Abstract
The objective of this study was to investigate leaching and transport of viruses, specifically those of an indigenous coliphage host specific to Escherichia coli ATTC 15597 (i.e., MS-2), from a biosolid-soil matrix. Serial extractions of 2% and 7% (solids) class B biosolid matrices were performed to determine the number of phage present in the biosolids and to evaluate their general leaching potential. Significant concentrations of coliphage were removed from the biosolids for each sequential extraction, indicating that many phage remained associated with the solid phase. The fact that phage was associated with or attached to solid particles appeared to influence the potential for release and subsequent transport of phage under saturated-flow conditions, which was examined in a series of column experiments. The results indicated that less than 8% of the indigenous coliphage initially present in the biosolids leached out of the biosolid-soil matrix. A fraction of this was subsequently transported through the sandy porous medium with minimal retention. The minimal retention observed for the indigenous phage, once released from the biosolids, was consistent with the results of control experiments conducted to examine MS-2 transport through the porous medium.
Land application is a widely employed means of use and disposal of class B biosolids. Sixty-six percent of the 5.6 million dry tons of biosolids disposed of annually in the United States is done so via land application (11). Land application of biosolids has proven to be an efficient means of disposal and serves as an inexpensive fertilizer source for nonfood agricultural crops (12). Biosolids contain pathogenic microbes, including bacteria and viruses that could pose a risk to human health. Thus, there is interest in the fate of pathogens associated with land-applied biosolids.
The transport and fate of viruses in porous media have received significant attention (for an example, see reference 17). Such studies have focused primarily on the adsorption, transport, and fate of viruses in aqueous (e.g., sewage effluent) systems (1, 2, 3, 6, 7, 8, 9, 16, 17). Conversely, minimal information in regard to the transport of indigenous coliphage associated with land application of biosolids is available. This study evaluated the potential for release of indigenous coliphage host specific to Escherichia coli ATCC 15597 (i.e., MS-2) from biosolids and their subsequent transport through a sandy porous medium via saturated flow.
MATERIALS AND METHODS
Soil.
Vinton fine sand, with a pH of 7.3 (1:1 soil:water suspension), was used in a model porous medium consisting of the following: 93.1% sand, 5.6% silt, 1.3% clay, and 0.01% total organic carbon content (21). The soil was sieved through a 3-mm screen prior to use.
Biosolids.
Samples of class B biosolids were obtained from the Ina Road wastewater treatment plant in Tucson, AZ. They were produced by anaerobic digestion. The solid content was analyzed by drying 10 ml of biosolids in an oven at 104°C for 24 h. The result was expressed as a percentage on a weight (g)-to-volume (ml) basis (13). The samples of biosolids were stored in a refrigerator at 4°C for up to 24 h prior to use.
Bacteriophage and assays.
Bacteriophages are often used as surrogates to evaluate the transport and fate of enteric viruses, due to the size and structural similarities of some bacteriophages to enteric viruses. Using bacteriophages as a model is less expensive than using enteric viruses. Furthermore, they do not pose a threat to human health. As a consequence, these viruses are used extensively in field studies, and MS-2 phage is often used as a representative of group I viruses, such as echovirus I and coxsackievirus B4 (7).
MS-2 is an icosahedral, male-specific coliphage of approximately 27 nm in diameter (14). It has a pI of 3.9 (6). MS-2 was obtained from the American Type Culture Collection (ATCC 15597-B1). It was grown to a titer of 1,010 PFU/ml in Trypticase soy broth (Difco, Sparks, MD) and stored at 4°C. New stocks of MS-2 were produced for each experiment. MS-2 was grown and assayed in E. coli (ATCC 15597). It was assayed using the plaque-forming-unit method described by Pepper and Gerba (15). A modified version of the plaque-forming-unit method was used to assay indigenous coliphage present in biosolids. Bacto Agar with m-Endo Broth (Difco, Sparks, MD) was used as the medium instead of Trypticase soy broth in order to prevent growth of fungi and other bacteria.
Extraction of phage from biosolids.
One goal of this study was to enumerate indigenous coliphage host specific to E. coli ATTC 15597 that is present in anaerobically digested biosolids. Three sets of serial extractions (i.e., triplicate experiments), incorporating 5, 10, and 17 extractions, were performed with samples of 2% biosolids. Multiple sequences of centrifugation (4,068 × g for 15 min) and resuspension were conducted for each set. The supernatant was removed after each centrifugation step and assayed for coliphage using the plaque-forming-unit method. Three subsamples were collected from each supernatant; data reported are the means of the analyses of the three subsamples. The pellet was resuspended with 10 ml (5- and 10-step extraction sets) or 50 ml (17-step extraction set) of 3% Tris buffer (Trizma base, NaCl, KCl, Na2HPO4 [pH 7.0]). The concentration of coliphage in the pellet was assayed by directly plating 100 μl of the resuspended pellet prior to performing the next centrifugation step.
A fourth set of sequential extractions was performed using a sample of 7% biosolids. The 7% biosolids contained a higher level of organic matter and thus required a stronger buffer to elute the coliphage. Therefore, a 10% beef extract buffer solution at a pH of 7 was used in place of the 3% Tris buffer. The centrifugation and resuspension sequence was performed 58 times for this set. After each extraction, the supernatant was assayed for coliphage. After the last centrifugation, the resuspended pellet was assayed for coliphage to estimate the number of coliphage that remained associated with the solids.
Column transport studies. (i) Apparatus.
A Plexiglas column of 10-cm length and 2.5-cm diameter was used for the column experiments. The column was equipped with pieces of Nitex, a nylon-monofilament screening fabric (Lab Pak [precision woven screening medium]; Tetko Inc.), at both ends to retain the porous medium. The column was packed incrementally (1-centimeter lifts) with Vinton sand. Once packed, the soil was saturated with dechlorinated, filter-sterilized tap water that was pumped through the bottom of the vertically oriented column. The water was dechlorinated using 0.2% sodium thiosulfate (Sigma, St Louis, MO) prepared in nanopure water (Ultrapure system; Barnstead, Irvine, CA). The dechlorinated water was then filtered through a 0.45-μm cellulose acetate membrane (Nalge Nunc International, Rochester, NY).
The solutions were pumped upward through the column using a single-piston liquid chromatography pump (Acuflow Series II; Fisher Scientific, Tustin, CA) at a rate of 0.17 ml/min (equivalent pore-water velocity of 2.3 cm/h). Teflon (Kimble-Kontes, Vineland, NJ) and stainless steel (Alltech, Deerfield, IL) tubing were used to connect the pump and the column. A fraction collector (RediFrac; Pharmacia, Uppsala, Sweden) was used to collect effluent samples in borosilicate glass tubes.
(ii) Nonreactive tracer tests.
Pentafluorobenzoic acid (PFBA) (Aldrich Chem. Co., Milwaukee, WI), a widely used nonreactive tracer, was employed to determine the hydrodynamic properties of the packed columns. A tracer test was conducted for each packed column. The tracer test consisted of pumping 3 pore volumes of PFBA solution through the column before flushing it with 5 pore volumes of dechlorinated, filtered tap water. The effluent samples were analyzed at a wavelength of 263 nm using a UV-visible spectrophotometer, Spectronic Genesys 2 (Milton Roy, Rochester, NY). The results were plotted as relative concentration (C/C0; measured concentration divided by injection concentration) versus pore volume (discharge volume divided by water retention volume of column).
(iii) MS-2 control experiments.
Two control experiments were performed to evaluate the transport behavior of MS-2 in the Vinton sand. For each experiment, 3 pore volumes of a solution containing 109 PFU/ml of MS-2 mixed in dechlorinated, filtered tap water was pumped through the column (from bottom to top), followed by 10 to 40 pore volumes of water containing no phage. Effluent fractions were analyzed within an hour of collection. At the end of the experiment, the column was disassembled, and the soil was separated into 1.25-cm slices. A gram of each slice was diluted in 9.5 ml of 3% Tris buffer, pH 7.4, and further diluted in 3% Tris buffer as needed. The appropriate dilution was assayed for phage. All viral assays were performed in triplicate. The bulk densities, porosities, resident pore volumes, and injection concentrations of MS-2 for these experiments are reported in Table 1.
TABLE 1.
Experiment properties and MS-2 recoveries for MS-2 control column experiments
| Property | Result from:
|
|
|---|---|---|
| Experiment 1 | Experiment 2 | |
| Bulk density (g·cm−3) | 1.41 | 1.44 |
| Porosity (Vw/Vc)a | 0.40 | 0.37 |
| Resident vol of water in column (ml)b | 18.5 | 17.2 |
| Injection concn of MS2 (PFU/ml) | 2.76 · 109 | 2.40 · 109 |
| Total PFU injected | 4.16 · 1011 | 2.24 · 1011 |
| Total PFU recovered from column effluent | 1.50 · 1011 | 1.20 · 1011 |
| Aqueous-phase recovery (%)c | 36.1 (±5.3) | 53.5 (±7.3) |
| Total PFU recovered from in soild | 3.27 · 108 | 2.57 · 108 |
| Soil phase recovery (%) | 0.08 | 0.11 |
Vw, volume of pore space; Vc, volume of porous medium.
A unit pore volume is calculated as follows: the mass of dry porous medium was subtracted from the mass of saturated porous medium.
This reflects the percent MS-2 recovered from the effluent phase.
This shows the total number of MS-2 recovered from the soil after the experiment was ended.
Two sets of data were obtained for each experiment. One set provided the amount of coliphage present in the effluent (i.e., aqueous concentration). The other set provided the amount of coliphage that remained in the column associated with the soil. A mass balance was performed to determine the percent recovery of MS-2 in the column effluent relative to the initial amount of coliphage injected into the column. The total number of PFU of coliphage in the effluent was determined by calculating the area under the breakthrough curve.
(iv) Biosolid-amended soil.
Four experiments were conducted to examine the release and subsequent transport of coliphage associated with biosolid-amended soil. In each experiment, a column was packed with Vinton sand and saturated, and a tracer test was conducted as described previously to evaluate column characteristics. The column was then unsealed to allow for the introduction of biosolids into the system. A layer of soil (1.3 cm) was removed and replaced with a mixture of the removed soil amended with 7% biosolids. The amount of biosolids relative to the entire amount of wet soil present in the column (including that present in the biosolid layer) was between 5 and 6% by weight.
After packing the soil/biosolid layer in the column, the column was resealed and oriented so that the layer containing the mixture of soil and biosolids was at the bottom of the column. Six to 14 pore volumes of dechlorinated, filtered tap water was pumped through the column from the bottom. Effluent samples were collected as described previously and analyzed within an hour of collection by use of a modified plaque-forming-unit method. All viral assays were performed in duplicate. A unit pore volume in this case represents the volume of water contained in the Vinton sand below the mixed biosolid-soil matrix. At the end of the experiment, the column was disassembled and the soil was sectioned. The biosolid-soil matrix layer was first removed, and the soil down gradient of it was sliced every 1.25 cm. The procedure used to assay the coliphage associated with each layer was the same as described for extraction of indigenous coliphage in 7% biosolids. Mass balances were conducted to determine the amount of coliphage recovered in the column effluent and the amount associated with the soil down gradient of the soil/biosolid layer. The relevant properties for these experiments are reported in Table 2.
TABLE 2.
Experimental properties for column studies conducted with biosolid-amended soil
| Experiment | % Solids in biosolids | Amt of wet biosolids placed in column matrix (g) | Equivalent amt of dry biosolids (g) | Original concn of indigenous coliphage in biosolid matrixa | Wet biosolid wtb | Porosity of down-gradient soil layerc |
|---|---|---|---|---|---|---|
| 1 | 8.8 | 4.12 | 0.36 | 9.97 · 104 | 5.2 | 0.29 |
| 2 | 9.1 | 4.25 | 0.37 | 1.03 · 105 | 5.7 | 0.30 |
| 3 | 7.8 | 4.44 | 0.34 | 9.48 · 104 | 5.8 | 0.26 |
| 4 | 4.8 | 4.91 | 0.23 | 6.43 · 104 | 6.3 | 0.29 |
This number (an estimate) was calculated as follows: the calculated number of indigenous coliphage in 8% biosolids (2.72 · 105 PFU/dry g biosolids [Table 3, experiment 4]) was multiplied by the amount (dry g) of biosolids in the biosolid-soil matrix.
Percentage of wet soil weight.
Down gradient of biosolid-soil matrix.
RESULTS
Coliphage extraction from biosolids.
The recovery of indigenous coliphage from the 2% biosolids is shown in Table 3 and Fig. 1. The results show that significant concentrations of coliphage are removed from the biosolids at every extraction. However, the majority of coliphage recovered from the supernatants was associated with the first two extractions. The recovery of coliphage in subsequent extractions amounted to less than 7% of the total extracted.
TABLE 3.
Indigenous coliphage recovery from serial extractions of biosolids
| Property | Experiment
|
|||
|---|---|---|---|---|
| 1 | 2 | 3 | 4 | |
| Biosolids (%) | 1.7 | 1.9 | 1.7 | 7.0 |
| Original sample vol (ml) | 10 | 10 | 25 | 15 |
| Total mass of dry biosolids (g) | 0.174 | 0.186 | 0.425 | 1.05 |
| No. of extractions performed | 5 | 10 | 17 | 58 |
| Total PFU recovered from supernatant per g dry biosolidsa | 8.68 · 104 | 1.19 · 105 | 4.24 · 104 | 2.65 · 105 |
| PFU recovered from last pellet per g dry biosolidsb | 5.11 · 103 | 1.86 · 104 | 2.39 · 103 | 8.10 · 103 |
| Total PFU per g dry biosolidsc | 9.19 · 104 | 1.38 · 105 | 4.48 · 104 | 2.72 · 105 |
Sum of PFU recovered for all extractions divided by the amount of dry g of biosolids.
PFU recovered in last pellet divided by the amount of dry g of biosolids.
Total PFU/g dry biosolids = (total PFU recovered in supernatant + PFU recovered in last pellet)/g dry biosolids.
FIG. 1.
Number of coliphage sequentially extracted from 2% biosolids. Closed symbols represent pellet concentrations. Open symbols represent supernatant concentrations. ∗, measure is likely an underestimate of the number of PFU associated with the pellet.
Prior research has shown that coliphage is either embedded in or attached onto surfaces of the biosolids (19, 20). The coliphage recovered in the second and subsequent extractions was thus likely either desorbed or released from the solid phase of the biosolid matrix. Assays of the resuspended pellet showed that a significant fraction of coliphage remained associated with the solids throughout the series of extractions (Fig. 1). The results obtained for the extraction experiment with 7% biosolids were similar (Fig. 2). It is not known whether all phage associated with the solid phase gives rise to plaques. In addition, some coliphage may remain associated with the solid component and thus remain undetected. Hence, the numbers presented are likely a conservative estimate of the total number of phage present in the biosolids. A conservative estimate based on the mass balance of coliphage in the 7% biosolids is 2.75 · 105 coliphage per dry gram of biosolids. This number will serve as an estimate of the concentration of coliphage initially present in the biosolid-soil matrix for the column studies.
FIG. 2.
Number of coliphage sequentially extracted from 7% biosolids.
Tracer and MS-2 transport studies.
A representative breakthrough curve for PFBA, the nonreactive tracer, is shown in Fig. 3A. PFBA appears after 1 pore volume has passed through the column. The relative concentration sharply rises to a value of 1 and plateaus over the next 3 pore volumes. Upon the switch to a PFBA-free solution, the PFBA relative concentration remains at a value of 1 for another pore volume followed by a sharp drop to a value of 0. The ideal transport behavior illustrated by these data indicates that the columns were packed uniformly.
FIG. 3.
Transport of pure-culture MS-2 through Vinton sand via saturated flow; results from duplicate experiments. (A) Transport expressed as C/C0 values. (B) Transport expressed on a log10 scale.
The breakthrough curves for MS-2 transport in Vinton sand are presented in Fig. 3. MS-2 appeared in the effluent upon the passage of approximately 1 pore volume (coincident with the nonreactive tracer), indicating that the transport of a large fraction of MS-2 was not retarded by Vinton sand. A sharp rise in concentration was observed after 1 pore volume and was followed by C/C0 values ranging from 0.6 to 0.8 relative concentration. The difference in concentrations observed is probably due to the imprecision of the plaque-forming-unit method of analysis.
The occurrence of steady-state effluent concentrations of less than 1 indicates that a fraction of the coliphage was irreversibly lost from solution. The loss of biocolloids from solution during transport in porous media is typically the result of some combination of attachment to solid-phase surfaces, straining, and inactivation. Given the relative sizes of the MS-2 phage and the Vinton soil grains (and associated pore sizes), straining is unlikely to be a significant factor. Thus, the loss observed in these experiments is likely due to a combination of attachment and inactivation. Totals of 1.5 · 1011 and 1.2 · 1011 PFU were recovered in the effluent for the two experiments. These values are 36% (±5%) and 54% (±7%) of the total amount of MS-2 injected into the column (Table 1). Approximately 0.1% of the coliphage was recovered from the soil in both experiments (Table 1).
Inspection of Fig. 3B shows that after the passage of 30 pore volumes, 104 PFU/ml was still being eluted from the column. This indicates that while inactivation may occur, some viable viruses remained attached to soil particle surfaces. A total of 3 · 108 PFU was recovered from the soil, indicating that there was a large amount of viruses available for detachment into the aqueous phase. However, this represents 0.1% of the coliphage introduced into the system. The low level of recovery from soil does not necessarily indicate a lack of virus within the soil but does possibly indicate the inability to extract them. Therefore, it is quite possible that a fraction of the phage injected into the column was in effect irreversibly attached to soil particles.
Transport of coliphage indigenous to biosolids.
The concentrations of indigenous coliphage in column effluents resulting from flushing water through a biosolid-soil matrix are presented in Fig. 4 for four replicate experiments. The concordance between the four sets of results indicates the reproducibility of the experiments. Coliphage was detected after the passage of 1 pore volume, after which concentrations increased to a maximum of 100 PFU/ml. Elution behavior is in the form of a pulse of higher concentrations of coliphage that spans the first 2 pore volumes and is followed by tailing at relatively low concentration (detection limit of 1 phage/ml). The concentrations comprising the tails are much lower than those observed in the MS-2 control studies. It is hypothesized that the initial elution pulse represented primarily those coliphage originally present in the aqueous phase of the biosolids as well as those more loosely attached and therefore flushed out in a manner similar to that seen in the previous pure-culture control studies done with MS-2. Conversely, the low-concentration tailing is attributed to leaching of coliphage embedded and/or strongly attached to the biosolids.
FIG. 4.
Leaching of indigenous coliphage able to infect host E. coli ATCC 15597 from biosolid-soil matrix in Vinton sand during saturated flow. Summary graph of four trials.
The results of the biosolid extraction studies discussed above indicated that most of the coliphage was embedded within or attached to the biosolids and was difficult to extract. The data reported in Table 4 show that most of the coliphage indeed remained in the biosolid-soil matrix layer for the transport studies. The percentages of coliphage recovered in the effluent ranged from 2 to 4% of the amount initially present in the biosolids (Table 4). Similar to what was seen in the MS-2 control studies, a low percentage of coliphage was recovered from the Vinton soil down gradient of the biosolid layer. Overall, the total recovered amount (soil plus effluent) of indigenous coliphage was less than 8% of the estimated amount of indigenous coliphage initially present in the biosolid-soil layer. Considering that the extraction studies produced an underestimate of the total coliphage number associated with the biosolids, as noted above, it is likely that the values used for the initial presence of coliphage in the biosolid-soil matrix are lower than the actual values. If so, the percentages reported in Table 4 for leachable fractions are overestimates.
TABLE 4.
Coliphage recoveries from column transport studies conducted with biosolid-amended soil
| % Coliphage recovery: | Experiment
|
|||
|---|---|---|---|---|
| 1 | 2 | 3 | 4 | |
| From effluent phasea | 2.3 | 3.6 | 2.4 | 4.0 |
| From sand down gradient of biosolid-soil matrixb | 3.0 | 0.9 | 3.4 | |
| Totalc | 5.3 | >3.6 | 3.3 | 7.4 |
The total amount of coliphage recovered in the column effluent, as a percentage of initial amount present in the biosolid-solid layer.
The total amount of coliphage recovered from the sand layer down gradient of the biosolid-soil layer at the end of the experiment, as a percentage of the initial amount present in the biosolid-soil layer.
The sum of rows one and two, representing the percentage of coliphage leached from the biosolid-soil layer.
DISCUSSION
The series of biosolid extractions showed that the majority of the indigenous coliphage is associated with the solids. The extraction data for biosolids are in concordance with the results obtained for the column studies conducted with biosolid-amended soil. These findings are in agreement with the results of prior research. Pancorbo et al. (13) seeded anaerobically digested sewage sludge with poliovirus type 1 to a concentration of 106 PFU/ml. They recovered 60% by use of a serial extraction protocol suitable for poliovirus. Their findings indicated that the seeded polioviruses were adsorbed to the solid component of the sewage. Wellings et al. (20) and Stagg et al. (19) both reported that indigenous viruses were solid associated in wastewater-activated sludge. Stagg et al. (19) demonstrated that 85% of the indigenous coliphage was adsorbed to solids present in sewage effluent.
Our experiments show that MS-2 has relatively low sorption potential for the sandy soil used in the study. These findings are in agreement with previously published articles on viruses in general (1, 2, 6, 7, 9) and MS-2 specifically (10, 18). A large percentage (36 to 54%) of MS-2 introduced into the column was not recovered. This is attributed to a combination of effectively irreversible attachment and inactivation. In addition, extended elution tailing occurred, with concentrations of 103 to 104 PFU/ml. After 40 pore volumes, coliphage was still detectable.
Indigenous coliphage leached from the biosolids and was available for transport during flushing with the first few pore volumes of water. After 6 pore volumes, the leaching level of indigenous coliphage approached zero. Indeed, leaching stopped despite the large numbers of coliphage that remained in the biosolids. Those left may have been strongly attached to or embedded within biosolid particles and therefore not available for transport (4, 5). The number of coliphage leached from the biosolid/soil layer was a small fraction of the initial content. Although transport studies of phage have been previously conducted, we are not aware of work that has specifically examined leaching of indigenous viruses from biosolids.
Conclusion.
The transport studies conducted with the biosolid-amended soil show that little leaching of coliphage occurred within a 10-cm column soil profile. The biosolid-soil matrix, despite being subjected to continuous saturated flow, retained most of the coliphage. The viruses that were leached from the column most likely represent primarily the coliphage present in the aqueous phase of the biosolids. Once the coliphage leaves the biosolid-soil matrix, it appears to behave in a manner similar to that seen with the transport of MS-2. Human enteric viruses are normally present in class B biosolids at concentrations less than 1 most probable number/g. This value is 6 orders of magnitude lower than corresponding phage concentrations. Therefore, the potential for leaching of human enteric virus from biosolids is likely to be less than that for phage. Field experiments examining the transport behavior of biosolid-associated virus are needed to quantify the actual risk for groundwater contamination posed by amendment of soil with biosolids.
Acknowledgments
This research was supported in part by funds provided by the University of Arizona National Science Foundation Water Quality Center and the U.S. EPA STAR program.
Special thanks to Janick Artiola and Karen Josephson for their help.
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