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Virus concentration from sewage
Water Research Pergamon Press 1973. Vol. 7, pp. 945-950. Printed in Great Britain
VIRUS CONCENTRATION FROM SEWAGE AKIRA HOMMA*, MARK D. SOBSEY, CRAIG WALLIS and JOSEPH L. MELNICK Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77025 U.S.A.
(Received 26 October 1972) AbstractwOptimal conditions for concentrating poliovirus from large volumes of raw sewage were established. Solids I t~m or larger, present in the raw sewage, were removed by textile clarifying filters without significant retention of virus. By acidification of the clarified sewage and addition of salts to enhance virus attachment to the adsorbent, virus in the sewage was concentrated on a fibre glass depth filter, with subsequent elution of virus into small volumes suitable for assay. An 80-95 per cent efficiency of virus concentration was effected. INTRODUCTION THE DEVELOPMENTof sensitive and quantitative methods for isolation of viruses from sewage and water is needed to evaluate the public health hazards of viruses in the aquatic environment (BERG, 1967). Several methods for concentrating viruses from sewage have been reviewed recently by HILL et al. (1972). However, current methods do not permit sampling of raw sewage in volumes larger than 1-5 gal. Recently, we have reported the concentration of virus from tap water (WALLIS et al., 1972c) and from sea and channel water (MEa'CALF et al., 1972) using an apparatus which can process up to 400 gal h - 1 . The current report is concerned with determining the parameters for concentrating virus from sewage with this apparatus. MATERIALS AND METHODS
Monkey kidney (MK) cells Kidneys obtained f r o m immature vervet monkeys were trypsinized and grown in Melnick's medium A containing 2 per cent calf serum. Virus and virus assays A plaque-purified line of poliovirus type 1 (strain LSc)--an attenuated poliovaccine strain--was used throughout this study. Stock virus was grown in M K cells and stored at --70°C. Virus assays were made by the plaque-forming unit (PFU) method.
Sewage The raw sewage used in this study was collected from the main influent at a Houston sewage treatment plant.
Virus concentrator apparatus A portable unit for virus concentration for use in the field has been described by WALLIS et al. (1972c) and was used throughout this study.
Clarifying filters Ten-inch "Fulflo" yarn-wound filters available in porosites from 100 to 0.8/~m were obtained from the Commercial Filter Division, Carborundum Company, *
Recipient of a World Health Organization/Pan American Health Organization fellowship, on leave from the School of Public Health, Institute Oswaldo Cruz, Rio de Janeiro, Brazil. 945
946
AKIRA HOMMA, MARK D. SOBSEY,CRAIG WALLI$ and JOSEPH L. MELNICK
Lebanon, Indiana. They were housed in see-through cartridge holders which are rated at 100 psi in normal temperature ranges (FIG. 1). These filters are in the form of tubes, and the depth filtration they provide traps particles not only on the surface, but also through the total depth of the filter elements. This gives progressively finer filtration through the filter tube and provides much greater capacity for retention of solids than can be achieved by surface filter media of the same dimensions. The precision winding pattern covers the entire depth of the filter tube with hundreds of funnelshaped tunnels which become gradually finer from the surface in to the center of the tube and trap progressively finer particles as the fluid travels to the center. This method of winding provides depth filtration areas of 3.5 ft 2, for each 10-in. filter. A 10-in. filter will retain approximately ½-1 lb of solids before it must be replaced. The orlon filters are electrostatically inert, and do not adsorb viruses or metallic complexes (WALLIS et al., 1972b). They are retainers of particulates. A series of orlon 10-in. cartridge filters of decreasing porosity (100, 10 and 1/~m) and a cellulose acetate filter (0.8 tzm) were used to clarify the raw sewage. The orlon cartridge filters were washed for 5 rain with tap water using full faucet pressure [about 10 gal rain-' ], and air-flushed for 2-3 rain, in order to remove detergents incorporated during the manufacture of these elements. The cellulose acetate cartridge filter was soaked in 1 1. of 1 per cent Tween-80 (polyosyethylene [20] sorbitan monooleate) for 1 h to prevent virus adsorption to this clarifier. The cartridge was then washed also with water for 5 rain and air-flushed as described above. The excess Tween-80 must be eliminated from the filter, because even slight traces of it may interfere with virus adsorption in further steps (WALLrSand M~LNICK, 1967).
Virus adsorbent This was a filter similar to that described above, but composed of fiber glass (K27), with a 3 tzm nominal porosity (Commercial Filter Division, Carborundum Company). Virus eluent Virus was eluted from the fiber glass filter with pH 11.5, 0.05 M glycine-NaOH buffer (WALLISet aL, 1972a). Buffers that have magnesium or other cations present (which react with NaOH to form a gel) should not be used, nor should buffers be used which contain anions which complex with aluminum ions. The eluate was neutralized by the addition of pH 1.0, 0.05 M glycine-HCl buffer. RESULTS
Clarification of sewage To adsorb viruses from sewage, the sewage must be clarified without loss of virus en route to the adsorbents. Various textile filters were tested, and orlon filters (100, 10 and 1 ~,m in series) were found capable of removing sewage particulates without adsorbing significant amounts of virus. However, the orlon-clarified sewage still clogged the virus adsorbent. Therefore, an additional filter was needed to remove 0.5-1.0 ~m particles. A cellulose acetate filter was found to entrap these small particulates in sewage, but the filter also removed 20-30 per cent virus by adsorption. Therefore, the filter was treated with Tween-80 as described in the Materials and Methods section, which precluded virus adsorption (WALLISet al., 1972b). TABL~ I shows a
947
Virus Concentration from Sewage TABLE 1. CLARIFICATIONOF SEWAGE Gallons of sewage filtered*
Average No. PFU ml-1 in filtrates
Virus removed by clarification ( ~ )
Unfiltered, control sewage-~ 5 10 15 25 35
1950~ 1850 1650 1600 1500 1500
-5 15 18 23 23
* Poliovirus was added to 35 gal of raw, influent sewage, and the sewage was filtered through 100, 10 and 1 /~m orlon filters and a Tween-80 treated cellulose acetate filter (0.5-1.0 t,m) as described in the Materials and Methods section. Filtration proceeded at 10 lb in -2. 1" Index of input virus. typical experiment in which p o l i o v i r u s was a d d e d to influent sewage a n d the sewage was clarified w i t h o u t significant r e m o v a l o f virus. The flow rate o f the sewage passing the filters b e g a n to decrease c o n s i d e r a b l y after 15 gal h a d been processed, b u t the clogging o f the clarifiers did not result in a large loss o f virus. A c t u a l l y , 23 p e r cent o f the virus was r e m o v e d by the clarifiers, b u t this can readily be m a d e up by s a m p l i n g a larger v o l u m e on l o c a t i o n ; the a p p a r a t u s can concentrate virus at a rate well over 100 gal o f s a m p l e p e r hour.
Effects of different salts on virus adsorption to a fiber glass depth filter In the past, MgCI2 has been used to enhance virus a d s o r p t i o n to m e m b r a n e s (WALLIS and MELNICK, 1967). Recently, WALLIS et al. (1972a) r e p o r t e d that trivalent ions were 100- to 250-fold m o r e efficient for enhancing virus a d s o r p t i o n to m e m b r a n e surfaces. Therefore, a c o m p a r i s o n o f MgCI2 a n d A1C13 was m a d e to d e t e r m i n e the salt which enhanced virus a d s o r p t i o n to a fiber glass filter in the presence o f sewage organics (see TABLE 2). TABLE 2. EFFECTS OF DIFFERENT SALTS ON VIRUS ADSORPTION TO A FIBERGLASS DEPTH FILTER*
Average no. PFU ml-1 pH
Salt
Final molarity
Unfiltered control
Fiber glass filtrate
Virus adsorbed (~)
8.0t 3.5 8.0 3.5 3.5
None None MgCI2 MgCI2 AICI3
--0,05 0.05 0.0005
1700 1600 1750 1850 1620
1600 730 1500 940 60
6 44 14 49 96
* Poliovirus was added to sewage which was then clarified through the system described in TABLE 1. One-gallon samples of the clarified sewage were then treated with HCI to obtain the pH levels shown and with the salts indicated; representative 1-gal samples were filtered through fiber glass (K27) virus adsorbents at the rate of 0.67 gal min- ~ and filtrates were assayed for unadsorbed virus. I" Clarified sewage, unadjusted for pH.
948
AKh'L~ HOMMA,
MARKD. SOBSEY,CRAIGWALLISand JOSEPHL. MELNICK
In the presence of excess sewage organics, 0.05 M MgCI2 did not efficiently enhance virus adsorption to the fiber glass filter. However, A1Cl3 at 100-fold less molarity than MgC12 effected a 94 per cent removal of virus on the adsorbent at p H 3.5. Since A1Cla will precipitate sewage components at p H levels higher than 4.0, this salt could only be used at p H 4 or less, and preferably at p H 3.5. It is of signal importance that the sewage first be adjusted to a p H value below 4.0 before addition of aluminum ions.
Effects of flow rates on virus adsorption to fiber glass filters One-gallon samples of sewage containing added virus were filtered at different flow rates through a fiber glass virus adsorbent. The experimental procedures and results are shown in TABLE 3. At flow rates up to 0.76 gal rain-1, 96 per cent of the virus was adsorbed by the glass filter. As the flow rate increased to 1.66 gal rain-~, only 76 per cent of the virus was adsorbed, and at 2.5 gal rain-a, 66 per cent was removed by the virus adsorbent. Therefore, subsequent experiments were performed using flow rates of 0.75 gal rain- x. TABLE 3. EFFECTS OF FLOW RATES ON VIRUS ADSORPTION TO FIBERGLASS
Flow rates of sewage-virus samples* (gal rain- 1)
Average No. PFU ml-1 in fiber glass filtrates
Virus adsorbed (%)
Control virus in sewage (input)~ 0.56 0.76 0.86 1.66 2.50
1610 70 71 210 390 550
-96 96 87 76 66
* Virus was added to influent sewage as described previously. The sewage was clarified as described in TABLE1, and then treated with HCI and AICIa (0.0005 M) as described in TABLE2. Onegallon samples were then filtered at the flow rates indicated and filtrates were assayed for unadsorbed virus.
Effects of membrane coating components (MCC) on virus adsorption to fiber glass filters Organic compounds, especially proteins, will compete with viruses for reactive sites on membranes or textile adsorbents (WALLIS et al., 1972b; WALLIS and MELNICK, 1967). These compounds have been termed MCC. When a virus adsorbs to a reactive site on an adsorbent, M C C will elute the virus by exchanging for it. To overcome this problem, M C C had to be removed by passing sewage through anion exchangers; the virus could then be concentrated on nitrocellulose membranes (WALLIS and MELNICK, 1967). However, since the depth-type fiber glass adsorbents have surface capacities far greater than membrane surfaces, the resins were not used to remove MCC. An experiment was conducted to determine the amount of sewage which could be processed through the virus adsorbent before the accumulating effects of M C C in the sewage prevented virus f r o m being adsorbed on the fiberglass filter. The experimental procedures and results are shown in TABLE 4. When a virus adsorbent was pretreated
FIG. l. Fulflo filter cartridge in holder. A transparent filter holder x~hich is rated at I O0 Ib in-2. c o n t a i n i n g a Fulflo filter cartridge. Water is forced from the outside of the filler into the center effluent channel which is 1 in. dia. and 10 in. long
(fachlg p. 946)
Virus Concentration from Sewage
949
with as much as 35 gal of sewage, the treated filter could still adsorb 74 per cent of the virus, with a 64 per cent recovery by elution. In view of the gross organic load present, these etticiencies are exceptionally significant. TABLE 4.
EFFECTS OF MEMBRANE-COATING COMPONENTS ADSORPTION TO FIBER GLASS FILTERS
Gallons of clarified sewage used to pretreat fiber glass virus adsorbent* Untreated filtrate--> 5 10 15 25 35
(MCC)
ON VIRUS
Virus (~) Adsorbent Eluted 97 80 65 63 74 74
87 69 60 50 50 64
* Sewage (virus-free) clarified as described in TABLE1 was treated with HCI and AIC13 as described in TABLE2, and the samples indicated were filtered through fiber glass virus adsorbents. Each adsorbent filter was rinsed with saline and pretreated with clarified sewage in the amount indicated. Virus added to 1 gal of clarified sewage at pH 3.5 containing 0.0005 M AICIa was then passed through the filter. The filtrates were examined for unadsorbed virus, and then after a rinse with 1 gal of saline, the virus was eluted with 800 ml of glycine buffer, p H I 1.5, as described in the Materials and Methods section, and the basic eluate was neutralized with HCI.
Concentration of exogenously added poliovirus from raw sewage using the portable virus concentrator Sixty liters of raw, influent sewage were processed using the virus concentrator and the optimal methods described above. Poliovirus was added to the 60 I. of sewage, and the virus-sewage mixture was processed through the virus concentrator as described in TABLE 5. The sewage passing the clarifiers was treated with aluminum salts and HCI by an injection system to optimize virus adsorption on the fiber glass filter. Only 6 per cent of the virus passed the virus adsorbent. Elution of virus off this adsorbent yielded 81 per cent of the total input virus in a 1-1. eluate. The total time for processing this volume and recovering the virus was 20 min. DISCUSSION The current study was concerned with concentrating viruses from sewage, by varying the methods previously developed for tap water and sea water, which have a minimal organic load. A 60-fold concentration was readily achieved. Other methods presently in use will concentrate virus from sewage and other waters with higher concentration factors, but with these methods only 1-5 gal of sewage can be examined (HILL et al., 1972). If some types of enteric viruses are present in sewage at levels not detectable in 1-5 gal, they would not be detectable by prior methods. Further, we are at present developing a reconcentration method which will reduce the 1-1. final eluate to 5 ml, which will then represent a concentration factor of 12,000-fold when 601. are processed, and a correspondingly higher concentration if the initial sample is larger.
950
AKIRAI-'IOMMA,MARKD. SOBSEY,CRAIGWALLISand JOSEPHL. MELNICK TABLE 5. CONCENTRATIONOF EXOGENOUSLYADDEDPOLIOVIRUSFROMRAW SEWAGE USINGTHE PORTABLEVIRUSCONCENTRATOR*
Sample Raw sewage -+- virus Fiber glass filtrate Saline wash Eluate
Volume (1.)
No. PFU in total volume ( )< 109)
Total PFU (~o)
60 60 4 1
1.6 0.1 0.035 1.3
100 6 2 81
* Undiluted stock poliovirus was added to 601. of raw sewage. The virus-containing sewage was processed through the portable virus concentrator at a flow rate of 0.75 gal rain- t as follows: (1) The sewage-virus mixture was clarified by filtration through 100, 10 and 1 tzm orlon filters, in series, and through a Tween-80 treated cellulose acetate (0.8/zm) filter. (2) An AICI3-HCI concentrate was driven into the clarified sewage-virus mixture at a dilution of 1:100 to give the mixture a pH of 3.5 and an AICIa concentration of 0.0005 M. The formulation of the A1CI3HC1 concentrate was predetermined by titration of an aliquot of clarified sewage with I N HCI. The injection of the concentrate was monitored by continuous measurement of pH and conductivity. (3) The treated sewage-virus mixture was then filtered through a fiber glass (K27) virus adsorbent. (4) After the entire 60 1. was processed by this procedure, 4 1. of physiological saline was filtered through the fiber glass adsorbent to remove residual aluminum ions. (5) Virus was eluted off the fiber glass cartridge with 800 ml of glycine-NaOH biaffer, pH 11.5, and the eluate was partially neutralized with 200 ml of glycine-HCl buffer, pH 1.0. A t present, the virus c o n c e n t r a t i n g a p p a r a t u s is being used for detection o f viruses in relatively clean waters (WALLIS et al., 1972c), c h a n n e l , estuary a n d sea waters (METCALF et aL, 1972), m u n i c i p a l solid waste leachates ( m a n u s c r i p t in preparation), a n d for the survey o f wastewaters a n d effluent receiving bodies. Once the second step---reconcent r a t i o n of the initial eluate to a final v o l u m e of 5-10 m l - - i s accomplished, the procedure will be o f p a r t i c u l a r value for public health studies concerning the spread o f sewageb o r n e viruses. Acknowledgements--Supported in part by research project No. R-801220 from the Environmental Protection Agency. The authors wish to thank CHARLESSTAGGfor his engineering assistance.
REFERENCES BERG G. (ed) (1967) Transmission of Viruses by the Water Route. Wiley, New York. HILL W. F. JR., AKINE. W., BENTONW. H. and METCALFT. G. (1972) Virus in water. II. Evaluation of membrane cartridge filters for recovering low multiplicities of poliovirus from water. AppL MicrobioL 23, 880-888. METCALET. G., WALLISC. and MELNICKJ. L. (1972) Concentration of viruses from seawater. Proc. 6th Int. Conf. on Water Pollution, Jerusalem, June 18-23, pp. 109-115, 1972 (in press). WALLISC., HENDERSONM. and MELNICKJ. L. (1972a) Enterovirus concentration on cellulose membranes. Appl. Microbiol. 23, 476-480. WALLI$ C., HOMMAA. and MELNICKJ. L. (1972b) Apparatus for concentrating viruses from large volumes. J. Am. Water Wks. Ass. 64, 189-196. WALLISC., HOIV~AA. and MELNICKJ. L. (1972c) A portable virus concentrator for testing water in the field. Water Research 6, 1249-1256. WALLISC. and MELNICKJ. L. (1967) Concentration of viruses from sewage by adsorption on Millipore membranes. Bull. WId Hlth Org. 36, 219-225.
VIRUS CONCENTRATION FROM SEWAGE AKIRA HOMMA*, MARK D. SOBSEY, CRAIG WALLIS and JOSEPH L. MELNICK Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Texas 77025 U.S.A.
(Received 26 October 1972) AbstractwOptimal conditions for concentrating poliovirus from large volumes of raw sewage were established. Solids I t~m or larger, present in the raw sewage, were removed by textile clarifying filters without significant retention of virus. By acidification of the clarified sewage and addition of salts to enhance virus attachment to the adsorbent, virus in the sewage was concentrated on a fibre glass depth filter, with subsequent elution of virus into small volumes suitable for assay. An 80-95 per cent efficiency of virus concentration was effected. INTRODUCTION THE DEVELOPMENTof sensitive and quantitative methods for isolation of viruses from sewage and water is needed to evaluate the public health hazards of viruses in the aquatic environment (BERG, 1967). Several methods for concentrating viruses from sewage have been reviewed recently by HILL et al. (1972). However, current methods do not permit sampling of raw sewage in volumes larger than 1-5 gal. Recently, we have reported the concentration of virus from tap water (WALLIS et al., 1972c) and from sea and channel water (MEa'CALF et al., 1972) using an apparatus which can process up to 400 gal h - 1 . The current report is concerned with determining the parameters for concentrating virus from sewage with this apparatus. MATERIALS AND METHODS
Monkey kidney (MK) cells Kidneys obtained f r o m immature vervet monkeys were trypsinized and grown in Melnick's medium A containing 2 per cent calf serum. Virus and virus assays A plaque-purified line of poliovirus type 1 (strain LSc)--an attenuated poliovaccine strain--was used throughout this study. Stock virus was grown in M K cells and stored at --70°C. Virus assays were made by the plaque-forming unit (PFU) method.
Sewage The raw sewage used in this study was collected from the main influent at a Houston sewage treatment plant.
Virus concentrator apparatus A portable unit for virus concentration for use in the field has been described by WALLIS et al. (1972c) and was used throughout this study.
Clarifying filters Ten-inch "Fulflo" yarn-wound filters available in porosites from 100 to 0.8/~m were obtained from the Commercial Filter Division, Carborundum Company, *
Recipient of a World Health Organization/Pan American Health Organization fellowship, on leave from the School of Public Health, Institute Oswaldo Cruz, Rio de Janeiro, Brazil. 945
946
AKIRA HOMMA, MARK D. SOBSEY,CRAIG WALLI$ and JOSEPH L. MELNICK
Lebanon, Indiana. They were housed in see-through cartridge holders which are rated at 100 psi in normal temperature ranges (FIG. 1). These filters are in the form of tubes, and the depth filtration they provide traps particles not only on the surface, but also through the total depth of the filter elements. This gives progressively finer filtration through the filter tube and provides much greater capacity for retention of solids than can be achieved by surface filter media of the same dimensions. The precision winding pattern covers the entire depth of the filter tube with hundreds of funnelshaped tunnels which become gradually finer from the surface in to the center of the tube and trap progressively finer particles as the fluid travels to the center. This method of winding provides depth filtration areas of 3.5 ft 2, for each 10-in. filter. A 10-in. filter will retain approximately ½-1 lb of solids before it must be replaced. The orlon filters are electrostatically inert, and do not adsorb viruses or metallic complexes (WALLIS et al., 1972b). They are retainers of particulates. A series of orlon 10-in. cartridge filters of decreasing porosity (100, 10 and 1/~m) and a cellulose acetate filter (0.8 tzm) were used to clarify the raw sewage. The orlon cartridge filters were washed for 5 rain with tap water using full faucet pressure [about 10 gal rain-' ], and air-flushed for 2-3 rain, in order to remove detergents incorporated during the manufacture of these elements. The cellulose acetate cartridge filter was soaked in 1 1. of 1 per cent Tween-80 (polyosyethylene [20] sorbitan monooleate) for 1 h to prevent virus adsorption to this clarifier. The cartridge was then washed also with water for 5 rain and air-flushed as described above. The excess Tween-80 must be eliminated from the filter, because even slight traces of it may interfere with virus adsorption in further steps (WALLrSand M~LNICK, 1967).
Virus adsorbent This was a filter similar to that described above, but composed of fiber glass (K27), with a 3 tzm nominal porosity (Commercial Filter Division, Carborundum Company). Virus eluent Virus was eluted from the fiber glass filter with pH 11.5, 0.05 M glycine-NaOH buffer (WALLISet aL, 1972a). Buffers that have magnesium or other cations present (which react with NaOH to form a gel) should not be used, nor should buffers be used which contain anions which complex with aluminum ions. The eluate was neutralized by the addition of pH 1.0, 0.05 M glycine-HCl buffer. RESULTS
Clarification of sewage To adsorb viruses from sewage, the sewage must be clarified without loss of virus en route to the adsorbents. Various textile filters were tested, and orlon filters (100, 10 and 1 ~,m in series) were found capable of removing sewage particulates without adsorbing significant amounts of virus. However, the orlon-clarified sewage still clogged the virus adsorbent. Therefore, an additional filter was needed to remove 0.5-1.0 ~m particles. A cellulose acetate filter was found to entrap these small particulates in sewage, but the filter also removed 20-30 per cent virus by adsorption. Therefore, the filter was treated with Tween-80 as described in the Materials and Methods section, which precluded virus adsorption (WALLISet al., 1972b). TABL~ I shows a
947
Virus Concentration from Sewage TABLE 1. CLARIFICATIONOF SEWAGE Gallons of sewage filtered*
Average No. PFU ml-1 in filtrates
Virus removed by clarification ( ~ )
Unfiltered, control sewage-~ 5 10 15 25 35
1950~ 1850 1650 1600 1500 1500
-5 15 18 23 23
* Poliovirus was added to 35 gal of raw, influent sewage, and the sewage was filtered through 100, 10 and 1 /~m orlon filters and a Tween-80 treated cellulose acetate filter (0.5-1.0 t,m) as described in the Materials and Methods section. Filtration proceeded at 10 lb in -2. 1" Index of input virus. typical experiment in which p o l i o v i r u s was a d d e d to influent sewage a n d the sewage was clarified w i t h o u t significant r e m o v a l o f virus. The flow rate o f the sewage passing the filters b e g a n to decrease c o n s i d e r a b l y after 15 gal h a d been processed, b u t the clogging o f the clarifiers did not result in a large loss o f virus. A c t u a l l y , 23 p e r cent o f the virus was r e m o v e d by the clarifiers, b u t this can readily be m a d e up by s a m p l i n g a larger v o l u m e on l o c a t i o n ; the a p p a r a t u s can concentrate virus at a rate well over 100 gal o f s a m p l e p e r hour.
Effects of different salts on virus adsorption to a fiber glass depth filter In the past, MgCI2 has been used to enhance virus a d s o r p t i o n to m e m b r a n e s (WALLIS and MELNICK, 1967). Recently, WALLIS et al. (1972a) r e p o r t e d that trivalent ions were 100- to 250-fold m o r e efficient for enhancing virus a d s o r p t i o n to m e m b r a n e surfaces. Therefore, a c o m p a r i s o n o f MgCI2 a n d A1C13 was m a d e to d e t e r m i n e the salt which enhanced virus a d s o r p t i o n to a fiber glass filter in the presence o f sewage organics (see TABLE 2). TABLE 2. EFFECTS OF DIFFERENT SALTS ON VIRUS ADSORPTION TO A FIBERGLASS DEPTH FILTER*
Average no. PFU ml-1 pH
Salt
Final molarity
Unfiltered control
Fiber glass filtrate
Virus adsorbed (~)
8.0t 3.5 8.0 3.5 3.5
None None MgCI2 MgCI2 AICI3
--0,05 0.05 0.0005
1700 1600 1750 1850 1620
1600 730 1500 940 60
6 44 14 49 96
* Poliovirus was added to sewage which was then clarified through the system described in TABLE 1. One-gallon samples of the clarified sewage were then treated with HCI to obtain the pH levels shown and with the salts indicated; representative 1-gal samples were filtered through fiber glass (K27) virus adsorbents at the rate of 0.67 gal min- ~ and filtrates were assayed for unadsorbed virus. I" Clarified sewage, unadjusted for pH.
948
AKh'L~ HOMMA,
MARKD. SOBSEY,CRAIGWALLISand JOSEPHL. MELNICK
In the presence of excess sewage organics, 0.05 M MgCI2 did not efficiently enhance virus adsorption to the fiber glass filter. However, A1Cl3 at 100-fold less molarity than MgC12 effected a 94 per cent removal of virus on the adsorbent at p H 3.5. Since A1Cla will precipitate sewage components at p H levels higher than 4.0, this salt could only be used at p H 4 or less, and preferably at p H 3.5. It is of signal importance that the sewage first be adjusted to a p H value below 4.0 before addition of aluminum ions.
Effects of flow rates on virus adsorption to fiber glass filters One-gallon samples of sewage containing added virus were filtered at different flow rates through a fiber glass virus adsorbent. The experimental procedures and results are shown in TABLE 3. At flow rates up to 0.76 gal rain-1, 96 per cent of the virus was adsorbed by the glass filter. As the flow rate increased to 1.66 gal rain-~, only 76 per cent of the virus was adsorbed, and at 2.5 gal rain-a, 66 per cent was removed by the virus adsorbent. Therefore, subsequent experiments were performed using flow rates of 0.75 gal rain- x. TABLE 3. EFFECTS OF FLOW RATES ON VIRUS ADSORPTION TO FIBERGLASS
Flow rates of sewage-virus samples* (gal rain- 1)
Average No. PFU ml-1 in fiber glass filtrates
Virus adsorbed (%)
Control virus in sewage (input)~ 0.56 0.76 0.86 1.66 2.50
1610 70 71 210 390 550
-96 96 87 76 66
* Virus was added to influent sewage as described previously. The sewage was clarified as described in TABLE1, and then treated with HCI and AICIa (0.0005 M) as described in TABLE2. Onegallon samples were then filtered at the flow rates indicated and filtrates were assayed for unadsorbed virus.
Effects of membrane coating components (MCC) on virus adsorption to fiber glass filters Organic compounds, especially proteins, will compete with viruses for reactive sites on membranes or textile adsorbents (WALLIS et al., 1972b; WALLIS and MELNICK, 1967). These compounds have been termed MCC. When a virus adsorbs to a reactive site on an adsorbent, M C C will elute the virus by exchanging for it. To overcome this problem, M C C had to be removed by passing sewage through anion exchangers; the virus could then be concentrated on nitrocellulose membranes (WALLIS and MELNICK, 1967). However, since the depth-type fiber glass adsorbents have surface capacities far greater than membrane surfaces, the resins were not used to remove MCC. An experiment was conducted to determine the amount of sewage which could be processed through the virus adsorbent before the accumulating effects of M C C in the sewage prevented virus f r o m being adsorbed on the fiberglass filter. The experimental procedures and results are shown in TABLE 4. When a virus adsorbent was pretreated
FIG. l. Fulflo filter cartridge in holder. A transparent filter holder x~hich is rated at I O0 Ib in-2. c o n t a i n i n g a Fulflo filter cartridge. Water is forced from the outside of the filler into the center effluent channel which is 1 in. dia. and 10 in. long
(fachlg p. 946)
Virus Concentration from Sewage
949
with as much as 35 gal of sewage, the treated filter could still adsorb 74 per cent of the virus, with a 64 per cent recovery by elution. In view of the gross organic load present, these etticiencies are exceptionally significant. TABLE 4.
EFFECTS OF MEMBRANE-COATING COMPONENTS ADSORPTION TO FIBER GLASS FILTERS
Gallons of clarified sewage used to pretreat fiber glass virus adsorbent* Untreated filtrate--> 5 10 15 25 35
(MCC)
ON VIRUS
Virus (~) Adsorbent Eluted 97 80 65 63 74 74
87 69 60 50 50 64
* Sewage (virus-free) clarified as described in TABLE1 was treated with HCI and AIC13 as described in TABLE2, and the samples indicated were filtered through fiber glass virus adsorbents. Each adsorbent filter was rinsed with saline and pretreated with clarified sewage in the amount indicated. Virus added to 1 gal of clarified sewage at pH 3.5 containing 0.0005 M AICIa was then passed through the filter. The filtrates were examined for unadsorbed virus, and then after a rinse with 1 gal of saline, the virus was eluted with 800 ml of glycine buffer, p H I 1.5, as described in the Materials and Methods section, and the basic eluate was neutralized with HCI.
Concentration of exogenously added poliovirus from raw sewage using the portable virus concentrator Sixty liters of raw, influent sewage were processed using the virus concentrator and the optimal methods described above. Poliovirus was added to the 60 I. of sewage, and the virus-sewage mixture was processed through the virus concentrator as described in TABLE 5. The sewage passing the clarifiers was treated with aluminum salts and HCI by an injection system to optimize virus adsorption on the fiber glass filter. Only 6 per cent of the virus passed the virus adsorbent. Elution of virus off this adsorbent yielded 81 per cent of the total input virus in a 1-1. eluate. The total time for processing this volume and recovering the virus was 20 min. DISCUSSION The current study was concerned with concentrating viruses from sewage, by varying the methods previously developed for tap water and sea water, which have a minimal organic load. A 60-fold concentration was readily achieved. Other methods presently in use will concentrate virus from sewage and other waters with higher concentration factors, but with these methods only 1-5 gal of sewage can be examined (HILL et al., 1972). If some types of enteric viruses are present in sewage at levels not detectable in 1-5 gal, they would not be detectable by prior methods. Further, we are at present developing a reconcentration method which will reduce the 1-1. final eluate to 5 ml, which will then represent a concentration factor of 12,000-fold when 601. are processed, and a correspondingly higher concentration if the initial sample is larger.
950
AKIRAI-'IOMMA,MARKD. SOBSEY,CRAIGWALLISand JOSEPHL. MELNICK TABLE 5. CONCENTRATIONOF EXOGENOUSLYADDEDPOLIOVIRUSFROMRAW SEWAGE USINGTHE PORTABLEVIRUSCONCENTRATOR*
Sample Raw sewage -+- virus Fiber glass filtrate Saline wash Eluate
Volume (1.)
No. PFU in total volume ( )< 109)
Total PFU (~o)
60 60 4 1
1.6 0.1 0.035 1.3
100 6 2 81
* Undiluted stock poliovirus was added to 601. of raw sewage. The virus-containing sewage was processed through the portable virus concentrator at a flow rate of 0.75 gal rain- t as follows: (1) The sewage-virus mixture was clarified by filtration through 100, 10 and 1 tzm orlon filters, in series, and through a Tween-80 treated cellulose acetate (0.8/zm) filter. (2) An AICI3-HCI concentrate was driven into the clarified sewage-virus mixture at a dilution of 1:100 to give the mixture a pH of 3.5 and an AICIa concentration of 0.0005 M. The formulation of the A1CI3HC1 concentrate was predetermined by titration of an aliquot of clarified sewage with I N HCI. The injection of the concentrate was monitored by continuous measurement of pH and conductivity. (3) The treated sewage-virus mixture was then filtered through a fiber glass (K27) virus adsorbent. (4) After the entire 60 1. was processed by this procedure, 4 1. of physiological saline was filtered through the fiber glass adsorbent to remove residual aluminum ions. (5) Virus was eluted off the fiber glass cartridge with 800 ml of glycine-NaOH biaffer, pH 11.5, and the eluate was partially neutralized with 200 ml of glycine-HCl buffer, pH 1.0. A t present, the virus c o n c e n t r a t i n g a p p a r a t u s is being used for detection o f viruses in relatively clean waters (WALLIS et al., 1972c), c h a n n e l , estuary a n d sea waters (METCALF et aL, 1972), m u n i c i p a l solid waste leachates ( m a n u s c r i p t in preparation), a n d for the survey o f wastewaters a n d effluent receiving bodies. Once the second step---reconcent r a t i o n of the initial eluate to a final v o l u m e of 5-10 m l - - i s accomplished, the procedure will be o f p a r t i c u l a r value for public health studies concerning the spread o f sewageb o r n e viruses. Acknowledgements--Supported in part by research project No. R-801220 from the Environmental Protection Agency. The authors wish to thank CHARLESSTAGGfor his engineering assistance.
REFERENCES BERG G. (ed) (1967) Transmission of Viruses by the Water Route. Wiley, New York. HILL W. F. JR., AKINE. W., BENTONW. H. and METCALFT. G. (1972) Virus in water. II. Evaluation of membrane cartridge filters for recovering low multiplicities of poliovirus from water. AppL MicrobioL 23, 880-888. METCALET. G., WALLISC. and MELNICKJ. L. (1972) Concentration of viruses from seawater. Proc. 6th Int. Conf. on Water Pollution, Jerusalem, June 18-23, pp. 109-115, 1972 (in press). WALLISC., HENDERSONM. and MELNICKJ. L. (1972a) Enterovirus concentration on cellulose membranes. Appl. Microbiol. 23, 476-480. WALLI$ C., HOMMAA. and MELNICKJ. L. (1972b) Apparatus for concentrating viruses from large volumes. J. Am. Water Wks. Ass. 64, 189-196. WALLISC., HOIV~AA. and MELNICKJ. L. (1972c) A portable virus concentrator for testing water in the field. Water Research 6, 1249-1256. WALLISC. and MELNICKJ. L. (1967) Concentration of viruses from sewage by adsorption on Millipore membranes. Bull. WId Hlth Org. 36, 219-225.