Fate of coxsackievirus B3 during aerobic and anaerobic sludge stabilization

Waste Management & Research (1983) 1, 237-247

FATE OF COXSACKIEVIRUS B3 DURING AEROBIC AND ANAEROBIC SLUDGE STABILIZATION$ Anne L . Nielsen* and Birgit Lydhohn* (Received 9 May 1983)

The survival of viruses has been measured in bench scale stabilization units . A strain of coxsackievirus B3 was seeded in sludge and recoveries were measured at intervals from 1 h to 70 days . Mesophilic anaerobic digesters were operated at 33°C with detention times of 15 and 33 days . Thermophilic digesters were operated at 50, 53 and 56°C with detention times of 4 to 10 days. Aerobic stabilization was carried out at 5, 20 and 33°C (with detention times of 30, 20 and 15 days, respectively . Controls were run at the same temperature in Hanks' balanced salt solution . Inactivation of virus was slower in thermophilic anaerobic digesters than in the controls and the rate of inactivation often fell off with time (tailing) . In mesophilic anaerobic digesters and aerobic stabilization units, the rates of inactivation were greater than in the controls . The rates are, nevertheless, very much faster at the higher temperatures . Up to 3 log units of destruction might be obtained if the interval between feeding and drawing at 53°C were 10 h and S log units if it were 24 h . Retention times would have to be serveral days at 33°C to obtain similar reduction in virus content . Key Words-Virus, sludge, aerobic stabilization, anaerobic digestion, thermophilic digestion, coxsackievirus B3 . 1 . Introduction One of the effects of the increased demands for treatment of wastewater in recent times is the vast production of sludge . The disposal problems of these large amounts of sludge have renewed the interest for land application and thereby the use of the fertilizing and soil conditioning capacity of the product . Before land application of sludge can be carried out without creating serious health hazards a number of problems need to be solved . Among these is the risk of dispersing viruses and other pathogens in the environment . Virus concentrations of up to 10 4-10 5 TCID 50 t 1 -1 have been found in raw mixed sludge in Denmark (Lydholm & Nielsen 1983) . Although further treatment of the raw sludge at the plants has been directed mainly towards sludge stabilization and volume reduction, some of the processes have been shown to reduce the level of viruses and other pathogens . Several examinations of virus inactivation during mesophilic anaerobic digestion have been carried out (Bertucci et al . 1977 ; Eisenhardt et al. 1977 ; Moore et al . 1977 ; Sanders et al. 1979; Berg & Berman 1980 ; Kabrick & Jewel 1982), but little interest has been paid to the effect of aerobic treatment on the virus content of wastewater sludge although this process is commonly practised . The aerobic process is known to reduce the virus content in wastewater in the activated sludge treatment (Lund et al. 1969) where some inactivation occurs, and in aerobic treatment of liquid manure *The Royal Veterinary and Agricultural University of Copenhagen, Department of Veterinary Virology and Immunology, 13, Bûlowsvej, 1870 Copenhagen V, Denmark . t Tissue Culture Infective Dose at 50 per cent level . $Supported by grants from Statens Teknisk-Videnskabelige Forskningsrâd . 0734-242X/83/030237 + 11 $03 .00/0

Q 1983 ISWA



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Anne L . Nielsen & Birgit Lydholm

(Srivastava & Lund 1980 ; Lund & Nissen 1983) . With the increasing amounts of sludge produced the interest in thermophilic anaerobic digestion (50-60°C), which can treat larger amounts than the mesophilic digestion (30-36°C) in the same reactor volume, is also increasing . A few experiments have been carried out with inactivation of virus during thermophilic digestion (Sanders et al . 1979) . The purpose of the present work was to examine and compare the rate of inactivation of coxsackievirus B3 and echovirus 11 during aerobic and anaerobic (mesophilic and thermophilic) sludge stabilization in controlled laboratory reactors at different temperatures and detention times, and compare the rate of inactivation with parameters commonly used to characterize sludge stabilization (Lund et al . 1982) . 2. Materials and methods 2 .1 . Cells and viruses

A coxsackievirus B3 and an echovirus 11 strain, both originally isolated from Copenhagen wastewater and passed in HeLa cells, were used in the experiments . Virus titrations were made in tube cultures of HeLa cells grown in Eagles medium with 2% calf serum, 200 IU benzyl-penicillin and 260 pg streptomycin ml - ' . Each tube contained 2 ml of medium, which was renewed twice during the incubation period of 7 days at 37°C . Just before inoculation mycostatin was added to the sample to a final concentration of 1000 IU ml -1 . Serial tenfold dilutions were carried out in Hanks' balanced salt solution and each dilution was inoculated into three tubes . The inoculum was 0.1 ml . 2 .2 . Recovery of virus from sludge

A Zetag precipitation method was used for recovering viruses from the sludge samples (Lydholm & Nielsen 1981) . To 20 ml sludge adjusted to pH 5 .5, 2 ml of 0 .2% Zetag 94 (cationic polyelectrolyte, Allied Colloids) was added . The sample was gently mixed for a few minutes and allowed to settle . After 15 min the sample was filtered through a 1 mm sieve mesh . The flocculate collected in the sieve was transferred to a centrifuge tube with 20 ml 10% beef extract, pH 7 (Difco) . The sample was subjected to a vigorous mechanical shaking for 1 h followed by 15 min of centrifugation at 16,000 g . The supernatant was adjusted to pH 7 .2 and decontaminated by filtration (Millex HA 0 .45 µm, Millipore SA) before titration . 2 .3 . Wastewater sludge

Raw sludge used to feed the laboratory digesters was collected one to three times a week at Lundtofte treatment plant in the suburban area of Copenhagen, during the experimental period, May to November 1980 . The raw sludge, mainly from domestic wastewater, was a mixture of primary and secondary sludge (from biological filter) . Before use bigger particles were strained off and the remaining sludge blended in a high speed laboratory blender for 5 min . The content of total volatile solids (TVS) in the sludge was adjusted to about 30 g 1 - ' by addition of water . During the experimental period the chemical oxygen demand (COD) was 20-80 g 1 -', the bicarbonate alcalinity was around 10 mEq 1 -1 and the pH essentially between 6 .0 and 6 .5 (COWIconsult 1981) . The total fat and oil content was 15-20% of the total dry matter (COWIconsult 1981) .



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Raw sludge used in the experiments with the aerobic sludge stabilization units was collected every second week at Taarnby treatment plant in the suburban area of Copenhagen in the experimental period April 1981 to April 1982 . The sludge originated from a mixture of domestic and industrial wastewater, and consisted of primary and secondary sludge (from activated sludge treatment) . Before use water was added to the sludge to obtain a concentration of total suspended solids (TSS) of about 3% and a content of TVS of 20-25 g 1 - ' . The COD was 30-50 g 1 -1 feed sludge and the pH essentially between 6 .0 and 6 .5 . 2 .4 .

Anaerobic stabilization units

Six bench-scale digesters set up in parallel in continuous systems, where temperature and detention time could be varied, were used for the examination of virus inactivation during digestion. Two of the digesters were run mesophilic at 33°C and detention time of 15 and 33 days during the experimental period . The remaining four digesters were thermophilic with temperatures varying from 50 to 56°C and detention times from 4 to 10 days . The digester units illustrated in Fig . 1 were made of Plexiglass with an overflow ensuring a constant volume of 4 1. The sludge inlet, the gas inlet, the gas outlet and the sampling device were all through the top of the digesters, which were placed in water thermostats . Mixing was carried out by gas recirculation . Raw sludge, which was kept stirred at 5°C, was pumped to the digesters at different intervals from one to 24 times a day dependent on the detention time . Initially the digesters were innoculated with previously produced, natural mesophilic or thermophilic cultures . Results from the physical/ Airing

Air valve Gas collector

Refrigerator with feed sludge

Thermostat

Treated sludge

Fig. 1 . Anaerobic digester system (After COWIconsult, 1981) .



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Anne L . Nielsen & Birgit Lydholm

chemical analyses of the operating digesters have been presented previously by COWIconsult (1981) . The gas production was 0 .35-0 .45 1 g - ' of TVS added to the thermophilic digesters and about 0 .35 1 g - ' added to the mesophilic . The reduction in TVS was 20-40% for the thermophilic and 30-40% for the mesophilic process . The COD reduction was higher by mesophilic, 40-50%, than by thermophilic digestion, 3040%. Thermophilic digestion reduced the total fat and oil content by 50-70% against 70-80% under mesophilic conditions . The pH was generally between 7 .0 and 7 .5 in both types of digesters . 2 .5 . Aerobic stabilization units

Three bench-scale stabilization units were set up at 5, 20 and 33°C with detention times of 30, 20 and 15 days respectively . The aerobic units illustrated in Fig . 2 were made of Plexiglass with an overflow ensuring a total volume of 4 1 . Air was pumped through four pumices in the bottom of the reactors, aerating and mixing the sludge . Raw sludge, which was kept at 5°C was added once a day . At the start the reactors were filled with 3 1 of activated sludge and 1 1 of raw sludge . The mean reduction in TVS was 12, 24 and 55% at 5, 20 and 33°C respectively . The average COD reduction was 4, 12 and 54% at 5, 20 and 33°C . The pH was generally between 6 .5 and 7 .5 in the three reactors . The average oxygen content in the reactors was 6 mg 02 1 - ' at 5°C, 1 .2 mg 02 1 - ' at 20°C and 0.4 mg 02 1 - ' at 33°C .

Fig . 2. Aerobic stabilization system.

As a measure for the microbial activity in the reactors the rate of oxygen consumption in the stabilization sludge was measured once a week . Air was bubbled through 200 ml of sludge from the reactors at 5, 20 or 33°C till a concentration of 6 mg 02 1- ' has obtained. No further oxygen was added and during mechanical stirring the time for reduction in oxygen concentration to 1 mg 0 2 1 - ' was measured. The rates observed at 5°C were essentially between 10 and 80 mg 0, 1 - ' h - ', at 20°C between 80 and 180 mg 02 1 - ' h -1 and at 33°C between 175 and 360 mg 02 1 - ' h - ' .



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3. Experiments and results 3.1 . Recovery of coxsackievirus B3 from seeded sludge by use of a Zetag precipitation method

One of the main problems in studies of inactivation of enteric viruses during wastewater and sludge treatment is the lack of a quantitatively reliable method usable for various types of samples . For the present work the Zetag preciptation method was chosen. Before use the efficiency of the method was examined on raw sludge . Four 20 ml samples of raw sludge seeded with coxsackievirus B3 to a final concentration of 10 5 TCID SO ml -1 were treated as described by the method . Virus recoveries found in the eluates were between 60 and 120%, which indicates that about all infectious coxsackievirus B3 present in raw sludge are recoverable by the used technique . 3.2 . Comparison of virus inactivation in sludge of free viruses and viruses preincorporated into sludge particles

As the naturally occuring viruses usually are present in raw sludge at a relatively low concentration and an irregular occurence no significant values for the rate of inactivation during sludge treatment can be obtained . Therefore it was found necessary in the present work to add laboratory strains of virus in high concentrations to the stabilization units . Indigenous viruses in sludge are mainly incorporated into the sludge particles . Therefore to simulate natural conditions it would seem appropriate to establish solidassociation before addition of viruses to the stabilization units . In this study a seeded sludge was prepared by aerating return sludge for 2 h before addition of untreated wastewater and coxsackievirus B3 suspension . Aeration was continued for 2 h and the virus seeded activated sludge filtrated to increase the amount of TSS (to 2%) . The concentration of virus was 10' TCID SO ml -1 . In a batch experiment run mesophilic (35°C) and another thermophilic (56°C) with equal volumes of raw and digested sludge the inactivation of coxsackievirus B3 in the prepared seeded sludge was compared to inactivation of virus added directly as a suspension . No difference in the rate of inactivation was observed . At 35°C a 2 log,, unit reduction was found in 7 days and at 56°C a 2-3 log,, unit reduction in 3 h . Consequently in the experiments with the bench-scale stabilization units it was decided to add virus directly as a suspension . 3.3 . Anaerobic sludge stabilization The rate of virus inactivation during anaerobic sludge stabilization was studied in a joint investigation, in which sludge reactions were characterized (COWIconsult 1981) and the rate of bacteria inactivation followed (Munch & Schlundt 1983) . The virus inactivation experiments were all carried out after the results showed that stable conditions had been attained in the digesters . The virus suspensions (concentration about 10° TCID 5O 1 -1 ), were added to the digesters in the proportion 1 : 100. Thus the inactivation could be followed through 5 log,,, units till the limit of the assay technique (5 TCID 5O ml - 1 ). Samples were taken with a few hours interval over a 24-h period from the thermophilic units and with a few days interval over 2 weeks from the mesophilic. The sludge samples were treated by the Zetag precipitation method before assaying in HeLa cells. During the sampling period unseeded raw sludge was added to the digesters and treated sludge removed to ensure the actual detention time .



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In parallel with the digester experiments virus inactivation in controls with Hanks' balanced salt solution (pH 7) at the respective temperatures was examined . The recoveries of coxsackievirus B3 at different temperatures in the thermophilic range are shown in Fig . 3(a) from which it can be seen that the inactivations mainly follow first order kinetics with a distinct connection between temperature and rate of inactivation . In the thermophilic digesters the inactivation was followed at three different temperatures, 50, 53 and 56 ° C . In Fig . 3(b) the results, corrected for dilution with the unseeded feed sludge added during the experiments, are shown . From Fig . 3 it is seen that the inactivation generally went faster in the controls than in the digesters at the same temperature in the thermophilic range . The rate of inactivation in the digesters was varying . Under a few conditions first order reactions were followed during 5 log,, units, but mostly the rate of inactivation deviated from first order reactions after a few hours by gradually slowing down . Under thermophilic conditions it was not possible to demonstrate a correlation between inactivation and temperature and/or detention time . o

10 -2

10 -4

10-5

10 -6

3 Time (h)

Fig . 3 .(a) Coxsackievirus B3 recovered at 50 ( •) , 53 (0) and 56°C (o) in Hanks' balanced salt solution . (b) Coxsackievirus B3 recovered in sludge during anaerobic sludge digestion at 50, 53 and 56°C . ∎, 50°C, DT 6 days, GP unknown ; A, 50°C, DT 8 days, GP 0 .351 g - 'TVS added ; 0, 53°C, DT4 days, GP 0 .301 g- ' TVS added; *, 53°C, DT 6 days, GP 0.46 1 g - ' TVS added; *, 53°, DT 8 days, GP unknown ; L, 53°C, DT 8 days, GP 0 .50 1 g - ' TVS added ; 0, 53°C, DT 10 days, GP 0 .38 1 g - ' TVS added; •, 56°C, DT 6 days, GP 0.34 1 g - ' TVS added . DT = detention time ; GP = gas production ; TVS = total volatile solids .

In an experiment carried out with echovirus 11 at 53°C [Fig . 4(a)] the inactivation found was much faster than what was obtained for coxsackievirus B3 . The gas production was of high intensity. In the mesophilic digesters kept at 33 ° C the inactivation of coxsackievirus B3 was



243

Fate of coxsachievirus during sludge stabilization 10 0

(a)

10

10 -2

T >

oo

10-3

4)

10 -4

10 -

10 -6

l 3

6

Time (h)

10

5

15

Time (days)

Fig. 4 .(a) Echovirus 11 recovered in sludge during digestion and in Hanks' balanced salt solution (0) at 53°C . 0, Digester, DT 6 days, GP 0 .40 1 g - ' TVS added. (b) Coxsackievirus B3 recovered in sludge during digestion and in Hanks' balanced salt solution (0) at 33°C . A, Digester, DT 15 days, GP 0 .35 1 g - ' TVS added ; A, digester, DT 30 days, GP 0 .37 1 g - ' TVS added .

followed at detention times of 15 and 30 days as shown in Fig . 4(b) . The results presented in the figure are corrected for dilution with the feed sludge added during the experiments . The virus inactivation in the digesters at 33°C was faster than in the control, contrary to what was found in the thermophilic range . For both digesters at 33°C the intensity of the gas production was of medium intensity when the experiments were carried out . A single experiment with echovirus 11 at mesophilic conditions gave results similar to the results for coxsackievirus B3 . 3 .4 . Aerobic sludge stabilization

The experiments carried out to follow the virus inactivation during aerobic sludge treatment were carried out when stable conditions, according to parameters measured, had been obtained in the stabilization units . Virus addition, treatment of sludge samples as well as the corresponding controls in Hanks' balanced salt solution were carried out as described for the anaerobic experiments . Samples were taken with a few days interval from the 33°C reactor, from the reactor at 20°C about once a week and from the 5°C reactor every second week . Two experiments were carried out at each temperature . The inactivation of coxsackievirus B3 in Hanks' balanced salt solution at the three temperatures, at which the reactors were run, are shown in Fig . 5(a) . As for the temperatures in the thermophilic range there is a clear connection between rate of inactivation and temperature . In Fig . 5(b) the results from the reactors kept at 33, 20 and 5°C, with detention times of respectively 15, 20 and 30 days, are shown . In the figure corrections are made for the dilution with feed sludge added after seeding with the virus suspension and also for the volume reduction caused by the stabilization process and the evaporation . In four of

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Fig . 5 . Coxsackievirus B3 recovered during aerobic sludge stabilization at 5, 20 and 33°C . A, 5°C, DT 30 days, OC 44 ± 10 mg 0 2 1 - ' h - ' ; 0, 5°C, DT 30 days, OC 28 ± 3 mg O Z 1 - ' h -1 ; ∎, 20°C, DT 20 days, OC 213 ± 35 mg0 2 1 - ' h -1 ; O, 20°C, DT 20 days, OC 121 ± 14 mgO 2 1 - ' h' 1 ; •, 33°C, DT 15 days, OC 212 ± 52 mg0 2 1 - ' h - ' ; 0, 33°C, DT 15 days, OC 302 ± 32 ng 0 2 1 -1 h - ' . OC = oxygen consumption (see text) .

the experiments the rate of inactivation seems to follow first order reactions, whereas in the other two a deviation from first order reactions is found after some days . At 33°C the rates of inactivation found at the two experiments were much alike and a bit faster than in the controls. The rate is very similar to the average rates found in the mesophilic digesters although these deviated from first order reactions [Fig . 4(b)] . One log,, unit reduction occurred in 2 .5-3 days . A difference in the inactivation pattern was found between the two experiments at 20°C . In one of the experiments the inactivation (1 log, o unit per 14 days) resembled that of the control, but in the other a fast inactivation was found during the first 2 weeks followed by a rate very much like the one found in the control and in the other experiment at 20 9C. The average reduction rate was 1 log,, unit per 8 days . At 5°C the rates of inactivation were considerably higher than in the salt solution where the inactivation was negligible during the 70 days of examination . The inactivation rates obtained were 1 log o unit per 27 and 17 days . 4. Discussion

As seen from the inactivation rates in the controls [Figs 3(a) ; 4(b) ; 5(a)] heat is of great importance for the inactivation of virus, even a small rise in temperature result in a higher rate . Obviously the composition and activity of the environment also influence the inactivation [Figs 3(b) ; 4(b) ; 5(b)] . In the mesophilic digesters (33 °C) and in the aerobic reactors (5, 20, 33°C) the inactivation of coxsackievirus B3 was faster than in the corresponding control, indicating the existence of factors in the digesting sludge, which increase viral destruction . Unlike this the inactivation in the thermophilic digesters (50-56°C) generally is slower than found in the controls, therefore in the thermophilic digesting sludge some virus protective factors must be present . In contrast to these results from eight experiments with coxsackievirus B3 [Fig . 3(b)] Sanders et al . (1979) found in two experiments equal rates of poliovirus 1 recovery in digesters and controls in salt solution at 50°C . For the thermophilic digesters in the present work the



Fate of coxsachievirus during sludge stabilization

245

inactivation pattern was varied independent of temperature . Digesters of up to 6°C difference in operating temperature were seen to have the same influence on the coxsackievirus B3 added to the sludge . Neither in the thermophilic nor in the mesophilic anaerobic digesters the operating detention time seems to have any significant effect on the virus inactivation . The characteristic high rate of inactivation during the first few hours after addition of virus to the thermophilic digesters [Fig . 3 (b)] and during the first day in the mesophilic anaerobic digesters [Fig. 4(b)] might be caused by the addition of unprotected virus in suspension. After some contact time in the active biomass in the digester virus will be embedded in the sludge flocs, which is known to protect the virions against inactivation . In most of the aerobic experiments this high initial rate can not be seen, so the aerobic environment must be less drastic for the virus in suspension than the anaerobic . The average inactivation rate found in the anaerobic mesophilic digesters (33°C) are very similar to the aerobic inactivation at the same temperature aside from the deviation from first order reactions . In batch experiments with porcine enterovirus (Talfan virus) in liquid manure Lund & Nissen (1983) found much higher activation rates rates during aerobic than during anaerobic conditions at 5 and 20°C . Different enteric viruses are known to have different sensitivity towards temperature, pH and other factors . In a mesophilic digester experiment with exhovirus 11 an inactivation rate similar to coxsackievirus B3 was obtained, whereas at 53°C the echovirus inactivation seemed much faster than even the fastest found for coxsackievirus [Fig . 4(a)] . Bertucci et al. (1977) found rates from 1 to 3 log o units per 48 h at 35°C for four different enteric viruses during anaerobic digestion . Echovirus 11 was the slowest, after this came poliovirus 1 and coxsackievirus B4, and the fastest was coxsackievirus A9 . The inactivation found for echovirus 11 during 48 h was slower than the initial rate obtained in the present experiment . This can be caused either by different experimental conditions, variation in the digesting environment as found with coxsackievirus B3 in the thermophilic range or variation in resistance between different strains of the virus . Parallel to some of the anaerobic experiments with virus the inactivation of Escherichia coli and Salmonella typhimurium was examined (Munch & Schlundt 1983) . Characteristic of the bacterial inactivation was in contrast to the virus inactivation relatively constant rates following first order reactions dependent of the temperature . A I log o reduction was obtained for S. typhimurium in 2-3 days at 33 °C, in 80-90 min at 50°C and in 50-70 min at 53°C . For E. coli a I log o reduction was obtained in 1 day at 33°C, in 80-90 min at 50°C and in 30-60 min at 53°C (Munch & Schlundt 1983) . The high rates found for virus in the first few hours in the thermophilic experiments were quite close to the ones found for bacteria, but as the virus inactivation in several cases was slowing down, viruses could be recovered in the digesters several hours after the bacteria had been inactivated . Thus none of the bacteria examined can be used to indicate presence or inactivation of viruses . As seen from the results obtained the virus inactivation during thermophilic digestion are measured in hours and during mesophilic digestion and aerobic treatment in days . If the sludge was held in the reactors the total detention time a considerable inactivation of the virus content would occur, but to ensure stable conditions within full scale reactors it is common practise to add and draw sludge several times a day for thermophilic digestion and at least once a day for mesophilic digestion and aerobic treatment . Therefore a part of the sludge removed has only been in the reactor I day or less dependent on the interval between feeding and drawing .



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TABLE 1 Estimated reduction in coxsackievirus B3 content in anaerobic and aerobic sludge treatment plants* Anaerobic Temperature Detention time (days) Rate of inactivation 0 .2 Interval 3h between feeding 10 h drawing 24 h

Aerobic

53°C

33°C

33°C

20°C

5°C

4

15

15

20

30

log h - It 0 .3 log day - 't 0 .4 log day -1 0 .1 log day - ' 0.05 log day - ' 2 log 3 log

5 log

1 .2 log

1 .4 log

0 .8 log

0 .6 log

* The inactivation is calculated from average inactivation rates obtained in laboratory scale reactors . t The inactivation rates used in the calculation is the one occurring between the fast initial and the slow final rate .

A rough estimation of the reduction in virus content which would occur in reactors with operating parameters as in some of the experimental units is given in Table 1 . It is possible with the thermophilic digestion to obtain a considerable reduction in virus concentration, but if the interval between feeding and drawing is less than 3 h the reduction will be less than 2 log units and approaching the inactivation obtained at 33°C during anaerobic or aerobic treatment . Therefore to obtain a higher reduction during thermophilic digestion than during mesophilic the interval between feeding and drawing must be of a certain length . To characterize a treated sludge product concerning presumed reduction in virus content from the operating parameters it is of utmost importance also to include the interval mentioned . Aerobic treatment at 5 and 20°C is calculated to result in reduction of less than I log o unit (Table 1) . As the reduction in virus content in the mesophilic anaerobic and the aerobic reactors is measured in days, variation within a day in the interval between feeding and drawing is less important than for the thermophilic treatment . Thus to increase the virus reduction during aerobic (unheated) and mesophilic anaerobic treatment more radical changes are needed to increase the actual detention time of the viruses in the reactors . References Berg, G . & Berman, D . (1980), Destruction by anaerobic mesophilic and thermophilic digestion of viruses and indicator bacterial indigenous to domestic sludge, Applied and Environmental Microbiology, 39, 361-368 .

Bertucci, J. J ., Lue-Hing, C ., Zenz, D ., and Sedita, S . J . (1977), Inactivation of viruses during anaerobic sludge digestion, Journal of the Water Pollution Control Federation, 49, 1642-1651 .

COWIconsult (1981), Termofil udrâdning of spildevandsslam og gylle. (Thermophilic digestion of wastewater sludge and farm slurry) . COWIconsult, Copenhagen. Eisenharat, A ., Lund, E . & Nissen, B . (1977), The effect of sludge digestion on virus infectivity . Water Research, 11, 579-581 .

Kabrick, R . M . & Jewell, W . J. (1982), Fate of Pathogens in thermophilic aerobic sludge digestion, Water Research, 16, 1051-1060 .

Lund, E ., Hedström, C . E . & Jantzen N . (1969), Occurrence of enteric viruses in wastewater after activated sludge treatment . Journal of the Water Pollution Control Federation, 41, 169174.



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Lund, E ., Lydholm, B . & Nielsen, A. L . (1982) . The fate of viruses during sludge stabilization, especially during thermophilic digestion. In Disinfection of Sewage Sludge : Economic, Technical and Microbiological Aspects (A . H . Bruce, A . H . Havelaar & P . Henife, Eds) . Proceedings of a Workship, Zurich 1982, pp . 114-125. D . Reidel, Dortecht . Lund, E . & Nissen B . (1983), The survival of various pathogens in aerated and nonaerated cattle and pig slurry . The inactivation of enteroviruses, Agricultural Wastes . Lydholm, B . & Nielsen, A . L . (1981), The use of a soluble polyelectrolyte for the isolation of virus from sludge . In Viruses and Wastewater Treatment (M . Goddard & M . Butler, Eds), pp . 85-90 . Pergamon Press, Oxford . Lydholm, B . & Nielsen, A . L . (1983), Inactivation of indigenous viruses by aerobic and anaerobic sludge stabilization . Waste Management and Research, 1, 227-235 . Moore, B . E ., Sagik, B . P . & Sorber, C . A . (1977), An assessment of potential health risk associated with land disposal of residual sludges . In Sludge Management, Disposal and Utilization, Proceedings of the 3rd National Conference, Information Transfer, Rockville, MD . Munch, B . & Schlundt, J . (1983), Reduction of various pathogens in slurry and sewage sludge subjected to chemical disinfection or to anaerobic digestion at mesophilic or thermophilic temperatures . In Hygienic Problems of Animal Manures (D . Strauch, Ed .), pp . 130-149 . University of Hohenheim, Stuttgart . Sanders, D . A ., Malina, J . F ., Moore, B . E ., Sagik, B . P . & Sorber, C . A . (1979), Fate of Poliovirus during anaerobic digestion . Journal of the Water Pollution Control Federation, 51, 333-343 . Srivastave, R . N . & Lund, E . (1980), The stability of bovine parvovirus and its possible use as an indicator for the presence of enteric viruses . Water Research, 14, 1017-1021 . Acknowledgements We wish to express our appreciation to Leon Langli, Margrethe Moller and Birte Nissen for excellent technical assistance .