature under one set of conditions. Experiments consisted of placing 0.75 g of unbonded or 0.100 g of hydrophobic bonded silica (Min-u-si1 30, PGS, Berkeley Springs, WV) in sterilized 4-mL glass vials with Teflon cap inserts. Phage stock was diluted in buffer to the desired titer and 2-mL aliquots were pipeted into silica-containing vials and silica-free control vials. Vials were hand-mixed and placed on a rotating Labquake Shaker (Labindustries, Berkeley, CA) at 12 rpm. One set of vials was assayed to give starting values. After 60 (unbonded) and 90 (bonded) min, vials were centrifuged at 3500 rpm and 1-mL aliquots of the supernatant removed for assay. Concentrations were corrected for inactivation and isotherms plotted. The pH of each vial was measured following an experiment; samples that varied more than 0.05 pH unit from the set pH were discarded. Vials, caps, and inserts were autoclaved for 20 min after each experiment and then cleaned by soaking vials and inserts in RBS-35 detergent (Pierce Chemical Co., Rockford, IL) for 24 h followed by rinsing and soaking in distilled water for 2 h. Vials were sterilized by baking for 30 min at 300 "C; caps and inserts were autoclaved for 20 min. In four sets of experiments, mean phage removals (k sample standard deviation) in sets of vials shaken for 30, 60, and 90 min were 97.7 f 1.7,99.3 f 0.4, and 97.9 f 1.370, respectively. As there was no statistically significant difference in adsorption a t the three times, subsequent
0013-936X/91/0925-2088$02.50/0
0 1991 American Chemical Society
teflon tubina
perista1ti.c pump
peristaltic tubing
I input reservoir
Figure 1 Column
Input sample test tube
1-
emuent sample test tube
experimental setup
experiments had a contact time of 60 min. Column Experiment Procedures. Four continuousflow column experiments were conducted with the bacteriophage PRD-1 at p H s 5.5 and 7.0 and three with MS-2 at pH 5. Experiments were done at 4 "C using a 15 cm X 0.9 cm i.d. precision-bore glass chromatography column (Spectrum Medical Industries, Inc., Los Angeles, CA) packed with 45-90-flm glass beads. Buffers and phage were stored in glass reservoirs and fed through the column by a peristaltic pump (Ismatec, Cole Parmer Instrument Co., Chicago, IL). Column fittings were Teflon, and Teflon tubing was used everywhere in the system except for a length of Tygon tubing in the pump. Phage were pumped through the peristaltic tubing for a minimum of 1 (MS-2) or 18 h (PRD-1) prior to beginning an experiment to reduce subsequent tubing losses. Columns were packed with new beads for each experiment by the tap and fill method (12). After columns were flooded from the bottom to remove air, a minimum of 40 pore volumes of the buffer was passed through the column prior to introducing bacteriophage to the column. In order to take column inlet samples throughout an experiment without disrupting the flow, a parallel feed tubing was set up (Figure 1). Feed-reservoir titers (from 0.9 t o 3.9 X lo5 plaque-forming units (PFU) mL-') remained constant during an experiment, within analytical uncertainty. Inlet and outlet samples were collected in sterile glass test tubes for 10-20 min and assayed the same day. Desorption solutions were fed to the column with separate, uncontaminated tubing. Outlet pH's were within 0.06 pH unit of the target values, with the exception of the pH 8 and pH 9 Tween-80 detergent and beef extract eluent phases of experiment 7, when pH's dropped from 8.00 to 7.86, and from 9.00 to 8.81. Few phage were eluted with these solutions, however. Flow rates, monitored continuously, remained fairly steady; occasional excursions by as much as +9.5% were quickly reset to target values. Column pore volumes were determined by flooding with 0.01 M NaCl overnight and then introducing a 0.03 M NaCl solution and monitoring outflow conductivity. When outlet conductivity became constant, 0.01 M NaCl was again introduced at the top of the column and conductivity monitored. Pore volumes calculated by conservative tracer breakthrough were greater than those calculated by weighing empty and fdled columns by as much as 4.6%. This difference may be due in part to variability in bead specific gravity, reported by the manufacturer to be 2.45-2.50 g cm 3; we used 2.475 g cm?. Materials a n d Chemicals. Sodium phosphate was used to buffer pH, with Ca added as CaC1,. In experiments 1 and 2 (pH 7.01, 0.08 M NaCl was added to raise ionic strength. Stock solutions of beef extract and Tween 80 detergent were made by adding deionized water to beef extract V (Becton Dickinson and Co., Cockeysville, MD) and polyoxyethylene (20) sorbitan monooleate (Tween 80) (J. T. Baker, Inc., Phillipsburg, NJ) to give 1.0 or 2.5%
solutions. These solutions were autoclaved for 20 min and cooled to 4 "C, and the buffer was made with NazHP04. Tris-buffered saline solution was prepared by dissolving 63.2 g of Trisma base (Sigma Chemical Co., St. Louis, MO), 163.6 g of NaC1,7.46 g of KCI, and 1.13 g of Na2HP04in 1600 mL of distilled water. To make experimental Tris solutions, 32 mL of this saline solution was added to 368 mL of distilled water; the mixture autoclaved for 20 min and distributed with a sterile Cornwall pipet in 2.7-mL aliquots into sterile glass test tubes that were then capped with sterile rubber stoppers and stored a t 4 "C. Trypticase soy broth (TSB) host medium was prepared by dissolving 30 g of Tryptic soy broth powder (Gibco Laboratories, Madison, WI;Difco Laboratories, Detroit, MI) in 1 L of distilled water. Aliquots (3 and 100 mL) of TSB were then dispensed into glass test tubes and Erlenmeyer flasks, respectively; these were capped, autoclaved for 20 min, and stored at 4 "C. Trypticase soy agar (TSA) overlay medium was prepared by dissolving 30 g of TSA powder and 10 g of Bactoagar (Difco Laboratories, Detroit, MI) in 1 L of distilled water. Three-milliliter aliquots were delivered into 14-mL glass test tubes; these were then capped, autoclaved for 20 min, and stored at 4 OC. TSA plates for phage growth and assay were prepared by dissolving 40 g of TSA in 1 L of distilled water, autoclaving the solution for 20 min, cooling it to 50 "C, and dispensing in 10-mL aliquots into sterile, 100 mm X 15 mm plastic Petri dishes. TSA plates were cooled overnight and stored at 4 "C in their original plastic bags. Bacteriophage a n d Assays. Bacteriophage MS-2 is an icosahedral phage with a diameter of
[email protected] nm (13) and pHiepof 3.9 (14). The surface of MS-2 contains hydrophobic and hydrophilic portions (14). MS-2 (ATCC 15597 B-1) was obtained from the University of Arizona Department of Microbiology and Immunology culture collection. Bacteriophage PRD-1 is an icosahedral lipid phage with a diameter of 62 nm (15). PRD-1 was obtained from JuiCheng Hsieh (Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ). Coliphage MS-2 was grown and assayed in Escherichia coli (ATCC 15597) and PRD-1 in Salmonella typhimurium LT2. Both were assayed by the plaque-forming-unit method described by Adams (16). Phage stocks were prepared by covering infected host bacterial lawns with 5-10 mL of Tris-buffered saline solution for 2 h a t room temperature. The buffer was then poured from the Petri dishes and collected in 250-mL centrifuge bottles. The eluent solutions were centrifuged at 10000 rpm for 10 min and removed, leaving behind and agar pellet. Bacterial fragments were removed by filtration through a 0.45-pm membrane filter (Millipore Corp., Bedford, MA). The phage solution was then purified by ultracentrifugation for 2 (MS-2) or 1.5 h (PRD-1) at 25000 rpm. The phage pellet was resuspended in Tris buffer by pipeting action and the concentrated phage removed and diluted to 10 mL with Tris buffer. Phage titers between 10" and lOI3 pfu mL-' were obtained. For microelectrophoretic experiments, PRD-1 was further purified in a sucrose gradient. Concentrated PRD-1 stock was placed a t the top of a set of filter-sterilized sucrose solutions with bands made of 20, 10, and 5% sucrose by weight in deionized water. The gradients were placed in a ultracentrifuge for 105 min at 45 000 rpm, after which the pellet was resuspended in 1 mL of buffer. A titer of approximately 10" pfu mL-' resulted. Samples to be assayed were sequentially diluted with Tris (or phosphate buffer) to appropriate concentrations, Environ. Sci. Technol., Vol. 25, No.' 12. 1991
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usually 100-300 pfu mL-', by pipeting 0.3 mL of diluent into 2.7 mL of Tris (or phosphate buffer) and vortexing the mixture for 5 s. One milliliter of host culture and 0.1 mL of the diluted sample were added to each of two to six overlay media tubes, the solutions vortexed for 5 s, and the contents poured onto TSA plates and allowed to solidify for 15-30 min. The plates were incubated overnight and individual plaques counted with a C-100 automatic plaque counter (New Brunswick Scientific Co., New Brunswick, NJ). Silica Sorbents. The silica for batch experiments (Min-u-si1 30, PGS) was first washed by placing 60 g of Min-u-si1 and 200 mL of 0.1 M NH40H in each of four 250-mL Teflon centrifuge bottles. The bottles were shaken for 5 min and centrifuged for 5 min a t 5000 rpm. Supernatants were discarded, and the process was repeated with 200 mL of 1.0 M HCl, then with 200 mL of 0.5 M HCl, and finally with eight rinses of deionized water. The final rinse pH was -5. The silica was then transferred to a glass beaker, covered with a glass watchglass, and oven-dried at 100 "C. To prepare organosilane-modified silica, 100 g of the cleaned Min-u-si1 was suspended in 150 mL of deionized water, 0.125 mL of octadecyltrichlorosilane added, and the resultant solution stirred with an eye-shaped magnetic stir bar for 2 h. The mixture was dried at 110 "C for 24 h, at which time 50 g of the bonded Min-u-si1 was washed with 150 mL of pentane. The mixture was centrifuged, the pentane discarded, and the procedure repeated with another pentane rinse, two methanol rinses, two rinses in 1 M HC1, and seven deionized water rinses, at which point the supernatant was at a pH of - 5 . The bonded Min-u-si1 was oven-dried in a glass beaker. In order to maintain control experimental conditions, unbonded silica used in batch experiments for comparison with the bonded silica was subjected to the same additional rinsing as the bonded silica. All silicas were stored in sterile glass jars with ground-glass or solid PTFE stoppers. The silicas for column experiments, Spheriglass 2530 beads (Potters Industries, Inc., Hasbrouck Heights, NJ), were washed to remove soluble oxides of calcium and sodium and other impurities from the surfaces. Beads were cleaned by first rinsing with distilled water, with ",OH, and then with deionized water until the rinse water pH dropped below 11. Beads were then refluxed in 2 M HC1 for 4 h, rinsed in deionized water, and refluxed in fresh 2 M HC1 for 2 h more; this second refluxing was repeated a third time with fresh HCl. The beads were next rinsed in deionized water until the rinse water pH rose above pH 4 and then refluxed in deionized water for 1 h. This last step was needed as electrophoretic mobility measured on beads following the acid refluxing was slow to reach a constant value. Last, the beads were rinsed in deionized water and oven-dried overnight a t 200 OC. Surface area, determined by single-point N2 adsorption on a Model QS-10 Quantasorb Sorption System (Quantachrome Corp., New York), was 2.0 m2 g-' for unbonded and 1.7 m2 g-' for bonded Min-u-si1 and 0.060-0.066 m2 g-' for beads. Bonded and unbonded silica were analyzed for organic carbon by elemental pyrolysis (Desert Analytics, Tucson, AZ). The mass-fraction organic carbon ),fC for unbonded Min-u-si1 was 0.000 04; for bonded silica it was 0.000 18. Assuming a silanol surface site density of 4.6 nm-2 (17) and the manufacturer's reported size distribution, this corresponds to a surface coverage of 0.065. The mass-fraction organic carbon for both batches of beads was 0.00002; the instrument error for measurements was *O.OOO 05. 2090
Environ. Sci. Technol., Vol. 25, No. 12, 1991
2
co. m-
-
M
log
c, pfu/ml
e d a
unbonded
bonded
6
2
0
4
0
6 8 log c. pfuimL
10
Figure 2. Batch experiment isotherms for MS-2 adsorption in (a, top) calcium phosphate buffer at pH 5 at two temperatures with unbonded silica and (b, bottom) calcium-free phosphate buffer at pH's 5 and 7 at 4 "C with bonded and unbonded silica. Each point is from a single vial and blank.
Electrophoretic mobility was measured using a Rank Brothers (Cambridge, England) Mark I1 microelectrophoresis apparatus equipped with a flat cell and platinum electrodes. A measurement was made by timing at least five particles at each polarity (constant voltage) at each stationary level. Reported mobility is the average velocity divided by the voltage gradient. The large beads used in experiments settled out of the field of view before measurements could be made. Smaller beads (Spheriglass 2900; 1 5 3 pm) were washed by the same process as the larger beads and measurements made on the nonsettling fraction. For phage measurements, a cylindrical cell and a He-Ne laser (Scientifica-Cook, Ltd., London, England) were used. Purified PRD-1 was suspended in calcium phosphate buffer M Ca) to give approximately 1.5 x 1O'O pfu mL-'.
Res u 1t s Batch Experiments. Isotherms for MS-2 adsorption to Min-u-si1 in calcium phosphate buffer (Figure 2a) were linear, with the partition coefficient [ (pfu,,,bed/gsorbent)/ (pfuwater/mLwater) = S / C = K,] for the 24 "C isotherm (K, = 580 cm3 g-l) twice that for the 4 "C isotherm ( K , = 270 cm3 g-l). Standard errors were 30 for each. Freundlich isotherm (C = KSn)exponents for the respective experiments were 0.99 and 1-01. In calcium-free phosphate buffer, MS-2 adsorbed to unbonded Min-u-si1 at pH 5 but did not adsorb a t pH 7; it adsorbed to bonded Min-u-si1 at both pH 5 and pH 7 (Figure 2b). A one-way, completely random analysis of variance (ANOVA) (Costat Statistical Software, CoHort Software, Berkeley, CA) with C as the variate showed the variation between the pH's 5 and 7 bonded silica to be not statistically significant (P = 0.25). MS-2 adsorption to bonded Min-u-si1was 400 times greater than to unbonded Min-u-sil. Fitting a Freundlich isotherm to the bonded and unbonded silica isotherms gave K s of 41 000 and 100, respectively, with r2 values of 0.97 and 0.84; values of n were 0.92 in each case. Linear isotherm partition coefficients (K,) were 8300 and 6.6 cm3 g-', respectively, with rz values of 0.90 and 0.53; respective standard errors were 800 and 0.6.
Table I. E q u i l i b r i u m Model Fits to D a t a
fitted parameters exp"
u,cm
3 ads 3 des
0.0037 0.0037 0.0037 0.0037 0.0035 0.0035
4 ads 5 ads
0.0057 0.0039
1 ads 1 des 2 ads 2 des
ssqc
Kpl,cm3 g-'
D, cm2 s-'
MS-2 Experiments at pH 5.0 and 4 "C 2.71 f 0.10 4.51 f 0.78 0.81 f 0.02 109 f 47 1.55 f 0.06 6.47 f 1.43 0.645 f 0.016 7.37 f 1.18 2.91 f 0.16 1.79 f 0.37 0.964 f 0.016 326 f 202
0.1571 0.0956 0.1087 0.0181 0.1101 0.1354
0.376
0.0127 0.00053 0.0089 0.00778 0.0295 0.00016
PRD-1 Experiments at pH 7.0 and 4 O 192 f 107 1.00 f 0.02 221 f 99 1.02 f 0.01
0.0650 0.0505
Rb
Co, pfu mL-'
s-1
2.62 x 104 3.49 x 104 1.59 x 104
1.05 x 105 9.15 x 104
P
0.121 0.420
C
3.1 x 10-5 2.7 x 10-5
"Adsorption (ads) and desorption (des) parts of breakthrough curves listed separately. bParameter estimate f standard error. 'Sum of squared errors. l,J,
Exp
;
EauiL model fits
,
ExP 1
1.5~ 1
.
.
0.5
0
0
-3
2
4
6
8
1
0
PH
10
1.5
11
Exp3
Flgure 3. (a, top) Mobility of Min-u-si1 as a function of pH in calciumfree phosphate buffer and of silica beads as a function of pH in phosphate buffer, both with M calcium (0)and without calcium (*). (b, bottom) Mobility of PRD-1 as a function of pH in phosphate buffer with lo-' M calcium.
0.5
OO
At pH 5 and 4 O C , adsorption to unbonded silica was on the order of 10-fold or more greater with (Figure 2a) versus without (Figure 2b) Ca2+, suggesting either a charge-neutralization or cation-bridging role for the ion. Both unbonded and bonded Min-u-si1 are negatively charged in the pH range studied (Figure 3a), with bonded silica being slightly less negative than unbonded silica. Column Experiments. Experiments 1-3 (MS-2, p H 5.0) show a slowly rising breakthrough curve to C/Co of 1.0 after 4.5-5.0 pore volumes (Figure 4). The jaggedness of the curve is due to the variability of the plaque assay procedure. Replicate assays of a sample differed by an average of 25%. The first phage were detected after 0.9-1.0 pore volume had passed through the column. There was bacteriophage retention in the column, as C/Co = 0.5 occurred after 2.2, 1.4, and 2.0 pore volumes in experiments 1-3, respectively. It is thought that the earlier breakthrough of the bacteriophage front in experiment 2 was due to not fully flushing phage from experiment 1 out of the column; new packing was used for each subsequent experiment. All three experiments showed steep declines in C/Co -1 pore volume after the feed was switched to the bacteriophage-free solution. The fractions of bacteriophage recovered in desorption were 0.85 and 0.69 in experiments 1 and 3, respectively; these include perturbations after 10 pore volumes, which were not shown on
5
Pore volume
.
5j .
R
j
. . : 5
10
Pore volume
Figure 4. Breakthrough curves for experiments 1-3 with MS-2 at pH 5.0. Experimental conditions are given in Table I. Pulse values (number of pore volumes of feed solution containing phage) were 4.6, 4.7, and 4.3 for experiments 1-3, respectively (vertical dotted lines). (a, left) Data with equilibrium model fits; parameters given in Table I. (b, right) First-order model fits; parameters given in Table 11. Vertical dotted lines indicate points at which column input conditions were changed.
Figure 4. Slow desorption apparently contributed to the fractions being less than 1.0. Uncertainty in the plaque assay, especially for short-term peaks, could also be a factor. The second MS-2 experiment was carried out without changing column packing, so mass balance was not calculated. After 20 pore volumes of desorption at pH 5 in experiment 3, C/Co dropped below 0.01; switching to a pH 7.0 Ca-free buffer resulted in a small peak of C/Co = 0.06 (not shown). After 4 days (158 pore volumes) of desorption in experiment 2 at pH 5.0, changing the buffer pH to 7.0 resulted in an outlet pulse with C/Co = 0.14. PRD-1 did not adsorb at pH 7.0 (experiments 4 and 5, Figure 5 and Table I) for either the 64- (0.0057 cm 9') or 94-min (0.0037 cm s-l) residence time. PRD-1 adsorbed slowly at pH 5.5 (experiments 6 and 7, Figure 6), but did not reach C/Co = 1.0; upon switching to bacteriophage-free Environ. Sci. Technol., Vol. 25, No. 12, 1991
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extract and (ii) desorption with the same mixture a t pH 9. After 135 pore volumes of desorption, 76% of the adsorbed phage was eluted.
pore volumes Figure 5. Breakthrough curve for experiments 4 and 5 with PRDl and conservative salt tracer; pH 7.0 Experimental conditions are given in Table I .
0
50
100
150
10-3
50 100 150 Pore volumes Figure 6. Breakthrough curve for experiments 6 and 7 with PRD-1: M Ca2+, (a, top) adsorption at pH 5.5 in phosphate buffer with u = 0.0039 cm s-'; Co= 2.15 X lo5 pfu mL-'; (b, bottom) adsorption at pH 5.5 in phosphate buffer with M Ca2+, u = 0.0039 cm s-'; C o = 3.89 X lo5 pfu mL-'. See text for explanation of desorption sequences, which began after 44 and 28 pore volumes of input pulse (number of pore volumes of feed solution containing phage) in experiments 6 and 7, respectively. Vertical dotted lines indicate points at which column input conditions were changed.
0
buffer the phage slowly desorbed. PRD-1 is negatively charged a t pH's 4.5-7.5 in a phosphate buffer in the presence of low4M Ca2+ (Figure 3b). In experiment 6, there was little desorption with calcium phosphate buffer a t pH 5.5, but a pulse with C / C , = 14 occurred upon switching to a calcium-free pH 7.0 phosphate eluent. A smaller but significant pulse occurred upon switching to a pH 7.0 calcium-free buffer with 1% beef extract. The beef extract addition provided two chemical changes, a higher ionic strength and introduction of organic, largely protein, molecules. Mass balance calculations indicate that 82% of the sorbed phage was eluted during desorption (including perturbations). The unaccounted-for bacteriophage were apparently either resistant to desorption or were inactivated. Phage were still detaching after 77 pore volumes of desorption. There was little desorption in experiment 7 with calcium phosphate buffer at pH 5.5 (pore volumes 28-36), or with calcium-free phosphate buffer a t pH 5.5 (pore volumes 36-52). A change from p H 5.5 to 8.0 eluent produced a large bacteriophage pulse, suggesting that changes in pH rather than changes in calcium concentration were responsible for the large desorption pulse seen in experiment 3. Only small pulses were observed in subsequent desorption steps with (i) calcium-free buffer a t pH 8 with 1.0% Tween 80 detergent (a surfactant) and 2.5% beef 2092
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Discussion Interpretation of Chemical Effects. The greater MS-2 adsorption to bonded silica is apparently due largely to hydrophobic factors rather than any surface charge reduction from bonded carbon chains reducing the total number of silanol sites. Note that the difference in surface charge of the bonded silica at pH's 5 and 7 is greater than the difference in surface charge between bonded and unbonded silica a t pH 5 (Figure 3a). The greater MS-2 adsorption to unbonded silica a t pH 5 versus pH 7 is consistent with electrostatic repulsion being important in that the surface charges of both the colloid and the immobile surfaces are nearer zero a t lower pH. Thus MS-2, which is relatively nonadsorbing in sandy soil (11), should be retained in soil with an appreciable organic matter content. Other indications of the importance of surface hydrophobicity come from batch experiments with the bacteriophage (PX-174 and five different soils in which there was a correlation between adsorption and soil organic carbon content (18). Farrah et al. (19) observed that a t pH 9, antichaotropic salts, which act to increase the structure of water, retarded or prevented elution of poliovirus from membrane filters. Chaotropic salts, which disrupt the structure of water, antagonized the effects of antichaotropic salts at pH 9. At pH 4 neither salt had any effect. It was concluded that electrostatic interactions dominate poliovirus adsorption to membrane filters at pH 4, but that hydrophobic interactions dominate a t pH 9. Similar results were also found with bacteriophage MS-2 (20). MS-2 adsorption to unbonded silica is apparently endothermic, and the temperature effect was opposite that expected for ion adsorption. Hydrophobic interactions are more stable a t higher temperatures (21);if sorption was partially driven by a partitioning of hydrophobic portions of phage onto silica surfaces by exclusion from water, sorption could increase with temperature. We have not observed this behavior for sorption of hydrophobic molecules onto the same bonded surfaces, however (22). Zittle (23) cited evidence that proteins tend to sorb to a greater extent a t higher temperatures than they do at lower temperatures. He noted that the processes of protein unfolding is endothermic and suggested that protein sorption may involve such unfolding on surfaces. If virus adsorption involves the unfolding of surficial protein groups, virus uptake should increase with increases in temperature. In that case, our temperature results would be important for virus and bacteria transport but would not be applicable to abiotic colloids. Different rates of adsorption a t 4 and 24 "C could also contribute to the observed temperature dependence. Zittle (23) noted that the rate of protein adsorption is faster at higher temperatures. It is possible that experimental variability could have masked nonequilibrium in the time curves, creating a situation in which kinetics resulted in more sorption a t 24 "C than a t 4 "6. However, that seems unlikely given the small changes a t later times and the time scales observed in the column experiments. Results of the column experiments are also consistent with electrostatic repulsion being important. Strong adsorption a t low pH and no adsorption a t higher pH is expected where both the colloids and immobile surface have low pHi,is (isoelectric pH's). The desorption of MS-2 and PRD-1 with an increase in pH is also consistent with the greater repulsion further away from the pHiep. However, the small pulses that followed pH changes suggested
that complete desorption requires significant chemical perturbations. There was greater MS-2 adsorption to unbonded silica with versus without Ca present; adsorption of PRD-1 was also lower in experiment 7 as compared with 6 (lo4 versus IO4 M). Assuming a bead density of 2.5 g cm-3 and a mean bead diameter of 2 Fm, the surface area of the beads is estimated to be 1.2 m2 g-l. Assuming a 100 mg L-I suspension and 4.6 silanol groups/nm2 of silica suggests that the concentration of surface silanol groups was lo4 M. M Thus, sufficient Ca2+was present at both lo4 and to influence phage adsorption. These are consistent with Mix (24),who proposed that cations may act as complexing agents in attachment, forming salt bridges between viruses and surfaces. Modeling and Kinetic Influences. We fit experimental breakthrough curves to three different one-dimensional advection-dispersion models (equilibrium, first-order kinetic, and two-site kinetic models) in order to estimate the magnitude of adsorption and time scales for reaching equilibrium. Governing equations for the most general model used, one-dimensional transport in a porous media with two types of adsorption sites, one of which is kinetically limited, have been given by various investigators (25, 26):
P = Lu/D
indicates the time scale for dispersion divided by the residence time in the system. The dimensionless masstransfer coefficient is a Damkohler number (27, 28): physical time scale = -L/u klL =w = (6) chemical time scale l / k l u where L is the length of the column. When w > 100, local equilibrium applies, and as w drops below about 0.1-0.5, adsorption is too slow to observe and the solute appears to be conservative. A fourth parameter, 8, related to the ratio of equilibrium to total adsorption, can be defined by
For no type 1sites, a three-parameter (Kpl = 0; p = 1/R) model (first-order model) can be used. For no type 2 sites (p = 1/R and w 1 loo), a two-parameter equilibrium model results. Parameter values were estimated using the nonlinear-least-squares curve-fitting routine of van Genuchten (29),with constant-flux lower boundary condition. Applying the equilibrium model to three NaCl breakthrough curves accompanying experiments 1-3, we found dispersion to be linearly related to pore-water velocity ( D of 4.8 X lo”, cm2 s-l for velocities of 0.0014, 8.7 X and 18.0 X 0.0030, and 0.0058 cm s-l, respectively):
D = 0.0303~+ 1.92 X (3) where C is the bacteriophage (or solute) concentration in the aqueous phase; S1 and S2 are the bound concentrations for fast and kinetically limited sites, respectively; I9 is porosity; 4 is the dry bulk density of the solid material; D is the longitudinal dispersion coefficient; u is the average interstitial velocity; kl is a pseudo-first-order rate coefficient (s-l) for attachment, which depends on the bacteriophage’s (solute’s) molecular diffusion coefficient and the sticking efficiency (ie., net energy of interaction between phage and silica); and k2 is a pseudo-first-order detachment rate coefficient, which also depends on the energy of phage-surface interaction. These rate coefficients do not depend on the surface site concentration, as only a very small fraction of the surface was covered by adsorbed phage. Equation 1 expresses the total change in concentration with time due to advection, dispersion, attachment, and detachment. Equation 2 expresses the linear adsorption equilibrium for the fast (type 1, equilibrium) adsorption sites. K,, and 8k1/4k2 are the equilibrium partition coefficients for the type 1 and type 2 sites, respectively. Letting Kp2 = 19k1/4k2, the overall, total equilibrium partition coefficient for t a is Kpl + K . Type 1 sites could correspond to colloids held near surface in a secondary minimum of the potential energy of interaction, with little or no energy barrier for detachment. In eq ‘3,the change in bacteriophage concentration bound to type 2 sites with time is the difference between the attachment and detachment rates. It is often useful to express the four model parameters in dimensionless terms. The total partition coefficient, Kpl + K,, is related to the retardation factor:
-
The Peclet number
t&i
(5)
(8)
( r 2 = 0.99)
Fitting the adsorption portion of the MS-2 breakthrough curves with the equilibrium model gave good fits (low sum of squared errors) but apparent dispersion values 150fold higher than for the salt tracer, indicating nonequilibrium behavior (Table I and Figures 4 and 5). There was adsorption of MS-2 to the glass beads; fitted R’s ranged from 1.6 to 2.9. The desorption portions of the same three breakthrough curves gave dispersion values nearly the same as for the salt tracer; however, the desorption curve R values were less than 1. The apparent R can be less than 1.0 if there is colloid exclusion from a part of the pore volume, i.e., the pore volume for colloid transport is smaller than the pore volume for salt transport; that is not plausible for our system. These two results-small D and R I 1-are an artifact of fitting a slow-desorption breakthrough curve with an equilibrium model; they suggest that the time scale for desorption was slower than those for sorption, and for flow through the column. From experiments 4 and 5 (PRD-l), which had no adsorption (R = l.O), D estimated from an equilibrium model fit averaged 2.9 X cm s-l for a velocity of 0.0039 cm s-l (Table I and Figure 6). This dispersion value is in the same range as that of the salt tracer; there was apparently no additional dispersion associated with PRD-1 in the absence of adsorption. The fitted Peclet number of 210 for PRD-1 was used for subsequent analysis of experiments 6 and 7. A corresponding Peclet number of 470-based on its smaller size-was used for MS-2 in later analyses to separate the effects of slow adsorption and desorption from dispersion. Bales et al. (11)obtained D values of (1.4-1.7) X lo-* cm’ s-’ in soil column experiments with MS-2 at velocities of 0.01 cm s?. Using a Peclet number based on the mean grain size for their heterogeneous soils, -1.5 mm, suggests that dispersion was similar to that observed in our PRD-1 experiments (Figure 7). Further modeling efforts concentrated on determining a range of values for kl and k,, the attachment and detachment rate coefficients, respectively. Adsorption and desorption portions of the curves were modeled separately
-
Environ. Sci. Technol., Vol. 25, No. 12, 1991
2093
Table 11. First-Order and Two-Site Model Fits to Data fitted parameters* exp" 1 ads 1 des 2 ads
2 des 3 ads 3 des
RC 2.66 f 0.07 1.62 f 0.05 2.72 f 0.09
6 ads 6 des 7 ads 7 des
210 f 42
6 ads 6 des 7 ads 7 des
230 f 51
115 f 22
118 f 24
p'
uc
KPl
ssqd
g Ifpz,
cm g
First-Order Model: MS-2 Experiments at pH 5.0 and 2.16 f 0.23 0.1270 0.365 0.160 f 0.043 0.0928 0.871 f 0.014 0.0547 0.136 0.1045 0.132 f 0.037 1.20 f 0.12 0.1041 0.378 0.0348 f 0.0120 0.0092 4.53 f 2.16 f 2.08 f 0.11 f
k z , s-l
k , , s-l
4 "C 5.33 x 3.95 X 2.15 x 3.26 x 2.80 X 8.12 X
First-Order Model: PRD-1 Experiments at pH 5.5 and 0.40 0.0063 46 0.40 0.0088 0.08 0.0513 26 0.01 0.0027
Two-Site Model: PRD-1 Experiments at pH 5.5 and 4.30 f 0.41 0.0080 f 0.0039 0.0060 50 0.82 f 11.43 0.0028 f 0.0425 0.0083 2.07 f 0.08 0.0089 f 0.0018 0.0501 26 2.34 f 0.17 0.0050 f 0.0004 0.0029
3.21 x 2.38 X 10-4 3.47 x 10-5 5.25 x low4 1.63 X 10" 4.72 X 104
4 "C 1.18 X 5.62 X lo4 5.41 X 10" 2.86 x 10-5
5.64 X 2.69 X 4.74 X 2.51 x
a
10"
1.1 x io-* 8X 4 x 10-3 7 x 10-4 6X 2X
10" 10" 10" 10-7
1.3 X 6X 6X 3 x 10-4
10-4 10-4
10-5
4 "C 4.90 X lo4 9.34 x 10-7 4.60 X 10" 5.18 x io"
11.2 X
2.13 x 10-4 5.38 X 6.08 x 10-4
1.3 X lo-' 2 x 10-3 6X 7 x 10-3
aAdsorption (ads) and desorption (des) parts of breakthrough curves listed separately. *Peclet number fixed at 210 for PRD-1 and 470 for MS-2. cParameter estimate f standard error. dSum of sauared errors. 0.15
adsorption
g
o
0 05
l
p
10
20
'
desorption
'
-
O D
100
10-3
100
103
106
0.3
adsorption
udDo
Flgure 7. Dispersion Peclet number for salt tracer and virus breakthrough curves; from equilibrium model fits, Table I. D o is the molecular diffusion coefficient and d is the bead diameter.
because of poor performance of the fitting algorithm when given both parts of the breakthrough curves to model as a unit, due in part to the lack of complete mass balance. Note that the adsorption portion of the breakthrough curve refers to the part during which bacteriophage-containing solution was fed to the column; the desorption portion refers to the second part, in which phage-free buffer was fed to the column. The first-order model gave good fits (low sum of squared errors) to the MS-2 breakthrough curves (Figure 4), with R values nearly the same as from the equilibrium model fits (Table 11). The low w values indicate nonequilibrium conditions, with kl and k2 values on the order of (0.3-3.0) X s. Time scales for equilibration (kC1) are thus on the order of several hours. For the PRD-1 experiments, the unusually high bacteriophage concentrations eluted with the first pore volume of desorption were removed from the data set; it is thought that the high values were caused by jarring of the virus tubing during the changeover from virus-containing tubing to virus-free tubing. The first-order and two-site models provided essentially the same parameter estimates and equally good fits for experiment 6 adsorption and desorption curves (Figure 8), as indicated by the sum of square errors (ssq). The w values for PRD-1 are in the same range as for MS-2, but the R values are nearly 100fold higher. Time scales for reaching equilibrium are thus on the order of 1 h for adsorption and several days for desorption. The low p values for the two-site-model fit indicate that bonding to type 1 (fast) sites contributes little to the total adsorption. That is, Kpl
