Vol. 57, No. 8
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1991, p. 2192-2196
0099-2240/91/082192-05$02.00/0 Copyright ©3 1991, American Society for Microbiology
Effects of Organic Matter
on
Virus Transport in Unsaturated Flow
DAVID K. POWELSON,* JAMES R. SIMPSON, AND CHARLES P. GERBA
Department of Soil and Water Science, University of Arizona, Tucson, Arizona 85721 Received 4 March 1991/Accepted 29 May 1991
The effects of natural humic material and sewage sludge organic matter (SSOM) derived from primary treated sewage sludge on virus transport by unsaturated flow through soil columns were evaluated. Bacteriophage MS-2 was applied to loamy fine sand columns 0.052 m in diameter and 1.05 m long. Virus concentrations in the influent and effluent were measured daily for 7 to 9 days. In the first experiment, virus transport through two fresh soil columns was compared with that through a column previously leached with more than four pore volumes (1) of well water. The soil water organic matter concentrations in the leachate of the fresh soil declined with time. Relative virus concentrations (CIC0) from one fresh soil column reached 0.82 in 0.9 T and then declined to 0.51 by 2.1 T. The other fresh soil column reached and maintained a steady-state relative virus concentration [(CIC0)s] of 0.47 from 1.5 to 2.5 T. The leached column reached and maintained a (CIC,)s of 0.05. Concentrations measured at 0.2-, 0.4-, 0.8-, and 1.05-m depths indicated that most virus particles were removed in the surface 0.2 m. In the second experiment, one leached column was pretreated with SSOM derived from primary treated sewage sludge and the other leached column was untreated. SSOM concentrations declined with depth. A suspension of virus and SSOM in well water was applied to both columns. Although the (CIC,)s values were similar (0.41 for the pretreated column and 0.47 for the untreated column), breakthrough was delayed for the untreated column. Both natural humic material and sewage sludge-derived SSOM increased the unsaturated-flow transport of MS-2.
the field. For example, rainfall may reduce salt concentration, thereby releasing soil organic matter. Duboise et al. (6) found that total organic carbon and poliovirus 1 concentrations peaked at the same time when soil columns were flooded with distilled water to simulate rainfall. Agricultural cultivation also increases the release of organic compounds in soluble forms (24). Furthermore, organic matter concentrations normally decline with depth. Many organic matter molecules are strongly retained by soils by hydrogen bonding, van der Waals attraction, or other forces (4). The sorbed organics may then biodegrade before reaching a greater depth. The purposes of the research reported here were (i) to evaluate the effects of natural humic material on virus transport through unsaturated soil with humic material concentrations declining with time, (ii) to evaluate the effects of sewage sludge-derived organic matter on virus transport through unsaturated soil with sludge concentrations declining with depth, and (iii) to test whether the latter effects were due to complexation of virus with sludge or to competition for adsorption sites. Virus concentrations with depth were fit to a mathematical model (12) to quantify specific decay rates. Possible mechanisms of virus adsorption and inactivation in unsaturated flow are discussed.
Land disposal of sewage is believed to be an effective means of removing most pathogenic viruses provided that the waste is exposed to several meters of unsaturated flow (21). Organic matter appears to interfere with virus removal. Stagg et al. (23) found that the presence of 3 mg of organic carbon per liter in a 0.05 M MgCl2 solution reduced adsorption of bacteriophage MS-2 to bentonite from 97% to 35%. Bixby and O'Brien (3) found that fulvic acid complexed with bacteriophage MS-2 and prevented adsorption of virus to soil. Humic acid decreases adsorption of poliovirus type 1 to membrane filters (11), but complexation of the virus with humic materials was not observed. Sobsey and Hickey (22) found that soluble organic matter reduced the effectiveness of virus collection by membrane filters. They performed a single-particle analysis and found a high percentage of single virus particles in suspension with humic and fulvic acids, indicating that soluble organic matter interfered with filter effectiveness by competing with the virus rather than by complexing with it. Lance and Gerba (13) found that organic compounds in sewage counteracted the effect of higher salt content and decreased the adsorption of poliovirus to soil. The only exception seems to be that of Landry et al. (14), who found that exposure to sewage effluent enhanced the ability of a very coarse soil to adsorb virus. The research reported above was conducted under watersaturated conditions, or the degree of saturation was not specified. Many waste disposal sites, however, have nonwater-saturated conditions at some depth or time. One meter of unsaturated flow, under low-organic-matter conditions, has been shown to reduce the concentration of bacteriophage MS-2 by a factor of 20 compared with saturated flow
MATERIALS AND METHODS Virus. Bacteriophage MS-2 was chosen as the model virus since the virion has structural properties similar to those of many human enteric viruses but is safer to use (27). It has a diameter of 25 nm and an isoelectric point of 3.9 (18). The virus was obtained from the American Type Culture Collection (ATCC 15597B1) and grown on lawns of Escherichia coli (ATCC 15597) as described by Adams (1). The virus was assayed by the PFU method (1) on Trypticase soy agar (Difco, Detroit, Mich.). Each reported virus concentration is the average concentration from two duplicate plates. To minimize virus inactivation, the experiments were con-
(19).
It is also important to understand virus transport by unsaturated flow under conditions of fluctuating organic matter concentrations because this is often the situation in *
Corresponding author. 2192
ORGANIC MATTER EFFECTS ON VIRUS TRANSPORT
VOL. 57, 1991 TABLE 1. Soil and water conditions' Parameter
Value or characteristic
Soil material
Textureb ............................. pHc .............................
Loamy fine sand
8.0 0.29% Organic carbond............................ Column bulk density ..................... 1,540 kg m3 (1.54 g cm3)
Influent solution Source .............................
pH ............................. Electrical conductivity .................. Organic mattere............................
Tucson, Ariz., groundwater 8.1 0.057 S m10 mg liter-'
Soil water Volumetric water content (0,,)........ 0.35 m3 m-3 Average linear velocity (v)............ 0.27 m day-' Water potential at 0.4 m depth ....... -3.4 J kg-' (-0.35 m) aAdditional data on the soil material may be found in reference 19. b Determined by the pipette method (9). In 0.01 M CaC12, mixed 2:1 with soil (15). d Determined by the Walkley-Black procedure (17). Organic matter in water, including the virus culture, 105'C-to-550°C weight loss (20). fv = qO,-', where q is infiltration (0.095 m day-').
ducted in a walk-in refrigerator at 4°C. Virus was added to the soil-column influent to an approximate concentration of 105 PFU ml-'. Soil columns. The soil material was obtained near the Flushing Meadows Wastewater Renovation Project on the floodplain of the Salt River near Phoenix, Ariz. It consisted of Vint loamy fine sand (a sandy, mixed, hyperthermic Typic Torrifluvent) mixed with recent alluvium (Table 1). Clear polyvinyl chloride cylinders (inside diameter, 0.052 m) were used to hold 1.05-m-long soil columns. Twelvemillimeter-diameter holes were drilled in each cylinder at 0.20-, 0.40-, and 0.80-m depths from the soil surface for water sampler and manometer tubes, and a 4-mm hole was drilled at 1.05 m for the column effluent tube. This tube extended 0.2 m below the bottom of the column to maintain negative water potential at the bottom of the column. Airdried soil was crushed, passed through a 2-mm-pore-size screen, poured down a tube into the cylinders in 50-mm increments, and stirred to prevent layering. The columns were weighed and tapped to settle the soil to the desired volume, creating a bulk density of 1,540 kg m-3. After the soil was moistened, cores were removed through the holes and the samplers were installed. The water samplers were made of sintered stainless steel cups (pore size, 2 ,um; length, 25.4 mm; outer diameter, 10 mm; air-entry potential, 12.7 J kg-') (Mott Metallurgical Corp., Farmington, Conn.). This soil column design was used in our previous virus transport study (19). The samplers and cylinders were tested to ensure that they did not retain virus. Water from a 70-m-deep well located in Tucson, Ariz., was used (Table 1). Organic matter concentrations in water samples were estimated by a modification of the method of Rabenhorst (20). A 120-ml water sample was dried at 105°C; the residue was then heated to 550°C for 20 min in air, and the weight lost was taken to be the organic matter in the sample. This method provided a relative measure of the organic matter concentrations, but the concentrations should not be considered absolute determinations. The influent stock was applied with a peristaltic pump, and water flow conditions were maintained as listed in Table 1. The
2193
water contents were determined by weighing, and the water potentials were monitored by measuring the water level in manometer tubes. Samples of the influent and effluent for each column were collected daily for 7 to 9 days and assayed for virus concentration. The first experiment compared virus transport through two fresh soil columns (Fresh 1 and Fresh 2) and one that had been leached with well water for more than four pore volumes (7): T = vtz-1, where v is average linear velocity (meters day-'), t is time (days), and z is depth (m). Additional leached columns were not used since the leached condition was the same as that for the four unsaturated columns reported by Powelson et al. (19), in effect providing replicates. Soil water samples were extracted at 0.20-, 0.40-, 0.80-, and 1.05-m depths daily for Fresh 1 and at the same depths at the end of the experiment for the leached column to evaluate the virus concentration profile with depth. Soil water sampling for Fresh 2 was done at a depth of 1.05 m only. A second experiment was conducted by using leached soil to test whether the effect of organic matter on virus loss was due to competition for adsorption sites or complexation with the virus. Sewage sludge organic matter (SSOM) was obtained from the supernatant of autoclaved primary sewage sludge from the Ina Road Sewage Treatment Plant, Tucson, Ariz. The supernatant was diluted with well water to produce an influent solution with an electrical conductivity of 0.057 S m-1 and an organic matter concentration of 203 mg liter-'. These conditions were nearly the same as those of the fresh soil effluent in the first experiment. One leached column was pretreated with this solution for 1 T; the other leached column was not treated. Virus mixed with this solution was then applied to both columns. Mathematical fitting. The calculated influent virus concentration (C0,) at a given time was taken from the linear regression of the concentrations of the inflow samples over the course of an experiment. Input virus concentrations were used as the dependent variable and linearly regressed against time as the independent variable, with CO thus representing the value of the regression function at the time a sample was taken. Relative virus concentrations (CIC0) were determined by dividing C, the concentration of virus detected at a given depth and time, by Co. Relative virus concentrations were considered to be at steady state when they appeared to reach a plateau. The steady-state relative virus concentration [(CICO),] was calculated by averaging CICO values (the number of values denoted by n) after steady state was first achieved. The profiles of (CIC0), with depth were consistent with a model developed by Jury et al. (12) for the biodegradation of pesticides and used by Powelson et al. (19) to describe virus removal with depth. To use this model, the soil profile was divided into two regions, a surface zone (0 < z c L) and a transition zone (L ' z < 1.05 m). In the surface zone, the steady-state concentration was:
(C/C0),,
exp (-uz/v) (1) where u (per day) is the specific removal rate, z is depth (meters), and v (meters per day) is the average linear velocity. The steady-state concentration in the transition zone was:
(C/C0),t
=
{(-uIGv) [1
exp (L where G (per meter) is the depth constant and L =
exp
+
GL
-
z)I} (2) (meters) is -
APPL. ENVIRON. MICROBIOL.
POWELSON ET AL.
2194
200
0.9 1-J
0.8 150~
0.7
Fresh Soil at 1.05 m
0
0.6
0)
rn
o5
100
(f9
0.5
0.4
(I)
0.3 50
Leached Soil at 1.05 m
0.2 0.1
Well Water Influent a
0
L 0
2
0.5
s
2
s
1.5
2
0
0
2.5
0.5
Pore Volumes
Pore Volumes FIG. 1. Soil water organic matter concentrations for fresh soil and soil leached with more than four pore volumes of well water.
the depth of the boundary between the surface and transition zones.
RESULTS AND DISCUSSION Fresh versus leached soil. Organic matter concentrations in the effluent water of fresh soil (Fresh 2) decreased from 190 mg liter-' to 100 mg liter-' in 2 T, and the leached soil effluent had a mean organic matter concentration of 68 mg liter-1 [SD = 10.9, n = 4, where SD is the unbiased (n - 1) standard deviation] (Fig. 1). Non-steady-state conditions of organic matter concentration similar to those of the fresh soil columns might be found in a freshly plowed or manured field, in soils where sewage sludge had been added, or during rainfall on a drained sewage infiltration basin. The measured concentrations are high for soil water and may have been due to: (i) the recent alluvial origin of the soil material, (ii) the breakup of soil aggregates during drying and sieving, or (iii) the method used to measure the organic matter. Relative virus concentrations at the 1.05-m depth in Fresh 1 increased to as much as 0.82 of the inflow and then declined to 0.51 (Fig. 2). Although the Fresh 1 relative virus concentrations were still declining slightly at the end of the experiment, (CIC.), appears to be about 0.5. Fresh 2 reached a (CIC.), of 0.47 (SD = 0.016, n = 3). The (CIC0), in leached soil was only 0.05 (SD = 0.017, n = 4), the same steady-state concentration as the mean of four unsaturated and leached columns reported by Powelson et al. (19). All concentrations declined after reaching the initial breakthrough. This tendency may be related to increased leaching of the soil. Relative virus concentrations versus depth are plotted in Fig. 3. The scatter in the data may be due in part to declining organic matter concentrations. Nevertheless, it is clear that virus concentrations maintain higher values under the higher-organic-matter conditions of fresh soil. At the 1.05-m depth, the fresh soil virus relative concentrations were more than 10 times that of the leached soil. Most of the virus removal takes place in the upper 0.2 m, possibly due to greater leaching in this zone.
2.5
2
1.5
1
FIG. 2. Relative virus concentrations versus pore volumes at the 1.05-m depth for two fresh soil columns and one leached soil column.
Pretreated versus untreated sludge. SSOM decreased during transit through the columns. The influent organic matter concentration from added SSOM was 203 mg liter-1, and in 1.7 T the effluent mean concentration was 38 mg liter-' (Fig. 4). A similar decline with depth in organic matter concentrations due to sorption and biodegradation might be expected after application of sewage to soil. The organic matter concentrations in the water leaving the columns in this experiment were lower than those leaving the leached col-
0.9 0.8 0
0.7 0.6
CD C!)
cn
0.5 0.4 0.3 0.2
0.1 0
0
0.2
0.4
0.6
0.8
1
1.2
Depth (m) FIG. 3. Relative virus concentration profile with depth for two fresh soil columns and one leached soil column. The curves were derived from equations 1 and 2 (see text), with v = 0.27 m day-' and L = 0.2 m. Parameters for the fresh soil: G = 6 m-1, u = 0.5 day-1; parameters for the leached soil: G = 2.2 m-', u = 1.8 day-1. Symbols: *, Fresh 1, days 4 and 5; A, Fresh 1, days 6 and 7; +, Fresh 1, days 8 and 9; x, Fresh 2, days 4 to 7; C1, Leached, days 3 to 6; O, Leached, day 7.
ORGANIC MATTER EFFECTS ON VIRUS TRANSPORT
VOL. 57, 1991 220 s-
200
Sludge Inf luent 180
E
160
140 o
a)
120
U
.-_
100
0' 80 0
CO
60
Untreated Soil at 1.05 m 40 20
'.*-MPretreated
0 0
Soil at 1.05 m
1
0.5
0
1.5
Pore Volumes FIG. 4. Soil water organic matter concentrations for non-sludgetreated and pretreated soil columns. umn in the first experiment (68.0 mg liter-1). This may have been the result of more leaching of the columns prior to beginning the sludge experiment, resulting in lower concentrations of resistant humic material in the column effluents. It was anticipated that pretreatment of a leached column with SSOM would result in higher CICO values in the effluent compared with an untreated leached column because of competition for adsorption sites. Both treatments presumably had the same extent of complexation because the influent virus stock contained SSOM. Instead, the steadystate value for the pretreated column [(CIC0), = 0.41, SD = 0.038, n = 4] was slightly less than that for the untreated
0.9
0.8 0.7 0
0.6 (-)
0.5
2195
column [(CIC0), = 0.47, SD = 0.055, n = 3] (Fig. 5). However, virus breakthrough was delayed in the untreated column compared with that in the pretreated column. Untreated (CIC0), was reached in 1.4 T, whereas pretreated (C/C0), was reached in 1.0 T. This may indicate that the initial front of virus was removed when added to the untreated column and that only after the influent organic matter began to compete with virus did steady-state virus concentrations appear in the effluent. Possible adsorption and inactivation mechanisms. Given the isoelectric point of MS-2 (3.9) and the pH of the soil (8.0), it is likely that hydrophobic effects were the most important adsorption mechanism (7, 16). Hydrophobic areas on the soil solids do not appear to be involved because no removal of MS-2 was observed in saturated flow through this soil (19). It is possible, under unsaturated conditions, that the partially hydrophobic virions are forced to the air-water interface. Baylor et al. (2) found that bacteriophages T2 and T4 were concentrated by a factor of 50 in droplets from the surface film of bursting bubbles. Organic material may interfere with this adsorption by competing with the virus for sites at the air-water interface. Very few of the virions removed by unsaturated flow can be recovered by elution of the soil (19). Apparently, removal is caused by inactivation rather than reversible adsorption. Specific removal rates (u) found by fitting the data to the model of Jury et al. (12) indicated that the rate in fresh soil was 0.28 of the rate under leached conditions (Fig. 3). Furthermore, the decline in concentration in the transition zone (represented by G in equation 2) in fresh soil was less steep (Fig. 3). Air-water surface tension may physically disrupt and inactivate virus. According to Cheo (5), when a tobacco mosaic virus suspension was aerated by bubbling, the virus underwent rapid degradation that was attributed to air-liquid surface force. Trouwborst et al. (26) found that phages T1, MS-2, and Semliki Forest virus were rapidly inactivated by bubbling air or nitrogen gas through the suspension. Inactivation was attributed to the net radial stress on a virion at the air-water interface, which is proportional to the air-water surface tension (26). The increased survival of virus when mixed with organic matter may result from a lower surface tension, since almost all organic substances found in natural waters reduce the interfacial tension (25). One implication of this study for wastewater treatment is that virus removal is improved when there is less organic matter in the soil and in the infiltrating water. A site that had been irrigated with sewage for 40 years had a buildup of organic coatings and decreased virus adsorption (8). Gerba et al. (10) found that poliovirus removal from wastewater effluent was greatly improved by reducing the amount of organics by lime coagulation.
0.4
ACKNOWLEDGMENTS This work was supported by the University of Arizona through graduate research assistantship funds. We thank R. C. Bales, I. L. Pepper, and A. W. Warrick for valuable editorial suggestions regarding this paper.
>3 0.3 0.2 0.1 0
0.5
1
1.5
Pore Volumes FIG. 5. Relative virus concentrations versus pore volumes at the 1.05-m depth for non-sludge-treated and pretreated soil columns.
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bacteriophage adsorption and complexation in soil. Appl. Envi-
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POWELSON ET AL.
ron. Microbiol. 38:840-845. 4. Bohn, H. L., B. L. McNeal, and G. A. O'Connor. 1985. Soil chemistry. Wiley, New York. 5. Cheo, P. C. 1980. Antiviral factors in soil. Soil Sci. Soc. Am. J. 44:62-67. 6. Duboise, S. M., B. E. Moore, and B. P. Sagik. 1976. Poliovirus survival and movement in a sandy forest soil. Appl. Environ. Microbiol. 31:536-543. 7. Farrah, S. R., D. 0. Shah, and L. 0. Ingram. 1981. Effects of chaotropic and antichaotropic agents on elution of poliovirus adsorbed on membrane filters. Proc. Natl. Acad. Sci. USA 78:1229-1232. 8. Fuhs, G. W., M. Chen, L. S. Sturman, and R. S. Moore. 1985. Virus adsorption to mineral surfaces is reduced by microbial overgrowth and organic coatings. Microb. Ecol. 11:25-39. 9. Gee, G. W., and J. W. Bauder. 1986. Particle-size analysis, p. 383-409. In A. Klute (ed.), Methods of soil analysis, part 1, 2nd ed. Agronomy monograph 9. American Society of Agronomy and Soil Science Society of America, Madison, Wis. 10. Gerba, C. P., M. D. Sobsey, C. Wallis, and J. L. Melnick. 1975. Adsorption of poliovirus onto activated carbon in wastewater. Environ. Sci. Technol. 9:727-731. 11. Guttman-Bass, N., and J. Catalano-Sherman. 1986. Humic acid interference with virus recovery by electropositive microporous filters. Appl. Environ. Microbiol. 52:556-561. 12. Jury, W. A., D. D. Focht, and W. J. Farmer. 1987. Evaluation of pesticide groundwater pollution potential from standard indices of soil-chemical adsorption and biodegradation. J. Environ. Qual. 16:422-428. 13. Lance, J. C., and C. P. Gerba. 1984. Effect of ionic composition of suspending solution on virus adsorption by a soil column. Appl. Environ. Microbiol. 47:484-488. 14. Landry, E. F., J. M. Vaughn, M. Z. Thomas, and C. A. Beckwith. 1979. Adsorption of enteroviruses to soil cores and their subsequent elution by artificial rainwater. Appl. Environ. Microbiol. 38:680-687. 15. McLean, E. 0. 1982. Soil pH and lime requirement, p. 199-223. In A. L. Page et al. (ed.), Methods of soil analysis, part 2, 2nd ed. Agronomy monograph 9. American Society of Agronomy
APPL. ENVIRON. MICROBIOL. and Soil Science Society of America, Madison, Wis. 16. Murray, J. P. 1980. Physical chemistry of virus adsorption and degradation on inorganic surfaces: its relation to wastewater treatment. U.S. Environmental Protection Agency report 600/ 2-80-134. U.S. Environmental Protection Agency, Cincinnati. 17. Nelson, D. W., and L. E. Sommers. 1982. Total carbon, organic carbon, and organic matter, p. 539-579. In A. L. Page et al. (ed.), Methods of soil analysis, part 2, 2nd ed. Agronomy monograph 9. American Society of Agronomy and Soil Science Society of America, Madison, Wis. 18. Overby, L. R., G. H. Barlow, R. H. Doi, M. Jacob, and S. Spiegelman. 1966. Comparison of two serologically distinct ribonucleic acid bacteriophages. J. Bacteriol. 91:422-448. 19. Powelson, D. K., J. R. Simpson, and C. P. Gerba. 1990. Virus transport and survival in saturated and unsaturated flow through soil columns. J. Environ. Qual. 19:396-401. 20. Rabenhorst, M. C. 1988. Determination of organic and carbonate carbon in calcareous soils using dry combustion. Soil Sci. Soc. Am. J. 52:965-969. 21. Reneau, R. B., Jr., C. Hagedorn, and M. J. Degen. 1989. Fate and transport of biological and inorganic contaminants from on-site disposal of domestic wastewater. J. Environ. Qual. 18:135-144. 22. Sobsey, M. D., and A. R. Hickey. 1985. Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration. Appl. Environ. Microbiol. 49:259-264. 23. Stagg, C. H., C. Wallis, and C. H. Ward. 1977. Inactivation of clay-associated bacteriophage MS-2 by chlorine. Appl. Environ. Microbiol. 33:385-391. 24. Stevenson, F. J. 1986. Cycles of soil: carbon, nitrogen, phosphorus, sulfur, micronutrients. Wiley, New York. 25. Stumm, W., and J. J. Morgan. 1981. Aquatic chemistry, 2nd ed. John Wiley & Sons, New York. 26. Trouwborst, T., S. Kuyper, J. C. Dejong, and A. D. Plantinga. 1974. Inactivation of some bacterial and animal viruses by exposure to liquid-air interfaces. J. Gen. Virol. 24:155-165. 27. Yates, M. V., C. P. Gerba, and L. M. Kelly. 1985. Virus persistence in groundwater. Appl. Environ. Microbiol. 49:778781.