APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1977, p. 564-570
Copyright © 1977 American Society for Microbiology
Vol. 34, No. 5 Printed in U.S.A.
Inactivation of Enteric Viruses in Wastewater Sludge Through Dewatering by Evaporation RICHARD L. WARD`* AND CAROL S. ASHLEY2 Sandia Laboratories, Albuquerque, New Mexico 87115,1 and University of New Mexico, Albuquerque, New Mexico 871062 Received for publication 6 May 1977
The effect of dewatering on the inactivation rates of enteric viruses in sludge determined. For this study, water was evaporated from seeded raw sludge at 21°C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.
was
CHAT was purchased from the American Type Culture Collection, coxsackievirus Bi was a gift of L. C. MacLaren (Department of Microbiology, School of Medicine, University of New Mexico, Albuquerque), and type 3 reovirus strain Dearing was a gift of A. J. Shatkin (Roche Institute of Molecular Biology, Nutley, N.J.). Stock preparations of poliovirus and coxsackievirus were made as previously described (20). Reovirus stocks were prepared in a similar manner except that viruses were harvested by scraping infected cells from the culture bottles about 48 h after infection, and these lysates were stored at -600C without low-speed centrifugation of cell debris. These procedural changes were made because reoviruses are more strongly cell associated than are enteroviruses. The line of HeLa cells used for the growth and plaquing of both poliovirus and coxsackievirus was a gift of R. Radloff (Department of Microbiology, School of Medicine, University of New Mexico, Albuquerque). L-929 cells, purchased from Microbiological Associates, Bethesda, Md., were used for reovirus. All cells were grown in monolayer cultures in Eagle medium containing either 5% newborn calf serum (HeLa cells) or 5% fetal calf serum (L-929 cells). Dewatering of sludge and infectivity assay of virus. Raw sludge obtained from the Albuquerque Sewage Treatment Plant was used for all dewatering experiments. Raw rather than anaerobically digested sludge was chosen for these experiments to eliminate the virucidal effects of ammonia, known to be present in digested sludge. These effects are expressed at the pH of digested sludge (>7) but not the pH of raw MATERIALS AND METHODS sludge, which is consistently about 6 or less (21). Viruses and cells. Three strains of viruses were Because the pH of raw sludge does not increase during used in these experiments. Poliovirus type 1 strain dewatering, viral inactivation occurring during this
Sewage sludge, like many of the other waste products of modem civilization, is a resource whose quantity is increasing rather than decreasing. Because the earth's supply of most natural resources is being rapidly depleted, the need to find safe and cost-effective methods of utilizing sludge is very apparent. Even so, the value of sludge as a fertilizer or an animal foodstuff is only beginning to be realized. One feature of sludge that limits its use for these purposes is that it contains pathogens. It is, therefore, of immediate importance to find low-cost methods of inactivating the pathogens in sludge without reducing its economic value. Enteric viruses are one group of pathogens commonly found in very high concentrations in wastewater sludge (12). Although most procedures employed in handling and treating sludge have been used for many years, their effects on the survival of indigenous viruses are not known. Dewatering, a procedure often used in sludge handling, is a case in point. There are numerous ways of dewatering sludge, but in many circumstances the most cost-effective method is evaporation. The purpose of this study was to determine the effect of reducing the moisture content of sludge by evaporation on the survival of enteric viruses.
564
VOL. 34, 1977
INACTIVATION OF ENTERIC VIRUSES
process should not be due to the virucidal action of ammonia. To study the fate of viruses in sludge upon dewatering, sludge was seeded with virus either before or after the dewatering process. In experiments where the virus was present during dewatering, a 100-fold dilution of a stock solution of either poliovirus, coxsackievirus, or reovirus was made directly into raw sludge containing 5% solids. After thorough mixing, the sludge was placed in a shallow pan to a depth of about 1 cm and allowed to air dry at 21°C. This temperature was chosen because it was sufficiently high to permit evaporation at a reasonable rate while assuring that little or no inactivation of virus occurred due to temperature alone. Samples were taken when the sludge reached various degrees of dryness and were placed in tightly stoppered containers. These samples were stored at 21°C until the last sample was taken (4 days) to guarantee that all samples were exposed to the same temperature for the same periods of time. The solids content of each sample was then determined, and all samples were assayed for infectious viruses. For this, a portion of each sample was returned to the original solids content of 5% by the addition of an appropriate volume of water and blended in 0.2% sodium dodecyl sulfate. The sodium dodecyl sulfate procedure was used to dissociate virus from sludge solids and was found to cause no detectable loss of infectious virus. The infectivities of poliovirus type 1 and coxsackievirus Bi were determined by plaque assay as previously described (20). The type 3 reovirus infectivity assay was carried out in a similar manner, except the adsorption period was extended to 2 h and the incubation period was increased to 70 h before staining the monolayer with 0.5% crystal violet. The dewatered samples were then stoppered and left for an additional 7 days at 21°C, at which time both the solids content and infectivities were again determined. No significant change in solids content of the samples occurred during the additional 7-day period. In experiments where the viruses were added to sludge after dewatering, the moisture content of airdried sludge, containing 95% solids, was adjusted to the appropriate value by blending with distilled water, and samples were seeded with poliovirus and again blended. These samples were then placed in tightly stoppered bottles and left at 21°C. After 7 days, portions of each sample were removed and returned to a solids content of 5% by the addition of water before blending in 0.2% sodium dodecyl sulfate. Finally, the infectivities of viruses in all samples were measured as described above. Analysis of radioactively labeled poliovirus and poliovirus RNA recovered from sludge after dewatering. Poliovirus strain CHAT was labeled either with [3H]uridine or "C-protein hydrolysate and extensively purified as previously described (20). [3H]uridine-labeled virus was diluted into raw sludge, and the mixture was allowed to air dry at 21°C as described above. The solids contents of the samples were determined, and each sample was prepared for analysis by the sodium dodecyl sulfate blending procedure. Sludge solids were then removed by centrifugation at 18,000 x g for 20 min, and the supernatant was analyzed for recoverable plaque-forming units
565
(PFU) and total radioactivity. The latter was measured in Bray scintillation fluid. The sedimentation profile of 3H-labeled poliovirus particles after dewatering in sludge was determined by using density gradient centrifugation. After sludge solids were removed by centrifugation, a portion of the supernatant was mixed with "C-labeled poliovirus, which marked the position of normal infectious virus (156S). This double-labeled material was then layered onto 15 to 30% glycerol gradients containing 0.1 M NaCl, 0.01 M tris(hydroxymethyl)aminomethane (pH 7.5), and 0.001 M ethylenediaminetetraacetate and centrifuged in an SW27 rotor at 27,000 rpm for 4 h at 4°C. Fractions collected from the bottom of the gradients were analyzed for total radioactivity in Bray scintillation fluid. [3H]uridine-labeled ribonucleic acid (RNA) released from poliovirus in dewatered sludge was further analyzed by centrifugation in glycerol gradients. A treated sample prepared as described above was layered onto a 5 to 30% gradient containing 0.1 M NaCl, 0.02 tris(hydroxymethyl)aminomethane (pH 8), and 0.005 M ethylenediaminetetraacetate and centrifuged at 45,000 rpm in an SW50.1 rotor for 2.75 h at 4°C. Fractions were again collected from the bottom of the gradient and analyzed for total radioactivity. RNA extracted with phenol from [3H]uridine-labeled poliovirus was analyzed in a separate but identical gradient to mark the position of intact RNA (35S). Analysis of radioactively labeled poliovirus after evaporation in water. Purified poliovirus labeled with [3H]uridine was diluted 300-fold with distilled water, and 15-ml fractions were distributed into identical 20-ml beakers. These samples were left at 21°C, and the water in the open beakers was allowed to evaporate. After the amount of water lost through evaporation was determined, the recoverable PFU and radioactivity in each beaker were measured and compared to those of the control sample incubated for the same period without evaporation. The state of the viral particles was again examined by glycerol gradient centrifugation.
RESULTS AND DISCUSSION Recoverability of poliovirus from sludge after dewatering by evaporation. Dewatering of wastewater sludge can mean increasing the percentage of sludge solids from a fraction of a percent to 100%. It has been reported that enteric viruses are normally quite unstable in a dry state (4, 9, 15), although this instability can be at least partially overcome if drying is carried out under special conditions (3, 13). It is possible that drying sludge by evaporation may provide just such a condition. It is also possible, however, that enteric viruses are readily inactivated in sludge by evaporating only a small amount of
water. To cover both possibilities, virus survival in sludge was determined after evaporating only a few percent to nearly 100% of the water content. The initial experiment was to seed raw sludge with poliovirus and allow natural evaporation to take place at 21°C. Samples taken throughout
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APPL. ENVIRON. MICROBIOL.
WARD AND ASHLEY
the drying period were stored at 21°C in tightly stoppered bottles. After 4 days, the percentage of solids and recovery of PFU in all samples were measured. The remaining portion of each sample was incubated for an additional 7 days at 21°C before repeating both analyses. The samples were incubated the extra week without further evaporation to measure the stability of poliovirus in sludge held at different levels of dryness. The results of this experiment show that a gradual loss of recoverable infectious polioviruses occurred with the loss of water until the solids content of the sludge reached 65% (Table 1). At this point, the total decrease in PFU just exceeded 75% when compared with a control sample that had been kept at 210C without evaporation. A further reduction in viral infectivity of more than three orders of magnitude occurred when the solids content of the sludge was permitted to increase to 83%. Little additional loss of recoverable infectious viruses was observed when the percentage of sludge solids was increased from 83 to 91%. After another 7 days of incubation at 21°C without further dewatering, the recoverability of infectious polioviruses decreased in all samples, but the loss appeared to be greatest in samples that contained more than 65% solids. For example, the sample containing 83% solids lost more than 1 log of viral infectivity during this week, while the titer of viruses in the sample containing 65% solids decreased only about 20%. Taken together, these results indicate that there was a breakpoint in the stability of poliovirus in sludge with between 65 and 83% solids. This finding suggests that it may be possible to efficiently inactivate poliovirus in sludge by simply increasing the solids content above 83% by evaporation. Breakdown of poliovirus particles through dewatering of sludge. Although the plaque-forming ability of poliovirus was lost in sludge as a result of dewatering, it was not proven that the infectivity of the virus was destroyed during this process. Quite possibly, polioviruses become immeshed in sludge solids during the evaporation of water and their infectivities are simply not detectable with the techniques used here. To test this possibility, the previous experiment was repeated with purified polioviruses radioactively labeled with [3H]uridine. For this experiment, however, the sludge solids were removed from the treated samples by centrifugation just before the determination of recoverable radioactivity and PFU. This step was necessary to avoid the tremendous quench-
ing effects of these solids on detection of tritium by liquid scintillation spectrometry. Centrifugation of solids caused only a small loss of radioactivity in sludge samples after dewatering (Table 2). However, the recovery of infectivity in these same samples decreased by the amounts expected as a result of evaporation of water. Thus, the decrease of viral infectivity in sludge after dewatering by evaporation was not caused by an inability to release viral particles from sludge solids pelleted during centrifugation. Although more than 75% of the radioactively labeled poliovirus particles were recovered from dewatered sludge after centrifugation of sludge solids, it is still conceivable that these particles TABLE 1. Recovery ofpoliovirus PFU from sludge after dewateringa Sludge solids (final %)
Recovery of PFU/ml 4 days 11 days
5 1.8 x 107 6.5 x 106 12 1.7 x 107 4.5 x 106 20 9.5 X 106 4.0 x 106 6.4 x 106 30 3.8 x 106 5.5 x 106 58 3.2 x 106 65 4.0 x 106 3.2 x 106 83 2.5 x 103
Copyright © 1977 American Society for Microbiology
Vol. 34, No. 5 Printed in U.S.A.
Inactivation of Enteric Viruses in Wastewater Sludge Through Dewatering by Evaporation RICHARD L. WARD`* AND CAROL S. ASHLEY2 Sandia Laboratories, Albuquerque, New Mexico 87115,1 and University of New Mexico, Albuquerque, New Mexico 871062 Received for publication 6 May 1977
The effect of dewatering on the inactivation rates of enteric viruses in sludge determined. For this study, water was evaporated from seeded raw sludge at 21°C, and the loss of viral plaque-forming units was measured. Initial results with poliovirus showed that recoverable infectivity gradually decreased with the loss of water until the solids content reached about 65%. When the solids content was increased from 65 to 83%, a further, more dramatic decrease in virus titer of greater than three orders of magnitude was observed. This loss of infectivity was due to irreversible inactivation of poliovirus because viral particles were found to have released their RNA molecules which were extensively degraded. Viral inactivation in these experiments may have been at least partially caused by the evaporation process itself because similar effects on poliovirus particles were observed in distilled water after only partial loss of water by evaporation. Coxsackievirus and reovirus were also found to be inactivated in sludge under comparable conditions, which suggests that dewatering by evaporation may be a feasible method of inactivating all enteric viruses in sludge.
was
CHAT was purchased from the American Type Culture Collection, coxsackievirus Bi was a gift of L. C. MacLaren (Department of Microbiology, School of Medicine, University of New Mexico, Albuquerque), and type 3 reovirus strain Dearing was a gift of A. J. Shatkin (Roche Institute of Molecular Biology, Nutley, N.J.). Stock preparations of poliovirus and coxsackievirus were made as previously described (20). Reovirus stocks were prepared in a similar manner except that viruses were harvested by scraping infected cells from the culture bottles about 48 h after infection, and these lysates were stored at -600C without low-speed centrifugation of cell debris. These procedural changes were made because reoviruses are more strongly cell associated than are enteroviruses. The line of HeLa cells used for the growth and plaquing of both poliovirus and coxsackievirus was a gift of R. Radloff (Department of Microbiology, School of Medicine, University of New Mexico, Albuquerque). L-929 cells, purchased from Microbiological Associates, Bethesda, Md., were used for reovirus. All cells were grown in monolayer cultures in Eagle medium containing either 5% newborn calf serum (HeLa cells) or 5% fetal calf serum (L-929 cells). Dewatering of sludge and infectivity assay of virus. Raw sludge obtained from the Albuquerque Sewage Treatment Plant was used for all dewatering experiments. Raw rather than anaerobically digested sludge was chosen for these experiments to eliminate the virucidal effects of ammonia, known to be present in digested sludge. These effects are expressed at the pH of digested sludge (>7) but not the pH of raw MATERIALS AND METHODS sludge, which is consistently about 6 or less (21). Viruses and cells. Three strains of viruses were Because the pH of raw sludge does not increase during used in these experiments. Poliovirus type 1 strain dewatering, viral inactivation occurring during this
Sewage sludge, like many of the other waste products of modem civilization, is a resource whose quantity is increasing rather than decreasing. Because the earth's supply of most natural resources is being rapidly depleted, the need to find safe and cost-effective methods of utilizing sludge is very apparent. Even so, the value of sludge as a fertilizer or an animal foodstuff is only beginning to be realized. One feature of sludge that limits its use for these purposes is that it contains pathogens. It is, therefore, of immediate importance to find low-cost methods of inactivating the pathogens in sludge without reducing its economic value. Enteric viruses are one group of pathogens commonly found in very high concentrations in wastewater sludge (12). Although most procedures employed in handling and treating sludge have been used for many years, their effects on the survival of indigenous viruses are not known. Dewatering, a procedure often used in sludge handling, is a case in point. There are numerous ways of dewatering sludge, but in many circumstances the most cost-effective method is evaporation. The purpose of this study was to determine the effect of reducing the moisture content of sludge by evaporation on the survival of enteric viruses.
564
VOL. 34, 1977
INACTIVATION OF ENTERIC VIRUSES
process should not be due to the virucidal action of ammonia. To study the fate of viruses in sludge upon dewatering, sludge was seeded with virus either before or after the dewatering process. In experiments where the virus was present during dewatering, a 100-fold dilution of a stock solution of either poliovirus, coxsackievirus, or reovirus was made directly into raw sludge containing 5% solids. After thorough mixing, the sludge was placed in a shallow pan to a depth of about 1 cm and allowed to air dry at 21°C. This temperature was chosen because it was sufficiently high to permit evaporation at a reasonable rate while assuring that little or no inactivation of virus occurred due to temperature alone. Samples were taken when the sludge reached various degrees of dryness and were placed in tightly stoppered containers. These samples were stored at 21°C until the last sample was taken (4 days) to guarantee that all samples were exposed to the same temperature for the same periods of time. The solids content of each sample was then determined, and all samples were assayed for infectious viruses. For this, a portion of each sample was returned to the original solids content of 5% by the addition of an appropriate volume of water and blended in 0.2% sodium dodecyl sulfate. The sodium dodecyl sulfate procedure was used to dissociate virus from sludge solids and was found to cause no detectable loss of infectious virus. The infectivities of poliovirus type 1 and coxsackievirus Bi were determined by plaque assay as previously described (20). The type 3 reovirus infectivity assay was carried out in a similar manner, except the adsorption period was extended to 2 h and the incubation period was increased to 70 h before staining the monolayer with 0.5% crystal violet. The dewatered samples were then stoppered and left for an additional 7 days at 21°C, at which time both the solids content and infectivities were again determined. No significant change in solids content of the samples occurred during the additional 7-day period. In experiments where the viruses were added to sludge after dewatering, the moisture content of airdried sludge, containing 95% solids, was adjusted to the appropriate value by blending with distilled water, and samples were seeded with poliovirus and again blended. These samples were then placed in tightly stoppered bottles and left at 21°C. After 7 days, portions of each sample were removed and returned to a solids content of 5% by the addition of water before blending in 0.2% sodium dodecyl sulfate. Finally, the infectivities of viruses in all samples were measured as described above. Analysis of radioactively labeled poliovirus and poliovirus RNA recovered from sludge after dewatering. Poliovirus strain CHAT was labeled either with [3H]uridine or "C-protein hydrolysate and extensively purified as previously described (20). [3H]uridine-labeled virus was diluted into raw sludge, and the mixture was allowed to air dry at 21°C as described above. The solids contents of the samples were determined, and each sample was prepared for analysis by the sodium dodecyl sulfate blending procedure. Sludge solids were then removed by centrifugation at 18,000 x g for 20 min, and the supernatant was analyzed for recoverable plaque-forming units
565
(PFU) and total radioactivity. The latter was measured in Bray scintillation fluid. The sedimentation profile of 3H-labeled poliovirus particles after dewatering in sludge was determined by using density gradient centrifugation. After sludge solids were removed by centrifugation, a portion of the supernatant was mixed with "C-labeled poliovirus, which marked the position of normal infectious virus (156S). This double-labeled material was then layered onto 15 to 30% glycerol gradients containing 0.1 M NaCl, 0.01 M tris(hydroxymethyl)aminomethane (pH 7.5), and 0.001 M ethylenediaminetetraacetate and centrifuged in an SW27 rotor at 27,000 rpm for 4 h at 4°C. Fractions collected from the bottom of the gradients were analyzed for total radioactivity in Bray scintillation fluid. [3H]uridine-labeled ribonucleic acid (RNA) released from poliovirus in dewatered sludge was further analyzed by centrifugation in glycerol gradients. A treated sample prepared as described above was layered onto a 5 to 30% gradient containing 0.1 M NaCl, 0.02 tris(hydroxymethyl)aminomethane (pH 8), and 0.005 M ethylenediaminetetraacetate and centrifuged at 45,000 rpm in an SW50.1 rotor for 2.75 h at 4°C. Fractions were again collected from the bottom of the gradient and analyzed for total radioactivity. RNA extracted with phenol from [3H]uridine-labeled poliovirus was analyzed in a separate but identical gradient to mark the position of intact RNA (35S). Analysis of radioactively labeled poliovirus after evaporation in water. Purified poliovirus labeled with [3H]uridine was diluted 300-fold with distilled water, and 15-ml fractions were distributed into identical 20-ml beakers. These samples were left at 21°C, and the water in the open beakers was allowed to evaporate. After the amount of water lost through evaporation was determined, the recoverable PFU and radioactivity in each beaker were measured and compared to those of the control sample incubated for the same period without evaporation. The state of the viral particles was again examined by glycerol gradient centrifugation.
RESULTS AND DISCUSSION Recoverability of poliovirus from sludge after dewatering by evaporation. Dewatering of wastewater sludge can mean increasing the percentage of sludge solids from a fraction of a percent to 100%. It has been reported that enteric viruses are normally quite unstable in a dry state (4, 9, 15), although this instability can be at least partially overcome if drying is carried out under special conditions (3, 13). It is possible that drying sludge by evaporation may provide just such a condition. It is also possible, however, that enteric viruses are readily inactivated in sludge by evaporating only a small amount of
water. To cover both possibilities, virus survival in sludge was determined after evaporating only a few percent to nearly 100% of the water content. The initial experiment was to seed raw sludge with poliovirus and allow natural evaporation to take place at 21°C. Samples taken throughout
566
APPL. ENVIRON. MICROBIOL.
WARD AND ASHLEY
the drying period were stored at 21°C in tightly stoppered bottles. After 4 days, the percentage of solids and recovery of PFU in all samples were measured. The remaining portion of each sample was incubated for an additional 7 days at 21°C before repeating both analyses. The samples were incubated the extra week without further evaporation to measure the stability of poliovirus in sludge held at different levels of dryness. The results of this experiment show that a gradual loss of recoverable infectious polioviruses occurred with the loss of water until the solids content of the sludge reached 65% (Table 1). At this point, the total decrease in PFU just exceeded 75% when compared with a control sample that had been kept at 210C without evaporation. A further reduction in viral infectivity of more than three orders of magnitude occurred when the solids content of the sludge was permitted to increase to 83%. Little additional loss of recoverable infectious viruses was observed when the percentage of sludge solids was increased from 83 to 91%. After another 7 days of incubation at 21°C without further dewatering, the recoverability of infectious polioviruses decreased in all samples, but the loss appeared to be greatest in samples that contained more than 65% solids. For example, the sample containing 83% solids lost more than 1 log of viral infectivity during this week, while the titer of viruses in the sample containing 65% solids decreased only about 20%. Taken together, these results indicate that there was a breakpoint in the stability of poliovirus in sludge with between 65 and 83% solids. This finding suggests that it may be possible to efficiently inactivate poliovirus in sludge by simply increasing the solids content above 83% by evaporation. Breakdown of poliovirus particles through dewatering of sludge. Although the plaque-forming ability of poliovirus was lost in sludge as a result of dewatering, it was not proven that the infectivity of the virus was destroyed during this process. Quite possibly, polioviruses become immeshed in sludge solids during the evaporation of water and their infectivities are simply not detectable with the techniques used here. To test this possibility, the previous experiment was repeated with purified polioviruses radioactively labeled with [3H]uridine. For this experiment, however, the sludge solids were removed from the treated samples by centrifugation just before the determination of recoverable radioactivity and PFU. This step was necessary to avoid the tremendous quench-
ing effects of these solids on detection of tritium by liquid scintillation spectrometry. Centrifugation of solids caused only a small loss of radioactivity in sludge samples after dewatering (Table 2). However, the recovery of infectivity in these same samples decreased by the amounts expected as a result of evaporation of water. Thus, the decrease of viral infectivity in sludge after dewatering by evaporation was not caused by an inability to release viral particles from sludge solids pelleted during centrifugation. Although more than 75% of the radioactively labeled poliovirus particles were recovered from dewatered sludge after centrifugation of sludge solids, it is still conceivable that these particles TABLE 1. Recovery ofpoliovirus PFU from sludge after dewateringa Sludge solids (final %)
Recovery of PFU/ml 4 days 11 days
5 1.8 x 107 6.5 x 106 12 1.7 x 107 4.5 x 106 20 9.5 X 106 4.0 x 106 6.4 x 106 30 3.8 x 106 5.5 x 106 58 3.2 x 106 65 4.0 x 106 3.2 x 106 83 2.5 x 103
