Vol. 36, No. 6
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1978, p. 898-905 0099-2240/78/0036-0898$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Heat Inactivation of Enteric Viruses in Dewatered Wastewater Sludge RICHARD L. WARD' * AND CAROL S. ASHLEY2 Division 4535, Sandia Laboratories, Albuquerque, New Mexico 87185,' and University of New Mexico, Albuquerque, New Mexico 871062 Received for publication 18 September 1978
The effect of moisture content on the rates of heat inactivation of enteric viruses in wastewater sludge was determined. The protective effect of raw sludge on poliovirus previously observed (R. L. Ward, C. S. Ashley, and R. H. Moseley, Appl. Environ. Microbiol. 32:339-346, 1976) was found to be greatly enhanced in sludge dewatered by evaporation. Other enteroviruses responded in a similar fashion. This effect did not appear to be due merely to the state of dryness of the sludge samples because in humus-deficient soil, a relatively inert material, the rate of poliovirus inactivation by heat was not significantly altered through dewatering. Instead, this effect appeared to have been caused by protective substances in the sludge, such as detergents, which are concentrated through dewatering. As reported previously (R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 34:681-688, 1977; R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 36:889-897, 1978) raw sludge is not protective of reovirus, but, instead, the ionic detergents in sludge cause the rate of heat inactivation of this virus to be accelerated. Dewatering of sludge, however, was found to partially reverse this virucidal effect. Evidence is presented indicating that this reversal is caused by an unidentified protective substance in sludge also concentrated through dewatering. Finally, it was shown that the effects of raw sludge on heat inactivation of poliovirus and reovirus are greatly reduced by composting, a result that correlated with the degradation of detergents. One of the major expenses associated with the disposal and utilization of wastewater sludge is its transport. This is especially true of sludge that has not been dewatered, in which normally more than 90% of the total mass is water, which makes it the major item being transported. The cost of transporting sludge can be considerably reduced if it is dewatered. Therefore, the development and implementation of inexpensive methods to dewater sludge play a crucial role in sludge management. Another factor that influences utilization of sludge is the presence of human pathogens of enteric origin. It has already been demonstrated that one of the most efficient methods of inactivating human enteric viruses in sludge is through dewatering by evaporation (10). However, for this method to be truly effective, the water content must be reduced to less than 70%. In many cases this is not technically feasible, and other means of dewatering and pathogen reduction must be used. One such method is dewatering with heat. If, during this process, the temperature is high enough for a sufficient period of time, complete inactivation of viruses and other pathogenic species will result.
In our accompanying paper it is shown that detergents contained in wastewater sludge have dramatic but opposite effects on the rates of heat inactivation of reovirus and enteroviruses (11). Because detergents are not volatile, they become concentrated in sludge through dewatering by evaporation. As a result, dewatered sludge may enhance the effects of detergents on the rates of heat inactivation of viruses. In addition, dewatering may modify or enhance the effects of other sludge components on the rates of heat inactivation of viruses. Finally, the state of dryness of the sludge itself may have significant effects on the rates of viral inactivation by heat. The purpose of this study was to examine the effects of dewatering on the rates of heat inactivation of representative enteric viruses in sludge. As will be shown, dewatering of sludge has a large influence on the inactivation rates of all viruses tested. The factors responsible for these effects and how they can be eliminated are discussed.
MATERIALS AND METHODS Cells and virus. All strains of enteric viruses stud898
VOL. 36, 1978
VIRUS HEAT INACTIVATION IN DEWATERED SLUDGE
ied were grown and plaqued as described previously (7, 10). The initial titers of these preparations were 3 x 109 to 8 x 109 plaque-forming units (PFU)/ml for the enteroviruses and about 4 x 108 PFU/ml for reovirus. Heat treatment and infectivity analyses of viruses in dewatered sludge and soil. Sludge and soil samples to be examined for their effects on the rates of heat inactivation of enteric viruses were totally dewatered by evaporation, adjusted to the desired moisture content by the addition of distilled water, and then seeded with viruses. After the samples were thoroughly mixed, 1-g quantities were sealed in plastic bags, heated for the designated time periods, and cooled in an ice bath. The samples were then diluted with enough distilled water to cause a total dilution of the original virus preparation to be 100-fold and the concentration of solids in samples containing sludge or soil to be about 5% by weight. After being blended for 2 min, the samples were assayed for recoverable PFU as previously described (7, 10). Recoverable infectivity was determined relative to unheated samples containing the same percentages of solids, and this value is presented as surviving fraction of PFU or percentage of recovery of PFU.
RESULTS Heat inactivation of poliovirus (strain CHAT) in dewatered raw sludge. To determine the effect of dewatering on the rate of heat inactivation of enteric viruses in sludge, samples were seeded with known amounts of viruses and the rates of heat inactivation were measured as a function of temperature and moisture content.
899
In a previous publication (10) it was shown that the infectivity of viruses in raw sludge seeded before drying was reduced approximately in proportion to the water lost through evaporation at 21°C. However, if sludge was seeded after drying and further evaporation did not occur, the moisture content of the sludge appeared to have no effect on the rate of virus inactivation at this temperature. Therefore, for all the experiments reported here, the sludge was dried, adjusted to the predetermined moisture content by the addition of distilled water, and then seeded with virus just before heat treatment. For all strains of enteric viruses studied by this procedure, the recoverable infectivity from the unheated control sample was routinely greater than 30% relative to unheated samples not mixed with sludge, and normally this value was very nearly 100%. The initial experiment to determine the effect of dewatering on the rate of viral inactivation with heat was carried out with poliovirus type 1, strain CHAT, in raw sludge. In agreement with previous results (12), raw sludge was found to be protective of this virus against heat (Fig. 1). However, this protective effect is greatly enhanced through dewatering. For example, a greater number of viruses were inactivated in just 2 min at 51°C in sludge containing 5% solids than in 100 min in sludge with 80% solids. There were two apparent explanations for this observation. The first was that one or more
10
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0.
0
I
10- \ 0
10-3 >b
A
*0 -.
0b4 4 47( -4
Time (min)
FIG. 1. Heat inactivation ofpoliovirus type 1, strain CHAT, in dewatered raw sludge. The moisture content of raw sludge dried by evaporation was adjusted by the addition ofdistilled water so that the final percentages of solids (by weight) were 5 (0), 40 (0), or 80 (A) after seeding with poliovirus. These samples, together with control samples without sludge (O), were heated for the times and at the temperatures shown before assaying for recoverable PFU.
900
WARD AND ASHLEY
APPL. ENVIRON. MICROBIOL.
protective substances in the sludge became concentrated by dewatering, which caused increased stabilization of virus particles. The second explanation was that this virus is less sensitive to heat inactivation in a reduced moisture environment. The latter explanation could be tested by measuring the effect of moisture content on the rate of heat inactivation of this virus in a relatively inert material. Because much of the soil in the Albuquerque area is essentially void of humic matter, it appeared to be a possible candidate for such material. Therefore, heat inactivation of poliovirus (CHAT) was studied in this soil as a function of moisture content. The calcareous soil used in this study slightly accelerated poliovirus inactivation at 44°C (Fig. 2). However, the effect was very similar whether the solids content of the soil was 5 or 80%. From this result it appeared that reduced moisture content alone may not be resporfsible for the great reduction in the rate of heat inactivation of poliovirus observed in dewatered sludge. In the previous manuscript it was shown that the detergents in sludge protect poliovirus against inactivation by heat (11). It is possible that stabilization of this virus against heat in dewatered sludge occurs as a result of decreased moisture content coupled with the effect of detergents. Therefore, the rate of poliovirus inac10 0
tivation was studied at 44°C in soil containing different percentages of solids but a constant concentration of sodium dodecyl sulfate (SDS), a standard detergent. SDS added to soil at a low concentration (0.005%) is quite protective of poliovirus against heat inactivation (Fig. 3). However, as was found in the absence of this detergent, the rate of inactivation appeared to be independent of the moisture content of the soil sample. This result supports the conclusion that moisture content itself has an insignificant effect on the rate of poliovirus inactivation by heat. Therefore, the most likely explanation for increased thermal stability of poliovirus in dewatered sludge is that some protective component of sludge is concentrated during dewatering. Because detergents are quite protective of poliovirus against heat at concentrations found in sludge and, moreover, are not lost through evaporation (11), they are probably at least one of the sludge components responsible for this effect. An increase in the protective effect of SDS on poliovirus in soil containing 80% solids which was observed when the SDS concentration was increased from 0.005 to 0.08% was consistent with this suggestion (results not shown). Heat inactivation of other enteroviruses in dewatered raw sludge. To determine the general applicability of these results, other strains of enteric viruses were tested under similar conditions. Because poliovirus belongs to
10°0
a.. 0 2
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lo-l
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-40
-
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C
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½
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15
20
Time (min) FIG. 2. Heat inactivation of poliovirus in dewatered soil. Dried soil was adjusted by the addition of distilled water to contain 5% (A) or 80%o (0) solids (by weight) after seeding with poliovirus type 1, strain CHAT. These samples were heated at 440C for the times specified and assayed for recoverable PFU along with controls not containing soil (-).
1U
20
40
Time
60
80
100
(min)
FIG. 3. Effect of SDS on heat inactivation ofpoliovirus in dewatered soil. Dried soil was seeded with poiovirus type 1, strain CHAT, and adjusted to contain 0.005% (wt/vol) SDS and 5% (A) or 80%o (0) solids (by weight). Samples were then heated at 44°C and assayed for recoverable infectivity along with controls not containing soil or SDS (-).
VIRUS HEAT INACTIVATION IN DEWATERED SLUDGE
VOL. 36, 1978
the enterovirus group, the initial comparisons were made with other members of this group. Raw sludge was found to protect all enteroviruses tested against inactivation by heat, and it was consistently more protective in the dewatered state (Fig. 4). It appears, therefore, that the conclusions drawn concerning heat inactivation of poliovirus, strain CHAT, in dewatered sludge are applicable to other enteroviruses. Heat inactivation of reovirus in dewatered raw sludge. Another type of enteric virus commonly isolated from sludge is reovirus. It has already been shown that raw sludge increases the rate of heat inactivation of this virus (9) and that the sludge agents responsible for this effect are ionic detergents (11). Because dewatering by evaporation increases the concentration of detergents in sludge, it was probable that the rate of heat inactivation of reovirus would increase in dewatered sludge. However, this was not found to be the case (Fig. 5). Although the rate of heat inactivation of reovirus at 51°C was more rapid in sludge containing 80% solids than in the absence of sludge, the rate was considerably less than in sludge with lower percentages of solids. To determine whether moisture content per se was responsible for this anomalous result, its effect on reovirus inactivation with heat was determined in soil. As was found with poliovirus, the rate of heat inactivation of reovirus was nearly identical in soil containing 5 and 80% solids (Fig. 6). This result was observed both in the presence of 0.005% SDS, which considerably accelerated the rate of heat inactivation of reovirus, and in the absence of this detergent.
100lo . .
.
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901
Therefore, it appears that the decreased rate of heat inactivation of reovirus caused by dewatering of sludge was not due to moisture content alone. Another possible explanation for these results is that sludge contains substances that both accelerate and retard heat inactivation of reovirus. When both are present in relatively low concentrations, as found in sludge containing a low percentage of solids, the effect of the accelerating agent may be more pronounced. How-
100 0.1 00 a 10-2
>~
-3
10~~~~~~~~~~~ 0
10
20
30
40
50
Time (min) FIG. 5. Heat inactivation of reovirus in dewatered raw sludge. Reovirus samples in distilled water (U) or dewatered raw sludge containing 5% (0), 40% (0), or 80%c (A) solids were heated at 51°C and assayed for recoverable infectivity.
. -5--;
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-\
A
; f4
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20
50 0
20
50 0
20
50 0
20
50
Time (min)
FIG. 4. Heat inactivation ofpoliovirus type 1, strain Mahoney (A), poliovirus type 2, strain 712-Ch-2ab (B), coxsackievirus A13 (C), and coxsackievirus Bl (D) in dewatered rau sludge. Virus samples were heated at 500 C in distilled water (U) or in raw sludge containing 5%c (*) or 80%K (A) solids and assayed for recouerable
infectivity.
902
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APPL. ENVIRON. MICROBIOL.
WARD AND ASHLEY
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Time (min) H ic inactivation FIG. 6. Heat dewatered of reovirus in dewa soil in t)de presence and absence of SDS. Reovirus samples in distilled water (U), in soil containing 5% (A) or 8()% (0) solids, or in soil containing 0.005% SDS and1 5% (0) or 80%o (0) solids were heated at 45°C and( assayed for recovterable infectivity.
Tionmofeovirusin
ever, wbten both become more concentrated, as occurs wvith the dewatering of sludge, the protective sub,stance may be able to partially reverse the effecA of the accelerating material. This type of effectt has already been demonstrated with polioviriLas (12), and the sludge components responsibl .e have been identified. Ammonia is the accelera ting agent (8, 9), and detergents are at least onre of the protective substances (11). In the case of reovirus, the agent that accelerates heat ina ctivation is ionic detergents (11), but a protecti' ve component has not been identified. Howeve r, it has been suggested that sludge does contain a substance that is protective of reovirus against heat (11). It appears that this component is at least partially removed with the supernatant fraction after centrifugation of sludge because the resuspended pellet has much more virucidal activity than the original sludge. To further establish that a protective component in sludge can reverse the effects of detergents on reovirus, the rate of heat inactivation of reovirus in tris(hydroxymethyl)aminomethane (Tris) buffer was compared with that occurring in samples of the supernatant fraction of sludge, in both the presence and absence of SDS. Although the sludge supernatant did not alter the amount of reovirus inactivation caused by 0.1% SDS, it totally overwhelmed the effect of 0.01% SDS in this experiment (Table 1). This result clearly demonstrates that sludge contains material that can reverse the virucidal effects of a detergent on reovirus. Because it was also found that this protective material is still present
in the sludge supernatant after drying (results
not shown), it should be concentrated in sludge dewatered by evaporation. Therefore, it appears
that this unidentified sludge component is at least partially responsible for the reduced rate of heat inactivation of reovirus caused by dewatering of sludge. Heat inactivation of enteric viruses in composted sludge. One method to heat and dewater sludge at moderate cost that is being investigated is composting. During this process the solids content of the sludge typically exceeds 50%, and temperatures in excess of 55°C are maintained for several days (13). This temperature over an extended period of time would normally be sufficient to inactivate enteric viruses. However, if the detergents and other protective substances found in raw sludge are not degraded, they may be extremely protective of poliovirus and other enteric viruses against heat. Therefore, the rate of heat inactivation of viruses was compared in raw and composted sludges. The relative effects of these sludges on heat inactivation of poliovirus (CHAT) was first determined. Composted sludge (obtained from the composting facility of the U.S. Department of Agriculture in Beltsville, Md.) clearly does not provide as much protection against heat as raw sludge at the same moisture content (Fig. 7). Furthermore, the rate of heat inactivation in compost is comparable to that found in a lysate of infected cells which served as a control in this experiment. This result indicates that detergents or other substances in raw sludge protective of poliovirus against heat inactivation are present in substantially reduced amounts in composted sludge. Further support for this suggestion is provided TABLE 1. Comparative effects of SDS on heat inactivation of reovirus in Tris buffer and in the liquid fraction of sludge Sample
% ofRecovery PFU'
Tris 92 Tris + 0.01% SDS 0.02 Tris + 0.1% SDS 0.002 Sludge supernatant 97 Sludge supernatant + 0.01% SDS 90 Sludge supernatant + 0.1% SDS 0.001 a Reovirus was diluted 10-fold either into 0.01 M Tris (pH 8.5), containing the designated concentrations (weight/volume) of SDS, or into the liquid fraction of anaerobically digested sludge (supernatant after centrifugation at 18,000 x g for 20 min) buffered with 0.01 M Tris at this same pH. After heat treatment at 45°C for 20 min, the samples were assayed for recoverable PFU. Percent recoveries were determined relative to unheated samples of the same solutions.
VIRUS HEAT INACTIVATION IN DEWATERED SLUDGE
VOL. 36, 1978
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1978, p. 898-905 0099-2240/78/0036-0898$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Heat Inactivation of Enteric Viruses in Dewatered Wastewater Sludge RICHARD L. WARD' * AND CAROL S. ASHLEY2 Division 4535, Sandia Laboratories, Albuquerque, New Mexico 87185,' and University of New Mexico, Albuquerque, New Mexico 871062 Received for publication 18 September 1978
The effect of moisture content on the rates of heat inactivation of enteric viruses in wastewater sludge was determined. The protective effect of raw sludge on poliovirus previously observed (R. L. Ward, C. S. Ashley, and R. H. Moseley, Appl. Environ. Microbiol. 32:339-346, 1976) was found to be greatly enhanced in sludge dewatered by evaporation. Other enteroviruses responded in a similar fashion. This effect did not appear to be due merely to the state of dryness of the sludge samples because in humus-deficient soil, a relatively inert material, the rate of poliovirus inactivation by heat was not significantly altered through dewatering. Instead, this effect appeared to have been caused by protective substances in the sludge, such as detergents, which are concentrated through dewatering. As reported previously (R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 34:681-688, 1977; R. L. Ward and C. S. Ashley, Appl. Environ. Microbiol. 36:889-897, 1978) raw sludge is not protective of reovirus, but, instead, the ionic detergents in sludge cause the rate of heat inactivation of this virus to be accelerated. Dewatering of sludge, however, was found to partially reverse this virucidal effect. Evidence is presented indicating that this reversal is caused by an unidentified protective substance in sludge also concentrated through dewatering. Finally, it was shown that the effects of raw sludge on heat inactivation of poliovirus and reovirus are greatly reduced by composting, a result that correlated with the degradation of detergents. One of the major expenses associated with the disposal and utilization of wastewater sludge is its transport. This is especially true of sludge that has not been dewatered, in which normally more than 90% of the total mass is water, which makes it the major item being transported. The cost of transporting sludge can be considerably reduced if it is dewatered. Therefore, the development and implementation of inexpensive methods to dewater sludge play a crucial role in sludge management. Another factor that influences utilization of sludge is the presence of human pathogens of enteric origin. It has already been demonstrated that one of the most efficient methods of inactivating human enteric viruses in sludge is through dewatering by evaporation (10). However, for this method to be truly effective, the water content must be reduced to less than 70%. In many cases this is not technically feasible, and other means of dewatering and pathogen reduction must be used. One such method is dewatering with heat. If, during this process, the temperature is high enough for a sufficient period of time, complete inactivation of viruses and other pathogenic species will result.
In our accompanying paper it is shown that detergents contained in wastewater sludge have dramatic but opposite effects on the rates of heat inactivation of reovirus and enteroviruses (11). Because detergents are not volatile, they become concentrated in sludge through dewatering by evaporation. As a result, dewatered sludge may enhance the effects of detergents on the rates of heat inactivation of viruses. In addition, dewatering may modify or enhance the effects of other sludge components on the rates of heat inactivation of viruses. Finally, the state of dryness of the sludge itself may have significant effects on the rates of viral inactivation by heat. The purpose of this study was to examine the effects of dewatering on the rates of heat inactivation of representative enteric viruses in sludge. As will be shown, dewatering of sludge has a large influence on the inactivation rates of all viruses tested. The factors responsible for these effects and how they can be eliminated are discussed.
MATERIALS AND METHODS Cells and virus. All strains of enteric viruses stud898
VOL. 36, 1978
VIRUS HEAT INACTIVATION IN DEWATERED SLUDGE
ied were grown and plaqued as described previously (7, 10). The initial titers of these preparations were 3 x 109 to 8 x 109 plaque-forming units (PFU)/ml for the enteroviruses and about 4 x 108 PFU/ml for reovirus. Heat treatment and infectivity analyses of viruses in dewatered sludge and soil. Sludge and soil samples to be examined for their effects on the rates of heat inactivation of enteric viruses were totally dewatered by evaporation, adjusted to the desired moisture content by the addition of distilled water, and then seeded with viruses. After the samples were thoroughly mixed, 1-g quantities were sealed in plastic bags, heated for the designated time periods, and cooled in an ice bath. The samples were then diluted with enough distilled water to cause a total dilution of the original virus preparation to be 100-fold and the concentration of solids in samples containing sludge or soil to be about 5% by weight. After being blended for 2 min, the samples were assayed for recoverable PFU as previously described (7, 10). Recoverable infectivity was determined relative to unheated samples containing the same percentages of solids, and this value is presented as surviving fraction of PFU or percentage of recovery of PFU.
RESULTS Heat inactivation of poliovirus (strain CHAT) in dewatered raw sludge. To determine the effect of dewatering on the rate of heat inactivation of enteric viruses in sludge, samples were seeded with known amounts of viruses and the rates of heat inactivation were measured as a function of temperature and moisture content.
899
In a previous publication (10) it was shown that the infectivity of viruses in raw sludge seeded before drying was reduced approximately in proportion to the water lost through evaporation at 21°C. However, if sludge was seeded after drying and further evaporation did not occur, the moisture content of the sludge appeared to have no effect on the rate of virus inactivation at this temperature. Therefore, for all the experiments reported here, the sludge was dried, adjusted to the predetermined moisture content by the addition of distilled water, and then seeded with virus just before heat treatment. For all strains of enteric viruses studied by this procedure, the recoverable infectivity from the unheated control sample was routinely greater than 30% relative to unheated samples not mixed with sludge, and normally this value was very nearly 100%. The initial experiment to determine the effect of dewatering on the rate of viral inactivation with heat was carried out with poliovirus type 1, strain CHAT, in raw sludge. In agreement with previous results (12), raw sludge was found to be protective of this virus against heat (Fig. 1). However, this protective effect is greatly enhanced through dewatering. For example, a greater number of viruses were inactivated in just 2 min at 51°C in sludge containing 5% solids than in 100 min in sludge with 80% solids. There were two apparent explanations for this observation. The first was that one or more
10
fi101I0 ~A
0.
0
I
10- \ 0
10-3 >b
A
*0 -.
0b4 4 47( -4
Time (min)
FIG. 1. Heat inactivation ofpoliovirus type 1, strain CHAT, in dewatered raw sludge. The moisture content of raw sludge dried by evaporation was adjusted by the addition ofdistilled water so that the final percentages of solids (by weight) were 5 (0), 40 (0), or 80 (A) after seeding with poliovirus. These samples, together with control samples without sludge (O), were heated for the times and at the temperatures shown before assaying for recoverable PFU.
900
WARD AND ASHLEY
APPL. ENVIRON. MICROBIOL.
protective substances in the sludge became concentrated by dewatering, which caused increased stabilization of virus particles. The second explanation was that this virus is less sensitive to heat inactivation in a reduced moisture environment. The latter explanation could be tested by measuring the effect of moisture content on the rate of heat inactivation of this virus in a relatively inert material. Because much of the soil in the Albuquerque area is essentially void of humic matter, it appeared to be a possible candidate for such material. Therefore, heat inactivation of poliovirus (CHAT) was studied in this soil as a function of moisture content. The calcareous soil used in this study slightly accelerated poliovirus inactivation at 44°C (Fig. 2). However, the effect was very similar whether the solids content of the soil was 5 or 80%. From this result it appeared that reduced moisture content alone may not be resporfsible for the great reduction in the rate of heat inactivation of poliovirus observed in dewatered sludge. In the previous manuscript it was shown that the detergents in sludge protect poliovirus against inactivation by heat (11). It is possible that stabilization of this virus against heat in dewatered sludge occurs as a result of decreased moisture content coupled with the effect of detergents. Therefore, the rate of poliovirus inac10 0
tivation was studied at 44°C in soil containing different percentages of solids but a constant concentration of sodium dodecyl sulfate (SDS), a standard detergent. SDS added to soil at a low concentration (0.005%) is quite protective of poliovirus against heat inactivation (Fig. 3). However, as was found in the absence of this detergent, the rate of inactivation appeared to be independent of the moisture content of the soil sample. This result supports the conclusion that moisture content itself has an insignificant effect on the rate of poliovirus inactivation by heat. Therefore, the most likely explanation for increased thermal stability of poliovirus in dewatered sludge is that some protective component of sludge is concentrated during dewatering. Because detergents are quite protective of poliovirus against heat at concentrations found in sludge and, moreover, are not lost through evaporation (11), they are probably at least one of the sludge components responsible for this effect. An increase in the protective effect of SDS on poliovirus in soil containing 80% solids which was observed when the SDS concentration was increased from 0.005 to 0.08% was consistent with this suggestion (results not shown). Heat inactivation of other enteroviruses in dewatered raw sludge. To determine the general applicability of these results, other strains of enteric viruses were tested under similar conditions. Because poliovirus belongs to
10°0
a.. 0 2
0
lo-l
0.
-40
-
C
1-6
16U
C
V
½
:
A
a
._
n
U
0
5
10
15
20
Time (min) FIG. 2. Heat inactivation of poliovirus in dewatered soil. Dried soil was adjusted by the addition of distilled water to contain 5% (A) or 80%o (0) solids (by weight) after seeding with poliovirus type 1, strain CHAT. These samples were heated at 440C for the times specified and assayed for recoverable PFU along with controls not containing soil (-).
1U
20
40
Time
60
80
100
(min)
FIG. 3. Effect of SDS on heat inactivation ofpoliovirus in dewatered soil. Dried soil was seeded with poiovirus type 1, strain CHAT, and adjusted to contain 0.005% (wt/vol) SDS and 5% (A) or 80%o (0) solids (by weight). Samples were then heated at 44°C and assayed for recoverable infectivity along with controls not containing soil or SDS (-).
VIRUS HEAT INACTIVATION IN DEWATERED SLUDGE
VOL. 36, 1978
the enterovirus group, the initial comparisons were made with other members of this group. Raw sludge was found to protect all enteroviruses tested against inactivation by heat, and it was consistently more protective in the dewatered state (Fig. 4). It appears, therefore, that the conclusions drawn concerning heat inactivation of poliovirus, strain CHAT, in dewatered sludge are applicable to other enteroviruses. Heat inactivation of reovirus in dewatered raw sludge. Another type of enteric virus commonly isolated from sludge is reovirus. It has already been shown that raw sludge increases the rate of heat inactivation of this virus (9) and that the sludge agents responsible for this effect are ionic detergents (11). Because dewatering by evaporation increases the concentration of detergents in sludge, it was probable that the rate of heat inactivation of reovirus would increase in dewatered sludge. However, this was not found to be the case (Fig. 5). Although the rate of heat inactivation of reovirus at 51°C was more rapid in sludge containing 80% solids than in the absence of sludge, the rate was considerably less than in sludge with lower percentages of solids. To determine whether moisture content per se was responsible for this anomalous result, its effect on reovirus inactivation with heat was determined in soil. As was found with poliovirus, the rate of heat inactivation of reovirus was nearly identical in soil containing 5 and 80% solids (Fig. 6). This result was observed both in the presence of 0.005% SDS, which considerably accelerated the rate of heat inactivation of reovirus, and in the absence of this detergent.
100lo . .
.
.e
901
Therefore, it appears that the decreased rate of heat inactivation of reovirus caused by dewatering of sludge was not due to moisture content alone. Another possible explanation for these results is that sludge contains substances that both accelerate and retard heat inactivation of reovirus. When both are present in relatively low concentrations, as found in sludge containing a low percentage of solids, the effect of the accelerating agent may be more pronounced. How-
100 0.1 00 a 10-2
>~
-3
10~~~~~~~~~~~ 0
10
20
30
40
50
Time (min) FIG. 5. Heat inactivation of reovirus in dewatered raw sludge. Reovirus samples in distilled water (U) or dewatered raw sludge containing 5% (0), 40% (0), or 80%c (A) solids were heated at 51°C and assayed for recoverable infectivity.
. -5--;
-l2 10f 10 10-2
-\
A
; f4
0
20
50 0
20
50 0
20
50 0
20
50
Time (min)
FIG. 4. Heat inactivation ofpoliovirus type 1, strain Mahoney (A), poliovirus type 2, strain 712-Ch-2ab (B), coxsackievirus A13 (C), and coxsackievirus Bl (D) in dewatered rau sludge. Virus samples were heated at 500 C in distilled water (U) or in raw sludge containing 5%c (*) or 80%K (A) solids and assayed for recouerable
infectivity.
902
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|
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-
.°0
10-
C
-
c
APPL. ENVIRON. MICROBIOL.
WARD AND ASHLEY
S^
10'2
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10 3 0
l
5
io
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20
20
Time (min) H ic inactivation FIG. 6. Heat dewatered of reovirus in dewa soil in t)de presence and absence of SDS. Reovirus samples in distilled water (U), in soil containing 5% (A) or 8()% (0) solids, or in soil containing 0.005% SDS and1 5% (0) or 80%o (0) solids were heated at 45°C and( assayed for recovterable infectivity.
Tionmofeovirusin
ever, wbten both become more concentrated, as occurs wvith the dewatering of sludge, the protective sub,stance may be able to partially reverse the effecA of the accelerating material. This type of effectt has already been demonstrated with polioviriLas (12), and the sludge components responsibl .e have been identified. Ammonia is the accelera ting agent (8, 9), and detergents are at least onre of the protective substances (11). In the case of reovirus, the agent that accelerates heat ina ctivation is ionic detergents (11), but a protecti' ve component has not been identified. Howeve r, it has been suggested that sludge does contain a substance that is protective of reovirus against heat (11). It appears that this component is at least partially removed with the supernatant fraction after centrifugation of sludge because the resuspended pellet has much more virucidal activity than the original sludge. To further establish that a protective component in sludge can reverse the effects of detergents on reovirus, the rate of heat inactivation of reovirus in tris(hydroxymethyl)aminomethane (Tris) buffer was compared with that occurring in samples of the supernatant fraction of sludge, in both the presence and absence of SDS. Although the sludge supernatant did not alter the amount of reovirus inactivation caused by 0.1% SDS, it totally overwhelmed the effect of 0.01% SDS in this experiment (Table 1). This result clearly demonstrates that sludge contains material that can reverse the virucidal effects of a detergent on reovirus. Because it was also found that this protective material is still present
in the sludge supernatant after drying (results
not shown), it should be concentrated in sludge dewatered by evaporation. Therefore, it appears
that this unidentified sludge component is at least partially responsible for the reduced rate of heat inactivation of reovirus caused by dewatering of sludge. Heat inactivation of enteric viruses in composted sludge. One method to heat and dewater sludge at moderate cost that is being investigated is composting. During this process the solids content of the sludge typically exceeds 50%, and temperatures in excess of 55°C are maintained for several days (13). This temperature over an extended period of time would normally be sufficient to inactivate enteric viruses. However, if the detergents and other protective substances found in raw sludge are not degraded, they may be extremely protective of poliovirus and other enteric viruses against heat. Therefore, the rate of heat inactivation of viruses was compared in raw and composted sludges. The relative effects of these sludges on heat inactivation of poliovirus (CHAT) was first determined. Composted sludge (obtained from the composting facility of the U.S. Department of Agriculture in Beltsville, Md.) clearly does not provide as much protection against heat as raw sludge at the same moisture content (Fig. 7). Furthermore, the rate of heat inactivation in compost is comparable to that found in a lysate of infected cells which served as a control in this experiment. This result indicates that detergents or other substances in raw sludge protective of poliovirus against heat inactivation are present in substantially reduced amounts in composted sludge. Further support for this suggestion is provided TABLE 1. Comparative effects of SDS on heat inactivation of reovirus in Tris buffer and in the liquid fraction of sludge Sample
% ofRecovery PFU'
Tris 92 Tris + 0.01% SDS 0.02 Tris + 0.1% SDS 0.002 Sludge supernatant 97 Sludge supernatant + 0.01% SDS 90 Sludge supernatant + 0.1% SDS 0.001 a Reovirus was diluted 10-fold either into 0.01 M Tris (pH 8.5), containing the designated concentrations (weight/volume) of SDS, or into the liquid fraction of anaerobically digested sludge (supernatant after centrifugation at 18,000 x g for 20 min) buffered with 0.01 M Tris at this same pH. After heat treatment at 45°C for 20 min, the samples were assayed for recoverable PFU. Percent recoveries were determined relative to unheated samples of the same solutions.
VIRUS HEAT INACTIVATION IN DEWATERED SLUDGE
VOL. 36, 1978
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