Publication No. 40

Removal of Viruses From Water by Magnetic Filtration

By

Gabriel Bitton, George E. Gifford and Oscar C. Pancorbo

Department of Environmental Engineering Sciences University of Florida

Gainesville

Removal of Viruses From Water by Magnetic Filtration

By

Gabriel Bitton, George E. Gifford and Oscar C. Pancorbo

PUBLICATION NO. 40

FLORIDA WATER RESOURCES RESEARCH CENTER

RESEARCH PROJECT TECHNICAL COMPLETION REPORT

OWRT Project Number A-030-FLA

Annual Allotment Agreement Numbers

14-31-0001-5009 14-34-0001-6010

Report Submitted: October, 1976

The work upon wh ich th is report is based was supported in part by funds provided by the United States Department of the

I nterior, Office of Water Research and TechnologyI' as Authorized under the Water Resources

Research Act of 1964 as amended.

TABLE OF CONTENTS

LIST OF TABLES .................................................. .

LIST OF FIGURES ................................................. .

ABSTRACT ........................................................ .

PUBLICATIONS .................................................... .

THESIS .......................................................... .

INTRODUCTION ...................................•.................

REVIEW OF LITERATURE .........................................•... ,

Adsorption of Viruses to Surfaces .......................... . Implication of Sorptive Phenomena in the Removal of Viruses. Use of Adsorbents in the Concentration of Viruses .......... . The Process of High Gradient Magnetic Separation ........... .

MATERIALS AND METHODS ........................................... .

Viruses Used and Their Assays .............................. . Magnetic Filtration of Adsorbed Viruses ....•................ Wastewater Effluents Used in Adsorption Experiments ........ . Organics Used in Adsorption Experiments .................... . Infectivity of Virus 'Adsorbed to Magnetite ................. . Elution of Adsorbed Poliovirus from Magnetite .............. . Concentration of Poliovirus by Magnetic Filtration ......... . Statistical Treatment of Data .............................. .

RESULTS AND DISCUSSION .......................................... .

Effect of Cation Valency and Concentration ................. . Effect of pH ............................................... . Kinetics of the Adsorption Process ......................... . Effect of Magnetite Concentration .......................... . Effect of Initial Virus Concentration ....•.................. Effect of Wastewater Effluents on the Adsorption of Viruses to Magnetite ............................................... . Effect of Organics on the Adsorption of Poliovirus to Magne-ti te ....................................................... . Infectivity of Virus Adsorbed to Magnetite ................. . Elution of Poliovirus from Magnetite ....................... . Concentration of Poliovirus by Magnetic Filtration ......... .

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19 30 30 34 34

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TABLE OF CONTENTS -- Continued

Page

CONCLUSIONS. . • . . . • . • . • . • . • • . • . . • • . • • • . • • . • . . . • . • • . • • • • • • . .. . . . . • . 58

ACKNOWLEDGEMENTS. . . . . . . • . . • . . . . . . . . . . . . . . . . . . . • • • • • • . . . . • . • • . . . . . 61

REFERENCES. . . . • • . • . . • . . . . . • . . . . . • . . • • • • • • • • • • • . • • • . • • . . • • . • • • . • . . 62

APPENDIX. • . . . . • • • • . . . . . . • • . • • . • • . • • . • . . • • . . • • . . . • . • . . • • • • • • • . • . . . 69

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Table

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LIST OF TABLES

Properties of viruses used in this study ............... .

Characteristics of the secondary effluents used in the magnetic filtration of viruses ......................... .

Effect of monovalent cations (Na+ and K+) on the removal of bacteriophage T2 by magnetic filtration ............. .

Effect of a monovalent cation (Na+) on the removal of Poliovirus Type I (Sabin) by magnetic filtration ....... .

Effect of divalent cations (Ca++ and Mg++) on the removal of bacteriophage T2 by magnetic filtration ..... .

Effect of divalent cations (Ca++ and Mg++) on the removal of bacteriophage T4R(II) by magnetic filtration.

Effect of divalent cations (Ca++ and Mg++) on the removal of bacteriophage MS2 by magnetic filtration .....

Effect of a divalent cation (Ca++) on the removal of Poliovirus Type I (Sabin) by magnetic filtration ....... .

Effect of a trivalent cation (Al+++) on the removal of bacteriophage T2 by magnetic filtration ................ .

Effect of a trivalent cation (Al+++) on the removal of bacteriophage T4R(II) by magnetic filtration ........... .

Effect of a trivalent cation (Al+++) on the removal of bacteriophage MS2 by magnetic filtration ............... .

Effect of a trivalent cation (Al+++) on the removal of Poliovirus Type I (Sabin) by magnetic filtration ....... .

Effect of time of shaking on the removal of bacterio-phage T2, bacteriophage MS2 and Poliovirus Type I (Sabin) by magnetic fil tration ......................... .

Effect'iof:'a divalent cation (Ca++) on the removal of bacteriophage T2 by magnetic filtration in the presence of dome water ......................•....................

Removal of bacteriophage T2 by magnetic filtration in the presence of dome water ............................. .

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Table

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LIST OF TABLES -- Continued

Removal of Poliovirus Type I (Sabin) by magnetic filtra-tion in the presence of dome water ....................... .

Removal of Poliovirus Type I (Sabin) by magnetic filtra-in the presence of activated sludge effluent ............. .

Interference by egg albumin, casein and dextran with the removal of Poliovirus Type I (Sabin) by magnetic filtra-tion ..................................................... .

Infectivity of bacteriophage T2, bacteriophage MS2 and Poliovirus Type I (Sabin) absorbed to magnetite .......... .

Elution of Poliovirus Type I (Sabin) from the magnetite surface .................................................. .

Concentration of Poliovirus Type I (Sabin) by magnetic fil tration ............................................... .

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LIST OF FIGURES

Figure

1 Particle size distribution of magnetite (Fisher) as mea-sured with the Coulter Counter Industrial Model B (Coul-ter Electronics, Illinois)............................... 10

2 High gradient magnetic filter used to separate the virus-magnetite complex from the water suspension .............. 11

3 High gradient magnetic filter supplied by Sala Magnetics, Cambridge, Mass.......................................... 17

4 Effect of pH on the removal of bacteriophage T2, bacterio-phage MS2 and Poliovirus by magnetic filtration ..... ..... 31

5 Effect of magnetite concentration on the removal of bac-teriophage T2, bacteriophage MS2 and Poliovirus Type I (Sabin) by magnetic filtration........................... 35

6 Freundlich isotherms for the adsorption of bacteriophage T2, bacteriophage MS2 and Poliovirus Type I (Sabin) to magnetite: variable virus concentration ............ ..... 37

7 Freundlich isotherms for the adsorption of bacteriophage T2 to magnetite in distilled water and dome water: vari-able magnetite concentration ...... ................. ...... 45

8 Freundlich isotherms for the adsorption of bacteriophage MS2 to magnetite in distilled water and activated sludge effluent: variable magnetite concentration .... .......... 47

9 Freundlich isotherms for the adsorption of Poliovirus Type I (Sabin) to magnetite in distilled water and waste-water effluents: variable magnetite concentration ....... 49

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ABSTRACT

The process of magnetic filtration was applied to the removal of T2 bacteriophage, MS2 bacteriophage, and poliovirus type 1 (Sabin) from water and wastewater. Since the effectiveness of this process is governed by the adsorption of the viruses onto the magnetic iron oxide seed, magnetite, those factors influencing adsorption were investigated.

It was found that the adsorption of the three viruses studied to magnetite at a concentration of 500 ppm increased in the pre-sence of cations, was not affected when the pH was varied from 5 to 9, and could be described by the Freundlich adsorption isotherm.

Viruses were suspended in two wastewater effluents and subse-quently removed by magnetic filtration. It was shown that organic color interfered with the adsorption process. The inter-ference was reduced by the addition of CaC12, by the dilution of the wastewater with distilled water or by removing the organic color with activated carbon. Competition for adsorption was also exerted by 100 ppm of egg albumin, casein and dextran resulting in a significant reduction in the removal of poliovirus.

Additionally, the infectivity of the magnetite-bound viruses was studied. Poliovirus, adsorbed to magnetite, was 100% infective whereas T2 phage displayed a lower and somewhat variable infec-tivity.

Attempts were made to desorb poliovirus from magnetite by re-suspension of the magnetite pellet in a variety of possible eluents. The best eluent was an isotonic, 10% solution of fetal calf serum buffered at the pH of 9 with Tris buffer. Preliminary results of the concentration of poliovirus by magnetic filtration are also presented and discussed.

The experimental data obtained in this work show that magnetite is a good adsorbent towards viruses and that magnetic filtration can be effectively used for the removal of viruses from water and wastewater.

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PUBLICATIONS

1. G. Bitton, O. Pancorbo and G. E. Gifford. 1976. Adsorption of Poliovirus Type 1 to Magnetite and Subsequent Removal by Magne-tic Filtration. Abstract, Amer. Soc. for Microbiology Ann. Meeting, Atlantic City, N.J.

2. G. Bitton, J. L. Fox and H. G. Strickland. 1975. Algae from Florida Lakes by Magnetic Filtration. 30:905-908.

Removal of ~. Microbiol.

3. G. Bitton, O. Pancorbo and G. E. Gi~gord. 1976. Factors Affect-ing the Adsorption of Poliovirus to Magnetite in Water and Waste-wate'r". Water Res." Vol. !Q. (in press) .

4. G. Bitton, J. L. Fox, G. E. Gifford and O. Pancorbo. 1976. Utilisation d'electroaimants dans l'elimination des virus et des algues presents dans les eaux de surface et les eaux usees. Annales de l' ACM'" 43: 133.

Pancorbo, O. C. 1976. wastewater by magnetic Dept. of Environmental Florida, Gainesville.

THESIS

The removal of viruses from water and filtration. 115 pp. M.S. thesis, Engineering Sciences, University of

vii

INTRODUCTION

Viruses are obligatory intracellular parasites ranging in size from 100 A to 4000 A in diameter (Pollard, 1953). They contain a single type of nucleic acid, either DNA or RNA, surrounded by a pro-tein coat. The ionization of certain protein coat groups results in viruses acquiring a negative charge at neutral pH. The interaction of viruses with their environment is determined by the nature of their protein coat and is influenced by their ability to adsorb to surfaces. Bitton (1975) reviewed the adsorption of viruses to sur-faces and its importance in the removal of viruses by water treat-ment processes and in the concentration and recovery of viruses. Sproul et al. (1969) proposed that probably the most important mechanism for virus "inactivation" in waste water treatment plants is adsorption.

Among the various adsorbents studied, iron oxides';have been shown,to display excellent sorptive capacity towards viruses (Bitton and Mitchell, 1974; Pearson and Metcalf, 1974; Rao et al., 1968; Warren et al., 1966). Using magnetite as an adsorbent-,-Bitton and Mi tchell(f974) removed the bacteriophage T7 by a "magnetic fil tra-tion technique.'" This process consists of adsorbing the virus onto the magnetic iron oxide seed, magnetite, and subsequently removing the virus-seed mixture from the water by pouring through a filter placed in a background magnetic field.

The purpose of this study was to delineate further the effec~ tiveness of magnetic filtration towards the removal of viruses, mainly those of animal origin, from water. Factors known to influence the adsorption of viruses onto surfaces and that could, therefore, affect the adsorption of viruses onto magnetite were investigated. These included pH, electrolyte valency and concen-tration, magnetitecondentratio~, virus type and concentration, time allowed for adsorption, and concentration and type of organic sub-stances in the suspending medium. It was felt that the knowledge gained from this study could be applicable in the removal of patho-genic enteric viruses present in water and wastewater.

REVIEW OF LITERATURE

AdsorpfionofViruses to Surfaces

Viruses are able to adsorb to biological (Tolmach, 1957) and non-biological surfaces (Bitton, 1975). The adsorption process is usually influenced by such factors as the type of virus, nature of the surface involved, pH, and electrolyte, and aTganic matter content of the sus-pending medium.

Due to their widespread occurrence in soil and aquatic environ-ments and to their large surface area, clay minerals have been studied for their sorptive capacity towards viruses. The adsorption of entero-

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viruses to such clays as kaolinite, illite, montmorillonite, and bentonite has been shown to be significant and to increase in the presence of cations with trivalent cations being more effective than divalent or monovalent cations (Carlson et al., 1968; Schaub and Sagik, 1975; Schaub et al., 1974). Virus-Sorption onto clays has also been found to be-independent of pH over the range of 3 to 9 (Bartell et al., 1960; Schaub et al., 1974) and to conform to the Freundlich:rsotherm (Schaub et al., 1974). Organic materials such as egg and bovine albumin, and~etal calf serum were also shown to interfere significantly with the adsorption of viruses to clays (Carlson et al., 1968; Schaub and Sagik, 1975). Varia-bility in the adsorptive capacity of different clays towards viruses was proposed by Carlson et al. (1968) to result from differences in clay surface exchange-capacity which is determined by the sur-face charge density and clay particle geometry.

Activated carbon is extensively used in water and wastewater treatment and consequently, its ability to adsorb viruses has been investigated. Cookson (1969) studied the adsorption of T4 bacterio-phage on activated carbon and determined that the adsorption rate was influenced by the pH and the ionic strength of the medium. It was proposed that the adsorption process involved an electrostatic interaction between amino groups on the virus and carboxyl groups on the activated carbon. At very high ionic strengths as well as at low pH's, the tail fibers were unavailable for adsorption (Lauf-fer and Bendet, 1962) and this decreased the rate of reaction. The adsorption was described as a diffusion-limited process (Cookson, 1967), conformed to the Langmuir isotherm and did not inactivate the virus (Cookson and North, 1967). Gerba et al. (1975) found that the adsorption of poliovirus type 1 (straiU-LSc) to activated carbon obeyed also the Freundlich isotherm and was reduced in the presence of wastewater effluents. DesQrption of viruses from an activated charcoal column occurred when the wastewater pH was in-creased or when glycine buffer adjusted to pH 11.5 was used to elute the virus.

The adsorption of viruses onto such surfaces as glass, celite, nitrocellulose, aluminum oxide and gold has been reported and was found also to be influenced by the presence of cations and organic materials (Bache and Quilligan, 1966; Shepard and Woodend, 1951; Valentine and Allison" 1959).

Implication of Sorptive Phenomena in the Removal of Viruses

Pathogenic enteric viruses mainly from human and animal origin are constantly present in our water systems and constitute a serious public health hazard. Consequently, a great deal of research has been devo1ted to finding efficient water treatments for their elimi-nation. The removal of viruses by water (Berg, 1973a; Berg, 1973b; Berg, 1975; Sproul, 1972) and wastewater (Berg, 1973a; Berg, 1973b;

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Berg, 1975; Grabow, 1968) treatment processes usually involves stor-age, biological and tertiary treatment followed by disinfection with chlorine, iodine, bromine or ozone.

Among the various secondary (biological) wastewater treatment processes, the activated sludge system has been the most intensively studied for the removal of viruses. Clarke et al. (1961) found that the adsorption of coxsackie A9 virus and poliovirus type 1 (Mahoney) to activated sludge conformed to the Freundlich isotherm and resulted in the removal of about 90% of the viruses. The virus-sludge complex was shown to be very stable since only a small percentage of adsorbed viruses could be recovered. However, the removal or inactivation of viruses in stabilization ponds has received little attention. Data from laboratory experiments did not show evidence of active removal of poliovirus and reovirus by algae, but there was a significant inactivation of viruses by sunlight at the surface of sewage matura-tion ponds (Malherbe and Strickland-Cholmley, 1967). Sobsey and Cooper (1973) observed that the reduction of poliovirus in stabili-zation pond water was due to adsorption to solids.

Tertiary wastewater treatment processes are designed to further polish the quality of secondary effluents with regards to suspended solids and nutrients containing nitrogen and phosphorus. Among the various tertiary wastewater treatment processes, activated carbon has been shown to remove viruses very poorly. The factors influencing the adsorption of viruses to activated carbon have already been dis-cussed. Sproul et al. (1969) reported that virus removal from waste-water by activated carbon is not dependable. Coagulation and floc-culation using cationic polyelectrolytes as prime coagulants or as coagulant aids in the presence of alum: has also been studied and was found to be effective in the removal of viruses (Amirhor and Engelbrecht, 1974; Chaudhuri and Engelbrecht, 1972; Thorup et al., 1970). This process was shown to be salt dependent (Thorup-et-al., 1970) and unfortunately, organic matter interfered with virus-r~ moval (Amirhor and Engelbrecht, 1974; Chaudhuri and Engelbrecht, 1972). Even though sand filtration plays a role as a tertiary treat-ment process, its standard and more important use in potable water treatment is the primary reason for the intensive investigation of its role in virus removal. Dieterich (1953) found that adsorption was the primary mechanism responsible for the removal of a bacterio-phage during sand filtration and that egg albumin significantly inter-fered with virus removal. Cations, on the other hand, were reported to enhance the retention of viruses by sand columns (Lefler and Kott, 1974). Although sand is a poor adsorbent due to its small surface area (Dieterich, 1953), significant virus removal can be achieved by combining sand filtration with alum flocculation (Gilcreas and Kelly, 1955; Robeck ~al., 1962). The removal of viruses from water by diatomaceous-earth filtration (Brown et al., 1974a; Brown et al., 1974b) and by the use of coal assorbent (Oza and Chaudhuri-,-1975; Oza et al., 1973) was also investigated, and the results were encou-

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raging. However, more research on the effectiveness of these two treatments is in order.

Use of Adsorbents in the Concentration of Viruses

Viruses occur in natural water bodies in very low concentrations and therefore, their detection is dependent upon their adequate con-centration. Various virus adsorbents have been investigated for their potential application in the concentration of viruses from di-lute suspensions (Hill et al., 1971). The membrane-adsorption tech-nique has been demonstrate~to effectively concentrate viruses. Wallis and Melnick (1967a) showed that the adsorption of viruses on membrane filters was enhanced in the presence of cations and inhibited in the presence of proteinaceous matter such as cell extracts or serum. The optimal pH for adsorption was found to be 5. This method was further used in the concentration of viruses from water (Hill et al., 1974) and.:wastewater (Wallis and Melnick, 1967b). Synthetic-insoluble polyelectrolytes (Wallis et al., 1970, 1971), cationic exchange resins (Muller and Rose, 1952;-Fuck and Sagik, 1953) and anionic exchange resins (LoGrippo, 1950; Puck and Sagik, 1953) have also been found to be effective virus adsorbents and consequently, to possess the ability to concentrate and purify viruses. Finally, precipitates of aluminum hydroxide, aluminum phosphate, and calcium phosphate have been used effectively to concentrate viruses (Wallis and Melnick, 1967c, 1967d). Viruses may also be concentrated by a variety of techniques which do not involve adsorption onto surfaces (Hill et al., 1971; Shuval et al., 1967) but these methods will not be discussed in this review-.---

Among the various adsorbents used for virus concentration and removal, iron oxides have received little attention. Warren et al. (1966) reported for the first time the adsorption of influenza-virus to an iron oxide, hematite. Concentration and purification of this virus were achieved by adsorption onto hematite and subsequent elu-tion with a 10% sodium phosphate solution at a pH between 7.5 and 8.5 resulting in 10-fold concentrates. Rao et al. (1968) later used an iron oxide (M.O. 2530) column to adsorb andconcentrate a strain of coxsackie virus A9. It was found that concentrations as high as 2 x 106 pfu/ml of this virus could be removed, without detecting any virus in the filtrate, by 25 grams of the iron oxide. Concentration of the coxsackie virus A9 by adsorption to the iron oxide and subsequent elution with foetal calf serum resulted in 5-fold concentrates with recoveries ranging from 55% to 95%, depending on the initial virus concentration. Pearson and Metcalf (l974) studied the adsorption of several enteroviruses to the same iron oxide, M.O. 2530, and found virus recovery most effective when elutions were made from thin layers of iron oxide under alkaline pH and in the presence of 3% isotonic beef extract. Unfortunately, the use of columns or thin layers of iron oxide may ultimately result in the clogging of the

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filter (Rao et al., 1968). Due to the open matrix of the filter used in High~radient Magnetic Separation (HGMS), the problem of clogging is greatly minimized.

The Process of High Gradient Magnetic Separation (HGMS)

Convential'magnetic separators have been used for many years to remove strongly magnetic iron-bearing particles larger than 100 mi-crons in size from non-magnetic media. These devices are used to remove magnetic impurities in a variety of feeds, and to concentrate magnetic materials such as iron ores for their beneficiation (Ober-teuffer, 1973).

Recently, a great deal of research has been devoted to the development of "High Gradient Magnetic Separation." This process, unlike convential magnetic separation, is able to separate weakly paramagnetic materials of micron size by maximizing the magnetic force on such particles. The magnetic force on a particle is given by the equation,

dH F = VM-x dx (1)

Where V is the volume of the particle, M is the magnetic moment of the particle in the field H, and ~ is the field gradient in the x direction (Oberteuffer, 1973). In the case of paramagnetic materials,

and M = XH

dH Fx = V XH dx

(2)

(3)

where X is the magnetic susceptibility. From equation 3, it can bedH seen that both a large magnetic field H and a large field gradient ~ will yield a maximum magnetic force Fx. A large magnetic force is x required in order to overcome competing forces (e.g., gravitational) that oppose the magnetic separation of particles (Oberteuffer, 1973). A high gradient magnetic separator consists of a filter containing a ferromm.g~re.:t±;c~ matrix (usually stainless steel wool) placed in a strong magnetic field. Such a matrix provides a very large number of trapping sites for susceptible particles and enables high flow rates (50-150 gal. min.- l ft.-2) due to its loosely packed structure. With the magnetic field on, the magnetic particles in the feed slurry are trapped at the points and edges of the matrix fibers while non-magnetic constituents of the slurry pass through the filter easily. Due to the low residual magnetization of the matrix fibers, magnetic particles trapped in the matrix during the feed. mode can be easily washed out by turning the magnetic field off and backflushing (Mit-chell et al., 1975a, 1975b).

Several industrial applications have been found for high gradient magnetic separation. In the clay industry, for example, this process is used to clean kaolin from micron size iron stained titaniferous

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materials (Oder, 1973). Such impurities can seriously reduce the use of kaolin as a paper coating material. High gradient magnetic separation has also been used in the beneficiation of semitaconites which are made up of very small particles of weakly magnetic iron oxides intermixed with gangue material such as slate and chert. Kelland (1973) has used successfully the high gradient magnetic separation technique to separate the iron minerals from the gangue and this process resulted in an increase in the iron grade (iron content) of the semitaconites. This technique has also been applied to the desulfurization of coal. Trindade and Kolm (1973) obtained a 50 to 75% reduction in the sulfur content of Brazilian coal.

High gradient magnetic separation has been shown to be effec-tive in the removal of various pollutants from water and wastewater (DeLatour and Kolm, 1976; Mitchell et al., 1975a). Removal may be achieved in one of two ways depending upon the nature of the pollu-tant. For magnetic contaminants, such as the fine iron oxide par-ticles found in steel mill effluents and boiler feed waters, high gradient magnetic separation may be used alone to obtain their removal. However, for the more common non-magnetic pollutants found in natural waters, a magnetic seeding technique is required. This technique involves the adsorption of non-magnetic contaminants onto a magnetic seed, magnetite, and subsequent passage of the mix-ture through a high gradient magnetic separator. The magnetite and adsorbed pollutants are trapped on the matrix of the separator and can be easily washed out when the magnetic field is turned off. This process has been called "magnetic filtration" and it has been shown to reduce effectively coliforms, color, turbidity (DeLatour, 1973; Mitchell et al., 1975a, 1975b), phosphate (Bitton et al., 1974; DeLatour, 1973)-,-algae in lake water (Bitton et al., 1975) and the phage T7 (Bitton and Mitchell, 1974). Bitton-an~Mitchell (1974) found that the removal of the bacteriophage T7 by this process was dependent on the presence of cations and independent of virus con-centration in the range of 30 pfu/ml to 14 x 103 pfu/ml. Although having many similar properties, bacteriophages and animal viruses are, nevertheless, inherently different. Therefore, an animal virus study should be included when assessing the virus removing effec-tiveness of any treatment. Unfortunately, no research has previously been done on the removal of animal viruses by magnetic filtration. Additionally, little is known about the effect of such factors as pH and the presence of organic materials on the removal of viruses by this process. The research reported in this:report attempts to fill in these gaps in the knowledge of virus removal by magnetic filtration. Finally, it can be said that high gradient magnetic filtration is an efficient process capable of removing a wide variety of pollutants from wastewater. Consequently, its use either as a replacement for small sewage treatment facilities or in large facili-ties as an advanced treatment process is not far off (Mitchell et al., 1974~ 1975a, 1975b). - -

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MATERIALS AND METHODS

All 'virological work was performed aseptically and in the case of poliovirus, all work was done in a room previously sterilized by ultraviolet irradiation.

All glassware which came into @vernight in Haemo-Sol (Scientific in tap water and distilled water. claving.

Viruses Used and Their Assays

contact with the virus was soaked Products) and subsequently rinsed Glassware was sterilized by auto-

The viruses used in this study were the T2 bacteriophage, T4R(II) bacteriophage, MS2 bacteriophage and poliovirus type 1 (Sabin strain). Table 1 shows some important properties of these viruses. Bacterio-phages were used in this study to obtain preliminary data on a par-ticular phase of research and to resolve any difficulties in the experimental procedure prior to initiating experiments with polio-virus. Representatives from two different groups of bacteriophages were used: MS2 phage representing the group of small, RNA phages and T2 and T4R(II) phages representing the large, DNA phages posses-sing a contractile tail. These two types of bacteriophages were selected in an attempt to compare their behavior during magnetic filtration with that of poliovirus and to determine which is a better model of enterovirus removal by magnetic filtration. The bacteriophage MS2 should be a better model of poliovirus behavior than T2 and T4R(II) bacteriophages due to its greater similarity to poliovirus in size, particle mass and nucleic acid type (Table 1). Preliminary investigations showed that T2 and T4R(II) bacterio-phages behaved similarly during magnetic filtration, and therefore, subsequent experimentation was only carried out with T2 phage. Polio-virus type 1 (Sabin) was used as a representative human enteric virus. It is generally agreed that the use of an animal virus in a viral adsorption study increases the reliability of the results obtained.

The T2 and T4R(II) bacteriophage stocks and their host Esche-richia coli B were obtained from Dr. Donna H. Duckworth, Department of Immunology and Medical Microbiology, University of Florida. The phages were refrigerated at 4~C in NW-1) medium (Adams, 1959) and the host, E. coli B, was maintained at room temperature on 2% nutrient agar (Difco) slants. A complete list of all media and solutions used in this study including their composition and sO~Fce is presented in the Appendix. These T-even bacteriophages were assayed by the plague technique as described by Adams (1959) using the double layer plating procedure.

The MS2 bacteriophage stock was obtained from Dr. Anthony Pfister, Department of Immunology and Medical Microbiology, Univer-sity of Florida. The host Escherichia coli C3000 (ATCC #15597) was

Table 1. Properties of viruses used in this study

Virus Nuc1eica Isoe1ectricb Sizec (mll) Dry Partic1ed pH Stabili tye Acid Point Mass Range

(x 10-16 g)

T2 phage DNN) 4.2 head - 65 x 95 3.3 5.0 - 9.0' tail - 25 x 100

T4 phage DNAb head - 65 x 95 3.3 5.0 - 9.0 tail - 25 x 100

MS2 phage RNAb 3.9 25 0.06

Poliovirus RNAt) 4.5 and 7.0 27 0.14 3.6 - 8.4

a From Davis et a1. 1973. b From: Matide1 , 1971; Overby et al. 1966; Sharp et a1. 1946. c From Overby et a1. 1966; Schwerdt and Schaffer, 1955; Williams and Fraser, 1953. d

e From Kellenberger, 1962; Overby et a1. 1966; S~hwerdt and Schaffer, 1955; Taylor et a1. 1955.

Frmm Bachrach and Schwerdt, 1952; Putnam, 1953; Sharp et a1. 1946.

I 00 I

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obtained from Miles Laboratories, Kankakee, Illinois. The phage was refrigerated at 4°C in Tris buffer (see Appendix) and the host, E. coli C3000, was maintained at room temperature on 2% nutrient agar (Difco) slants. Assay of this virus was by the plaque technique using the double layer procedure (Adams, 1959).

The Poliovirus type 1 (Sabin strain) stock suspension used in this study was prepared by infecting a monolayer culture of AV3 cells in a 32 ounce bottle. After a 1 hour adsorption period with tilting at 15 minute intervals, 40 m1 of Eagle's Minimal Essential Medium (MEM) plus 10% fetal calf serum (see Appendix) were added. After two days of incubation at 37fiC, the overlay medium containing poliovirus was decanted, centrifuged for 15 minutes at 1500 rpm to remove debris and then distributed to 1 m1 ampules which were im-mediately frozen at -70°C. The poliovirus was assayed by the plaque assay technique on AV3 (human amnion) cell mono1ayers. This assay was performed by inoculating a drained AV3 monolayer with 0.2 m1 of the virus suspension which had been diluted in Eagle's MEM + 5% calf serum + 0.03 M Hepes buffer (see Appendix) to yield 100-300 plaques per bottle. Following inoculation, a @ne hour adsorption period with tilting at 15 minute intervals was allowed. The infected cells were then overlayed with 4 m1 of methyl cellulose overlay (see Appendix). After incubation at 37°C for 48 hours, the methyl cellulose overlay was decanted and the monolayer was stained with crystal violet (see Appendix). PPlaques were sub-sequently counted using an Omega Enlarger B22 (Simmon Bros., Wood-side, N.Y.).

Magnetic Filtration of Adsorbed Viruses

The salts used in this study were NaC1 (Fisher Scientific, Fairlawn, N.J.), CaC12 (Fisher) and A12(S04)3 . 18 H20 (Ma11inc-krodt, St. Louis, Mo.). The magnetite was supplied by Fisher Scientific (catalog no. 1119) and its particle size distribution was determined using a Coulter Counter Industrial Model B (Coulter Electronics, Illinois). Figure 1 shows that the particle size varied from 3 to 30 ~ with a maximum count between 3 and 5 ~.

The adsorption of viruses to magnetite and the subsequent removal of the magnetite-virus complex from the water suspension by filtration through a magnetic separator was undertaken as described below. Appropriate volumes from concentrated stock solutions of magnetite, salt, and virusew~~e added successively in batch experiments to samples of water resulting in a final volume of 100 m1. The pH of each solution was measured using a Beckman Expandomatic SS-2 pH meter and the resulting pH's were usually between 6 and 7. If required, pH adjustments were made using 0.1

5

4

.jJ

t: :::l ci U

0 3 r-

O'l l \~ I 0 ~ ....J 0 I

2

1 o 10 30

Particle diamete~

Figufe 1. Particle size distribution of magnetite (Fisher) as measured with the Coulter Counter Industrial Model B (Coulter Electronics, Illinois)

-11-

Figure 2. High gradient magnetic filter used to separate the virus-magnetite complex from the water suspension

This filter consists of a stainless steel wool ma-trix (compaction equal to lag/50 cm3) placed in a background magnetic field of 2000 gauss.

~12-

-13-

N NaOH or HCl. These mixtures were then shaken at room temperature at approximately 170 oscillations/min and poured through a filter placed in a background magnetic field of 2000 gauss (see Figure 2). The filter was made of a matrix of stainless steel wool (supplied by Dr. E. Maxwell, Francis Bitter National Magnet Laboratory, M.l.T., Cambridge, Mass.) having the compaction of 10 g/50 cm3 and magnetized ~n a background magnetic field. Each filtration experiment was carried out in triplicate and one control without magnetite was included in each experimental condition. Samples were removed and assayed for virus immediately after the shaking period (i.e., just prior to filtration) and after filtration through the magnetic separator. The controls without magnetite were included in order to account for loss of infectivity due to adsorption to containers or inactivation. The work described above was all done aseptically.

Wastewater Effluents Used in Adsorption Experiments

The wastewater effluents used in the adsorption experiments were "dome water" and an activated sludge effluent. "Dome water" is a secondarily treated effluent sampled at the edge of a cypress dome, located north of Gainesville, Florida. This effluent originated from a package treatment plant and became highly colored after standing in the dome site. When required, the removal of these coloring materials was undertaken by filtration through an activated carbon column, 11 cm in length and 3 em in diameter. The column con-tained 10 cm of Filtrasorb 400 (Calgon Co.) and 1 cm of fine particles of Hydrodarco B (Atlas Powder Co., N.Y.). The flow rate through the activated carbon column was 0.37 ml/min.

The activated sludge effluent was sampled, after secondary settling and prior to chlorination, at the University of Florida campus sewage treatment plant.

The effluents were immediately brought back to the laboratory and subsequently clarified by filtration through a Whatman filter no. 41, sterilized by autoclaving and stored at 4°C until used. Total organic carbon, turbidity, color, pH and conductivity were determined in the laboratory using a Beckman 915 total organic carbon analyzer, a Hach model 2l00A turbidimeter, a Bausch and Lomb. Spectronic 88, a Beckman Expandomatic SS-2 pH meter and a conductivity bridge Model RC l6B2, respectively. The values for the various characteristics of the effluents are shown in Table 2.

Table 2. Characteristics of the secondary effluents used in the magnetic filtration of viruses

Parameter

pH

Color (mg/1 as Pt)

Turb idi ty (JTU)

TOC (mg/1)

Conductivity (~mho/cm at 25°C)

Dome Watera

6.20 - 7.65

350.0 - 750.0

3.0 - 4.0

26.5 - 37.0

227.3 - 385.0

Dome Water #lb

7.10

575.0

4.0

31.0

385.0

Dome Water #1 After Activated Carbon Treatment

8.25 (adjusted to 7.10 with 0.1 N HC1)

8.0

4.0

7.0

320.9

Activated Sludge

EffluentC

7;40 - 7.73

30.0 - 35.0

0.6 - 2.0

4.0 - 12.0

385; 0 - 472.7

a Various batches of dome water were collected and the values given for each parameter represent the high and low measurements obtained.

b This is the first batch of dome water collected.

c Various batches of activated sludge effluent were collected and the values given for each parameter represent the high and low measurements obtained.

I i-' ..,. I

-15-

Organics Used in Adsorption Experiments

The interference by organics with the adsorption of poliovirus to magnetite and consequently, with the removal of the virus by magnetic filtration was investigated. The organic substances used were egg albumin (cat. no. B2SS), casein (cat. no. B337), both supplied by Difco Laboratories (Detroit, Mich.) and dextran-S x 105 MW (cat. no. 05251) supplied by Sigma Chemical Co. (St. Louis, Mo.). Stock solutions (0.1%) of these substances were prepared in dis-tilled water and were then sterilized by autoclaving and stored at 4°C.

Infectivity of Virus Adsorbed to Magnetite

In order to determine the infectivity of virus adsorbed to magnetite, an experiment was undertaken as described below. Viruses were shaken (~ 170 oscillations/min.) for 20 min. in the presence of 500 ppm of magnetite and 1610 ppm of CaC12' Low virus concen-trations were used so that samples might be plated directly with-out requiring dilution. Samples were removed and directly plated immediately after addition of the virus and after 20 min. adsorp-tion period. Following the adsorption period, the magnetite was pelleted by placing the mixtures next to a magnet and the supernatants were subsequently assayed for virus. Controls without magnetite were studied in order to account for loss of infectivity due to adsorption to containers or inactivation.

Elution of Adsorbed Poliovirus from Magnetite

Attempts were made to elute the adsorbed poliovirus from mag-netite. The procedure employed is described below. Poliovirus was shaken (~ 200 oscillations/min.) for 20 min.; lhn the presence of 500 ppm of magnetite and 1610 ppm of CaC12' Ten ml aliquots were then removed and distributed into sterile test tubes. The magnetite in each test tube was then pelleted by placing the mixture next to a magnet. Supernatants were removed for assay and then discarded. The magnetite pellets were resuspended in 10 ml of the eluents shown in Table 20 (the composition and source of these eluents appear in the Appendix). After a 1 hour elution period with periodic ~haking at 4°C (except for glycine buffer which was allowed only 1 min of con-tact time), the magnetite was pelleted again. The supernatants were then assayed for desorbed viruses.

Concentration of Poliovirus by Magnetic Filtration

Recovery of poliovirus from 4 liters of distilled water contain-ing initial virus concentrations ranging from 1.0 x 103 to 1.3 x 10 3 pfu/ml was undertaken as described below. Poliovirus was shaken (~ 200 oscillations/min.) for 20 min. in the presence of 1000 ppm of magnetite and 1610 ppm of CaC12' The mixtures were subsequently

-16-

poured through the magnetic filter in order to separate the polio-virusffiillagnetite complex from the water suspension. Poliovirus was then eluted from the magnetite surface with a small volume of 10%, isotonic fetal calf serum, Tris buffered, pH = 9. The procedure used for the recovery of poliovirus varied with each experiment performed.

In a first experiment, 100 ml of the eluent was passed twice through the filter with the filter in the magnetic field and once (the last passage) with the filter outside the magnetic field. The eluauee (separated from any magnetite) was assayed for virus after each passage through the filter. The Sala magnet pictured in Figure 3 was used in this experiment (and only this experiment).

In a second experiment, 50 ml of the eluent was introduced into the filter and then the filter was shaken manually for 1 min and then shaken mechanically overnight at 4°C. After each shaking period, the eluate was separated from magnetite and then assayed for viruses.

In the third experiment, the matrix of the filter was removed (along with all the magnetite) and placed in a sterile beaker con-taining 100 ml of the eluent. The matrix was then sonicated in an ice bath for 10 min. using the standard probe of a Branson Sonifier (Danbury, Conn.) ~del S-75 which has a power output of 75 watts and an ultrasonic frequency of 20 kc/sec. The eluate was then assayed for viruses.

Statistical Treatment of Data

The two statistical procedures used to treat data were: 1. the small-sample, t test for comparing two means, and 2. linear regres-sion analysis to determine the least squares regression line for a set of points and the coefficient of determination r2 which is a measure of the strength of the relationship represented by the regres-sion line. The Hewlett-Packard Calculator Model 98l0A and Statistics PackageV~6 were used to perform the statistical analysis.

-17-

Figure 3. High gradient magnetic filter supplied by Sa1a Magnetics, Cambridge, Mass.

This filter was used in the concentration of Poliovirus, experiment 1.

-18-

-19-

RESULTS AND DISCUSSION

The purpose of this study was to determine the effective-ness of magnetic filtration in removing viruses from water. Consequently, factors that could influence the adsorption of viruses onto magnetite were investigated. These included cation valency and concentration, pH, time allowed for adsorp-tion, magnetite concentration, virus type and concentration and organic substances in the suspending medium.

Effect of Cation Valency and Concentration

The effect of monovalent (Na+, K+), divalent (Ca++, Mg++) and trivalent (Al+++) cations on virus adsorption to magnetite and on the subsequent virus removal by magnetic filtration was studied (Tables 3-12). It was observed that, in the presence of any salt under consideration, the removal of poliovirus and MS2 phage approached or exceeded the 99% level when the salt concen-tration was at or above 20 ppm. The percent removal of T2 phage was below that of poliovirus or MS2 phage for any cation used and at any concentration. The highest removal of T2 phage (95.9%) was achieved in the presence of 16100 ppm of CaC12 (see Table 5). The removal obtained for the T4r (II) phage in the presence of 1610 ppm of CaC12 or 1380 ppm of MgC12 (Table 6) was similar to that found for the T2 phage (Table 5). In the presence of 100 ppm of alum (see Tables 9 and 10), however, the removal of the T4r (II) phage (99.2%) greatly exceeded that displayed by the T2 phage (85.4%). There was no significant difference in the removal of the phage T2 in the presence of K+ OT Na+ cations (Table 3). Calcium cations (Ca++) were found more effective than Mg++ cations at ionic strengths (~) equal to 4.35 x 10- 2 and 4.35 x 10-1 (see Tables 5 and 6) .

It has been shown by other workers that the electrolyte content of the suspending medium is important for the adsorption of viruses to particles such as iron oxides, clays, activated carbon, polyelectrolytes, sand and soil (Bitton andMi tchell, 1974; Carlson et al., 1968; Cookson, 1969; Thorup et al., 1970; Lefler and Kott-,-1974; Drewry and Eliassen, 1968). Furthermore, it is known that, on a concentration or molar basis, trivalent cations are more efficient than divalent cations which in turn are more efficient than monovalent ones. Viruses are colloidal particles having a net negative surface charge at neutral pH. Most of the adsorbents studied also carry a net negative surface charge at neutral pH. An increase in the ionic strength of the suspending medium leads to a reduction of the thickness of the double-layer around the particles which are then bound by attractive forces, namely, London-Van der Waals forces (Clark et al., 1971; Osipow, 1962). The adsorption of viruses to magnetite-,-as seen in Tables 3 through 12, follows the trends discussed above with the exception

-20-

Table 3. Effect of monovalent cations (Na+ and K+) on the removal of bacteriophage T2 by magnetic filtration

Ionic Strength (]l) Concentration (ppm) % Removal of T2a by Magnetic Fi1trationb of the Salt Used of the Salt Used

NaCL or KC1 NaC1 K~n:;; NaC1 KC1

4.35

4.35

4.35

4.35

b

x 10-4 25.4 3Z244 62.1 46.8

x 10-3 254 ,'}2~24 56.1 55.9

x 10- 2 \~940 '·'3240 72.9 79.5

x 10-1 25Zl!00 Z~gi)OO 65.2 61.0

The initial bacteriophage T2 concentration was 2000 pfu/m1.

The bacteriophage T2 was shaken (~130 oscillations/min) for 20 min in the presence of 300 ppm of magnetite and various concentrations of NaC1 or KC1. These mixtures were subse-quently poured through the magnetic filter.

-21-

Table 4. Effect of a monovalent cation (Na+) on the removal of Poliovirus Type I (Sabin) by magnetic filtration

Ionic Strength Concentration % Removal of Poliovirus a of NaCl ( ]1) of NaCl (ppm) by Magnetic Filtrationb

4.35 x 10-4 25.4 93.0

4.35 x 10-3 254 98.7

4.35 x 10- 2 2540 99.1

4.35 x 10-1 25400 98.9

a The initial Poliovirus concentration was 15 x 103 pfu/ml.

b The Poliovirus was shaken (~ 200 oscillations/min) for 20 min in the presence of 500 ppm of magnetite and various concentra-tions of NaCl. These mixtures were subsequently poured through the magnetic filter.

-22-

Table 5. Effect of divalent cations (Ca++ and Mg++) on the removal of bacteriophage T2 by magnetic filtration

Ionic Strength (~) Concentration (ppm) % Removal of T2a by of the Salt Used of the Sal t Used Magnetic Filtrationb

CaC12 or MgC12 CaC12 MgC12 CaC1 2 MgC12

4. • t;Jg X l(,D-4 16.1 13.8 26.1 35.4

4.35 x 10- 3 161 138 82.1 80.7

4.35 x 10-2 1610 1380 93.1 81.2

4.35 x 10-1 16100 13800 95.9 82.3

a The initial bacteriophage T2 concentration was 2000 pfu/ml.

b The bacteriophage T2 was shaken (~ 130 oscillations/min) for 20 min in the presence of 300 pp~ of magnetite and various concentrations of CaC12 or MgC1 2 . These mixtures were sub-sequently poured th~~ugh the magneti~ filter.

-23-

Table 6. Effect of divalent cations (Ca++ and Mg++) on the removal of bacteriophage T4R(II) by magnetic filtration

Ionic Strength (~) Concentration (ppm) % Removal of T4R(II)a by Magnetic Fi1trationb of the Salt Used of the Salt Used

CaC12 or MgC12 CaC12 MgC1 2 CaC12 Mg0l;2

4.35

4.35

4.35

4.35

a

b

x 10-4 16.1 13.8 62.6 63.3

x 10-3 161 138 60.1 69.8

x 10-2 1610 1380 85.1 71.2

x 10-1 16100 13800 92.5 76.4

The inttia1 bacteriophage T4R(II) concentration was 2000 pfu/m1.

The bacteriophage T4R(II) was shaken (~ 130 oscillations/min) for 20 min in the presence of 300 ppm of magnetite and various concentrations of CaC12 or MgC1 2 . IIhae mixtures were subse-~uent1y poured through the magnetic filter.

-Z4-

Table 7. Effect of divalent cations (Ca++ and Mg++) on the removal of bacteriophage MSZ by magnetic filtration

Ionic Strength (~) of the Salt Used

Concentration (ppm) of the Salt Used

% Removal of MSZa by Magnetic Filtration b

CaC1 Z or MgC1 Z CaC1 Z MgCl z CaC1 Z MgCl z

4.11 x 10-Z 1610 1380 99.6 99.6

b

ID[b initial bacteriophage MSZ concentration was 7000 pfu/ml.

~me bacteriophage MSZ was shaken (~ 130 oscillations/min) for ZO min in the presence of 500 ppm of magnetite and the concen-tration of CaC1Z or MgC1Z sho~n above. These mixtures were subsequently poured through the magnetic filter.

-25-

Table 8. Effect of a divalent cation (Ca++) on the removal of Poliovirus Type I (Sabin) by magnetic filtration

Ionic Strength Concentration % Removal of Poliovirusa of CaC12 (11) of CaC12 (ppm) by Magnetic Filtrationb

4.35 x 10-4 16.1 16.0

4.35 x 10-3 161 99.8

4.35 x 10-2 1610 99.6

4.35 x 10-1 16100 99.1

a The initial Poliovirus concentration was 15 x 103 pfu/ml.

b The Poliovirus was shaken (rv 200 oscillations/min) for 20 min in the presence of 500 ppm of magnetite and various concentrations of CaC12. These mixtures were subsequently poured through the magnetic filter.

-26-

Table 9. Effect of a trivalent cation (Al+++) on the removal of bacteriophage T2 by magnetic filtration

Ionic Strength of A12(S04)3 . l8H20 (~)

Concentration of A1 2(S04)3 . l8H20 (ppm)

% Removal of T2a by Magnetic Filtrationb

4.35 x 10-4

13.05 x 10-4

21.75 x 10-4

20

60

100

64.8

43.6

85.4

a

b

The initial bacteriophage T2 concentration was 5600 pfu/ml.

The bacteriophage T2 was shaken (~ 130 oscillations/min) for 20 min in the presence of 300 ppm of magnetite, various concentrations o-f A12(S04)3 . l8H20 and pH's adjusted to 6.0 ~ 0.1 with 1.ON NaHC03' These mixtures were subsequently poured through the magnetic filter.

-27-

Table 10. Effect of a trivalent cation (Al+++) on the removal of bacteriophage T4R(II) by magnetic filtration

Ionic Strength of Concentration of % Removal of T4R(II)a by Magnetic Filtrationb A1 2(S04)3 . l8H20 (~) A1 2(S04)3 . l8H20

(ppm)

,.g~ x L@-4 20 41.7

13.05

21.75

a

b

x 10-4 60 99.5

x 10-4 100 99.2

The initial bacteriophage T4R(II) concentration was 3000 pfu/ml.

The bacteriophage T4R(II) was shaken (~ 130 oscillations/min) for 20 min in the presence of 300 ppm of magnetite, various concentrations of A1 2( S04)3 . l8H20 and pH's adjusted to 6.0 ~ 0.1 with 1.ON NaHC03. These mixtures were subsequently poured through the magnetic filter.

-2S-

Table 11. Effect of a trivalent cation (Al+++) on the removal of bacteriophage MS2 by magnetic filtration

Ionic Strength of Concentration of % Removal of MS2a bb A1 2(S04)3 . lSH20 (~) A1 2(S04)3 . lSH20 Magnetic Filtration

(ppm)

4.35 x 10-4 20 99.6

13.05

21. 75

a

b

x 10-4 60 100.0

x 10-4 100 100.0

The initial bacteriophage MS2 concentration was 3700 pfu/ml.

The bacteriophage MS2 was shaken ('V 130 oscillations/min) for 20 min in the presence of 500 ppm of magnetite, various concentrations of A12(S04)3 . lSH20 and pH's adjusted to 6.0 + 0.1 with O.lN NaOH. These mixtures were subsequently poured through the magnetic filter.

-29-

Table 12. Effect of a trivalent cation (Al+++) on the removal of Poliovirus Type I (Sabin) by magnetic filtration

T@1lI.9:c Strength of Concentration of pHa of % Removal of POliovirusb by A12(S04)3 l8H2O Al~~SO\) 3 .

tppm) l8H20 Solution

(ll) Magnetic Filtrationc

4.35 x 10-4 20 4.64 81.6

13.05 x 10-4 60 4.40 98.5

21.75 x 10-4 100 4.29 90.4

4.35 x 10-3 200 4.14 91.7

a

b

c

The pH's reported represent the actual measured pH's of the solutions; no pH adjustmen$sw~~re made.

The initial Poliovirus concentration was 15 x 103 pfu/ml.

The Poliovirus was shaken (~ 200 oscillations/min) for 20 min in the presence of 500 ppm of magnetite and various concentrations of A12(S04)3 . l8H20. These mixtures were subsequently poured through the magnetic filter.

-30-

of the 93% removal of poliovirus and 6Z.l% removal of TZ phage obtained in the presence of only Z5.4 ppm of NaCl (Tables 3 and 4). The adsorption of poliovirus was also found to decrease when the alum concentration was increased to above 60 ppm (see Table lZ). As no pH adjustments were made when adding the alum, this decrease in sorption was probably due to the lowering of the pH by 100 and ZOO ppm of alum to pH values of 4.Z9 and 4.14, respectively. These values are below the isoelectric point of 4.5 reported by Mandel (1971) for poliovirus type 1. We also noted that in the pH experiment described in the next section, the adsorption of poliovirus was also weak below pH 5. One may aiso add that, among the three bacteriophages studied, MSZ is the only one which has an adsorption pattern similar to that of poliovirus.

Effect of pH

The pH of the suspending medium has been reported to be significant in the adsorption of viruses to such surfaces as soil, membrane filters and synthetic insoluble polyelectrolytes (Reece, 1967; Wallis and Melnick, 1967a; Wallis et al., 1971). In order to simulate the pH of most natural waters,~he adsorp-tion of TZ phage, MS~ phage and poliovirus to magnetite was studied in the pH range of 4 to 9. Figure 4 shows that, above pH 5, the removal of poliovirus was i:limthe 98-99% level and did not vary significantly. However, below pH 5, a decrease in removal was observed for this virus. For the phages TZ and MSZ, the removal (98-99% level) did not vary significantly in the pH range of 4 to 9. Bacteriophages TZ and MSZ, and poliovirus type 1 have isoelectric points of 4.Z, 3.9 and 4.5, respectively (see Table 1). Consequently, the studied pH 4 was above the isoelec-tric point of MSZ, only slightly below the isoelectric point of TZ and significantly below the isoelectric point of poliovirus. It is postulated that the adsorption of poliovirus onto magne-tite was inhibited by lowering the pH of the suspending medium sufficiently below the isoelectric point of the virus. Unfortu-nately, no data was collected on the electrophoretic mobility of magnetite particles in order to draw more definite conclusions.

Kinetics of the Adsorption Process

The kinetics of the adsorption process was investigated and it was found that 10 to ZO minutes of shaking were sufficient to allow an optimum removal of poliovirus (see Table 13). For the phage MSZ, equilibrium was reached,wi;thl8.s little,as5to 10 min-utes of shaking (Table 13). On the other hand, the removal of TZ phage increased with time in the range of shaking times studied and did not reach an equilibrium value (see Table 13). From the above results, i ttmrssdecided to allow ZO minutes shaking time for the adsorption experiments. This period of shaking was suf-

Figure 4. Effect of pH on the removal of bacteriophage T2, bacteriophage MS2 and Poliovirus by magnetic filtration

Bacteriophage T2 (5 x 103 pfu/ml), bacteriophage MS2 (5 x 103 pfu/ml) and Poliovirus Type I (15 x 103 pfu/ml) were each shaken (~ 170 oscillations/ min) for 20 min in the presence of 500 ppm of magnetite, 1610 ppm of CaC12 and varying pH (adjusted with O.lN NaOH and O.lN HCl). These mixtures were subsequently poured through the magnetic filter.

I Vol ..... I

o o

-32-

T I I I ~

OJ en rO

..c: 0...

N l-

s a sn.A ~ A J 0 LEA 0 ill a ~%

OJ en rO

..c: 0...

N V) 0;:-.

"""

til ::::l So-

'r-

> ·0 'r-

0 c..

co

~ :c 0...

L()

o

-33-

Table 13. Effect of time of shaking on the removal of bacterio-phage T2• bacteriophage MS2 and Poliovirus Type I (Sabin) by magnetic filtration

Time

a

b

c

d

e

of Shakinga % Removal by Magnetic Fil trationb (min) T2c Msit PoUoviruse

1 56.3 90.4 66.8

5 80.9 99.2 94.7

10 83.2 100.0 100.0

20 95.3 98.5 99.5

30 97.1 99.8 98.9

The flasks we~e shaken at ~ 170 oscillations/min.

The:mmggBtic filtration was undertaken in the presence of 500 ppm of magnetite and 1610 ppm of CaC12.

The initial bacteriophage T2 concentration was 7 x 103 pfu/ml.

The initial bacteriophage MS2 concentration was 4 x 103 pfu/ml.

The initial ~oliobirus concentration was 3 x 103 pfu/ml.

-34-

ficient for the optimum removal of poliovirus and MS2 phage but apparently not for T2 phage. Nevertheless, a longer period of shaking was not allowed for the phage T2 in order to avoid the large inactivation of the virus which wOl!l,ld occur when shaken for periods longer than 20 minutes.

Effect of Magnetite Concentration

The concentration of magnetite, used as a seed material for magnetic filtration, is an important factor to consider in light of the economic feasibility of the process. Figyre 5 shows that 300 ppm of magnetite wasrsufficient for the optimum removal of the phage MS2 (6 x 103 ~ml) and poliovirus (8 x 103 pfu/ml) and 500 ppm is sufficient'for the phage T2 (4.5 x 103 pfu/ml). Consequently, adsorption experiments were carried out in the presence of 500 ppm of magnetite in order to insure optimal conditions for adsorption and subsequent removal by magnetic fiI tration.

Effect of Initial Virus Concentration

The Freundlich adsorption isotherm (Fair et al., 1968) has often been used to show that removal of a solute (e.g., virus) from an aqueous solution by solid media was an adsorption pro-cess. This empirical relation is expressed as

y/m = kcl / n , (4)

where, for the specific case in which the adsorbate is a virus, y/m is the quantity of virus removed per unit weight of adsor-bent (e.g., magnetite) and c is the concentration of virus remaining in solution at equilibrium. The constant k has been described as a measure of the surface area of the solid phase and the constant n as an indicator of the intensity of adsorp-tion (Reece, 1967). The Freundlich equation is usually used in the logarithmic form,

log (y/m) = log It + l/n log c, (5)

which indicates a linear variation of log (y/m) with log c. Con-sequently, a double-logarithmic plot of data conforming to the Freundlich isotherm should give a line with slope l/n and log k as the / y-intercept.

The interaction of viruses with such surfaces as activated carbon, activated sludge, stabilization pond solids and soil (Gerba et al., 1975; Clarke et al., 1961; Sobsey and Cooper, 1973; Drewry andJ6liassen, 1968) has been shown to obey the Freundlich isotherm. This general tendency of virus-surface systems to con-form to the Freundlich isotherm shows the importance of adsorption

Figure 5. Effect of magnetite concentration on the removal of bacteriophage T2, bacteriophage MS2 and Poliovirus Type I (Sabin) by magnetic filtration

Bacteriophage T2 (4.5 x 103 pfu/m1), bacteriophage MS2 (6 x 103 pfu/m1) and Poliovirus (8 x 103 pfu/m1) were each shaken (~ 170 oscillations/min) for 20 min in the presence of 1610 ppm of CaC12 and various concentrations of magnetite. These mixtures were subsequently poured through the magnetic filter.

I Vl Ul I

U1 (J)

U1 :::s s...

>

4-0

0 E (J)

0:::

~

100 1 ("\~ K: me :a: _______ ~

80

60

T 2 phage .. -----e MS2 phageO 0

Poliovirus rJ ()

200 400 600 800 1000

Magnetite concentration (ppm)

I V-l Q'\ I

-37-

Figure 6. Freundlich isotherms for the adsorption of bacterio-phage T2, bacteriophage MS2 and Poliovirus Type I (Sabin) to magnetite: variable virus concentration

Ba6te:tH:(\1]ilhllgegf£2 r bacteriophage MS2 and Poliovirus were each shaken (~ 170 oscilla-tions/min) for 20 min in the presence of 500 ppm of magnetite and 1610 ppm of CaC12. These mixtures were subsequently poured through the magnetic filter.

Linear regression analysis yielded the least squares regression lines drawn:

Slope y-Intercept r2

T2 phage 1.035 1.354 0.99

MS2 phage 1.166 2.314 0.98

Poliovirus 0.736 2.743 0.99

OJ +-> ',-

+-> OJ s::: en to E

4-o

en E

~

OJ Cl.

"0 OJ > o E OJ ~

',-

>

o

7

6

5

4

3

2

o

...;.38-

2

T2 phage .. --- .... MS2 phageO----~O

Poliovirus()~--~()

3 4

Log lo virus concentration in effluent (pfu/ml)

5

-39-

in removing viruses from the water suspension. In order to determine the nature of the removal process by magnetite, an experiment was performed in which the T2 phage, MS2 phage and poliovirus concentration was varied but the magnetite concen-tration was held constant. Figure 6 shows that the results obtained for the three viruses conform to the Freundlich iso-therm. The Freundlich isotherm for the T2 phage was found to be below the isotherms for poliovirus and the MS2 phage (Figure 6). This could be explained by the larger size of the T2 phage in comparison to poliovirus and the phage MS2 (see Table 1). Due to its larger size, less T2 phage can be adsorbed per mg of magnetite when compared to a smaller virus such as poliovirus or the MS2 phage. Also, notice that the y-intercept ("log k" in the Freundlich equation) is less for the T2 phage 0.354) than for poliovirus (2.743) or the phage MS2 (2.314). Since the constant k in the Freundlich equation has been described as a measure of the surface area of the adsorbent, then a decrease in its value, as seen for the phage T2, may indicate a reduction of the surface area available to viruses for adsorp-tion onto magnetite. In this case, the surface area is not decreased but the size of the virus is increased. The dif-ference in the adsorption pattern may also be due to the fact that T2 phage has a tail whereas MS2 phage and poliovirus are tailless.

Effect of Wastewater Effluents on the Adsorption of Viruses to Magnetite

Wastewater effluents contain organic materials which may compete with viruses for adsorption onto solids (Amirhor and Engelbrecht, 1974; Carlson et al., 1968; Dieterich, 1953; Gerba et al., 1975) and may lead to a-decreased removal of the infective particles. Consequently, experiments were undertaken to deter-mine if the adsorption of viruses to magnetite was influenced by organics present in two wastewater effluents, "dome water" and University of Florida campus activated sludge effluent (see Table 2 for characteristics of these effluents). Table 14 shows that, in the absence of CaC1 2, dome water strongly inter-feres with the removal of the phage T2 (0.0% removal) and that this interference could be reduced (but not completely eliminated) with the addition of CaC12' It can be seen in Table 15 that the removal obtained in the presence of dome water and 1610 ppm of CaC1 2 for the phage T2 can be increased by diluting the waste-water effluent (and the organics present) in distilled water. Removal of the organic color present in dome water with an acti-vated carbon treatment greatly reduced the interference exerted by this effluent (94.9% removal of T2 -- see Table 15). However, the removal of T2 phage found in distilled water (98.3%) could not be obtained in dome water regardless of previous treatment. Apparently, the activated carbon did not remove all the interfer-ing substances from the water. Table 16 shows clearly that dome

-40-

Table 14. Effect of a divalent cation (Ca++) on the removal of bacteriophage TZ by magnetic filtration in the pre-sence of dome water

Ionic Strength of CaClZ (]l)

~

4.35 x 10-4

4.35 x 10-3

4.35 x 10-Z

4.35 x 10-1

Concentration of CaCl Z (ppm)

0

16.1

161

1610

16100

% Removal of TZa by Magnetic Filtrationb in the presence of Dome

WaterC

0.0

16.4

44.8

76.0

86.9

a The initial bacteriophage TZ concentration was 5.6 x 103 pfu/ml.

b The bacteriophage TZ was shaken (~ 130 oscillations/min) for ZO min in the presence of dome water, 500 ppm of magne-tite and various concentrations of CaClZ. These mixtures were subsequently poured through the magnetic filter.

c The dome water was sampled at a cypress dome located north of Gainesville, Florida.

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Table 15. Removal of bacteriophage T2 by magnetic filtration in the presence of dome water

Description

Dome WaterC

Dome Water I1il1lltibed 1:2 in Distilled Water

Dome Water kiiill.1lJ[1ten 1: 4 in Distilled Water

Activated Carbon Filtered Dome Water

Distilled Water

% Removal of T2a by Magnetic Filtrationb

74.3

75.7

88.6

94.9

98.3

a The initial bacteriophage T2 concentration was 4 x 103 pfu/ml.

b

c

The bacteriophage T2 was shaken (~ 130 oscillations/min) for 20 min in the presence of 500 ppm of magnetite, 1610 ppm of CaC12 and the various solutions described above. These mix-tures were subsequently poured through the magnetic filter.

The dome water was sampled at a cypress dome located north of Gainesville, Florida.

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Table 16. Removal of Poliovirus Type I (Sabin) by magnetic filtration in the presence of dome water

Description

Dome WaterC

Dome Water + 1610 ppm CaC1 2

Distilled Waterd

Distilled Water + 1610 ppm CaC12

Activated Carbon Filtered Dome Water

Activated Carbon Filtered Dome Water + 1610 ppm CaC12

% Removal of Poliovirusa by Magnetic Filtrationb

0.0

96.3

99.4

99.6

99.2

99.1

a The initial Poliovirus concentration was 15 x 103 pfu/ml.

b

c

d

The Poliovirus was shaken (~ 200 oscillations/min) for 20 min in the presence of 500 ppm of magnetite and the various solutions described above. These mixtures were subsequently poured through the magnetic filter.

The dome water was sampled at a cypress dome located north of Gainesville, Florida.

The distilled water was adjusted to the same .conductivity as the dome water (conductivity equal to 385.0 ~mho/cm at 250 C) with CaC1 2.

-43-

water also interferes with the removal of poliovirus. This interference was significantly reduced by adding 1610 ppm of CaC12 (96.3% removal) or by removing the organic color with activated carbon treatment (99.2% removal). The interfer-ence of dome water with the adsorption of viruses on magnetite is probably due to the ful vic acid fraction released in the water by the decomposition of cypress needles and other leaves within the cypress dome. It is generally known that fulvic acids are soluble in water whereas humic acids are not although some low molecular fractions may be (Flaig, 1960; Prakash and Rashid, 1968). It has been found that dome water also inter-feres with the adsorption of poliovirus on soil and may be efficient for the elution of soil-adsorbed viruses (Bitton, et a1., 1976).

The other effluent used in this study was the University of Florida campus activated sludge effluent. The interference exerted by the organics present in this effluent was slight. When this effluent was used as the suspending medium for polio-virus, the removal was 96.7% in the absence of any salt and was enhanced to 99.4% with the addition of 1610 ppm of CaC12 (Table 17). This removal was similar to the one observed in distilled water adjusted to the conductivity of the activated sludge effluent (385 ~mho/cm at 250 C) or containing 1610 ppm of CaC12 (Table 17).

It is possible to ~re the two wastewater effluents, dome water and campus activated sludge effluent, for their inter-ference with the adsorption of poliovirus onto magnetite (Tables 16 and 17). The interference was mostly apparent with the dome water and was completely eliminated by filtering the dome water through an activated carbon column. In the presence of CaC12, the campus activated sludge effluent displayed a better virus removal (99.4%) than the dome water (96.3%). The main difference between the two types of effluents (st:fe Table 2) was the color content. The dome water had a color ranging from 350.0 to 750.0 units, whereas the campus activated sludge effluent had a color ranging from 30.0 to 35.0 units. Therefore, it could be postu-lated that the organic color was responsible for the observed interference. It should be added that dome water is not a typical wastewater effluent due to its high organic color. Consequently, the results of the campus activated sludge effluent should be considered when assessing the virus removing potential of magnetic filtration in the presence of wastewater effluents.

Additional experiments were carried out in which the virus concentration was held constant and the magnetite concentration was varied. The data in Figures 7, 8 and 9 shows that, in the presence or the absence of secondary wastewater ~ff£llents, the adsorption of bacteriophage T2, bacteriophage MS2 and poliovirus

-44-

Table 17. Removal of Poliovirus Type I (Sabin) by magnetic filtration in the presence of activated sludge effluent

Description % Removal of Po1iovirus a by Magnetic Fi1trationb

Activated Sludge Eff1uentC 96.7

Activated Sludge Effluent + 1610 ppm CaC12

99.4

Distilled Waterd 99.4

Distilled Water + 1610 ppm CaC12

99.6

a

b

c

d

The initial Poliovirus concentration was 8 x 103 pfu/m1.

The Poliovirus was shaken (~ 200 oscillations/min) for 20 min in the presence of 500 ppm of magnetite and the various solutions described above. These mixtures were subsequently poured through the magnetic filter.

The activated sludge effluent was sampled at the University of Florida campus sewage treatment plant.

The distilled water was adjusted to the same conductivity as the activated sludge effluent (conductivity equal to 385.0 ~mho/cm at 2S0 C) with CaC12.

Figure 7. Freundlich isotherms for the adsorption of bacteriophage T2 to magnetite in distilled water and dome water: variable magnetite concentration

Bacteriophage T2 (4.5 x 103 pfu/ml) was shaken (~ 130 oscillations/min) for 20 min in the presence of 1610 ppm of CaC12, various concentrations of magnetite (ranging from 100 ppm to 1000 ppm) and distilled .. Water of dome water. These mixtures were subse-quently poured through the magnetic filter.

Linear regression analysis yielded the least squares regression lines drawn:

Distilled Water Dome Water

slope

0.595 0.718

y-intercept

2.889 1.167

r2

0.86 0.99

I .j:>.

c.n I

-46-

T ..--.. I ..... I E I

.......... ::l

~ 4-0-S- .f-I a; c:

+> a) to ::l

:3: M ..... S- 4-

-0 a; 4-a; .f-I a; ..... to r- :3: c:

\ ..... ..... , .f-I a; • III E c: , ..... 0 0 , Cl Cl ..... +> , to s... , • .f-I , c: a; , U c: , 0 , N U

'. N I-\ , a; O"l ,. to

oJ:: , 0-•• 0 . .... S-a; .f-I U ·to ..a

0 r- r-.

O"l 0 -I

o

. (6wjnJd) cl:j. ~ lau6ew JO 6w .,lad paAOWa.A G 1 a6eljdo ~iapeq 0 LOOl

Figure 8. Freundlich isotherms for the adsorption of bacteriophage MS2 to magnetite in distilled water and activated sludge effluent: variable magnetite concentration

Bacteriophage MS2 (6.3 x 103 pfu/ml) was shaken (~ 130 oscillations/min) for 20 min in the presence of 1610 ppm of CaC12' various concentrations of magnetite (ranging from 100 ppm to 1000 ppm) and distilled water or activated sludge effluent. These mixtures were subsequently poured through the magnetic filter.

Linear regression analysis yielded the least squares regression lines drawn:

Distilled Water Activated Sludge Effluent

slope

0.769 1.021

y-intercept

3.082 1.272

r2

0.99 0.95

I ..,. ---.] I

0)

E -;- 5 4-0..

Q) ~ ...... ~ Q)

s:: 0)

ro E

4-o 0)

E ~

~ 4 -0 Q)

> o E Q) ~

N Vl :E: Q) 0)

ro ..s:: 0.. o ~

'" ",e /

/ /

'" W' [eJ

'" . .,/ /

'" / ..I'

[oJ

2 3 I Distilled Water .. ---~ i L Activated Sludge Effluent 0 0 o III 2 3 4

Log lo bacteriophage MS2 concentration in effluent (pfu/ml)

I .j::> 00 I

Figure 9. Freundlich isotherms for the adsorption of Poliovirus Type I (Sabin) to magnetite in distilled water and wastewater effluents: variable magnetite concentration

Poliovirus (8 x 103 pfu/ml) was shaken (~ 200 oscillations/min) for 20 min in the presence of 1610 ppm of CaC12, various concentrations of magnetite (ranging from 100 ppm to 1000 ppm) and distilled water, dome water or activated sludge effluent. These mixtures were subsequently poured through the magnetic filter.

Linear regression analysis yielded the least ~quares regression lines drawn:

Distilled Water Activated Sludge Effluent Dome Water

slope

0.715 1.395 2.590

y-intercept

2.897 1.011

-3.313

r2

0.98 0.94 0.99

I ..j:>. \D I

5

01 [.] E '- (J ::::s 4-a.

'-'"

Q) .jJ .... ". +-l Q) / c 01 " tt1 ", E ,.4 4-

4 e''''' 0 01 E [.yo [OJ S-Q) 0.. I (J1

-0 0 Q) I

> [0] 0 E Q) S-

III ::::s S-.... >

°1 Distilled Water .. ---... .... ~. 3 . Activated Sludge Effluent 0 0 o· ~ Dome Water 0 0 r-01

.0 -l

~/I I I I o . I 1 2 3 Il LoglO Poliovirus concentration in effluent (pfu/ml)

-51-

onto magnetite conformed to the Freundlich isotherm. However, in the presence of wastewater effluents, the Freundlich iso-therms had a lower y-intercept ("log kIf in the Freundlich iso-therm equation) than that found in distilled water. For example, in Figure 7, for the phage T2, the y-intercept of the Freundlich isotherm was as high as 2.889 in the absence of organic materials and decreased to 1.167 in the presence of dome water. For the phage MS2 (see Figure 8), the y-intercept was 3.082 in distilled water and dropped to 1.272 in the presence of activated sludge effluent. Figure 9 shows that, for poliovirus, the y-intercept was as high as 2.897 in distilled water and decreased to 1.011 in the presence of campus activated sludge effluent and -3.313 in the presence of dome water. This consistent drop in the value of the y-intercept ('I'ihQg kIf) when the viruses are suspended in wastewater effluents aan be explained in the following manner. The constant k in the Freundlich isotherm equation has been des-cribed as a measure of the surface area of the solid phase (Reece, 1967) and, therefore, a decrease in its value may indicate a reduction in ~he surface area of magnetite available to viruses for adsorption. This reduction is due to the occupation of adsorption sites on the magnetite by competing organics present in wastewater.

Effect of Organics on the Adsorption of Poliovirus to Magnetite

A variety of organic substances such as bovine albumin, egg :m.lbuniin. meat infusion broth and cell extracts have been shown to compete with viruses for adsorption onto solids (Carl-son et al., 1968; Shepard and Woodend, 1951; NRililis and Melnick, 119)67a). In order to determine if organics would also interfere with the adsorption of poliovirus to magnetite, an experiment was undertaken using egg albumi~,caasein and dextran. Table 18 shows that 1000ppm of these materials significantly :iiutterfered with the removal of poliovirus. Casein is known to be a highly efficient eluent of virus from cellulose membranes (Wallis and Melnick, 1967b) and therefore, its complete interference with the removal of poliovirus (0.0%) is not surprising (Table 18). Moreover, it should be noted that 100 ppm of these organic materials is an extremely high concentration. For example, Carlson et al. (1968) showed that as little as 2ppm of egg albumin reduced the adsorption of the phage T2 to the clay KaoIilTite 4 from 93% to 27%. It can be concluded that a reduction in the removal of viruses by magnetic filtration in the presence of high concentrations of an interfering substance is to be ~ecmd~

Infectivity of Viruses Adsorbed to Magnetite

The previous sections have dealt with the factors influenc~ ing the adsorption of viruses to magnetite. It was learned that

-52-

Table 18. Inn~e by egg albumin, casein and dextran with the removal of Poliovirus Type I (Sabin) by magnetic fil-tration

Description

100 ppm Egg Albumin

100 ppm Casein

100 ppm Dextran (5 x 105 MW)

% Removal of Poliovirusa by Magnetic Filtrationb

42.4

0.0

7.1

99.6

a The initial Poliovirus concentration was 1.0 x 104 pfu/ml.

b The Poliovirus was shaken (~ 200 oscillations/min) for 20 min in the presence of 500 ppm of magnetite, 1610 ppm of CaC1 2 and the various solutions described above. These mixtures were subsequently poured through the magnetic filter.

-53-

under appropriate conditions, viruses can be efficiently ad-sorbed to magnetite and subsequently removed by magnetic fil-tration. Experiments were then performed to determine the infectivity of the adsorbed virus. We used low concentrations of viruses so that samples might be directly plated and there-by, avoid dilutions which could lead to the desorption of viruses. Table 19 shows that T2 phage (97.5%), MS2 phage (~OO.O%) and poliovirus (98.9%) were effectively adsorbed to magnetite. The infectivity of adsorbed poliovirus and MS2 phage was >100.0%. The elevated titer (>100.0%) of the adsorbed virus could have resulted from virus disaggregation when shaken for 20 minutes. Schaub and Sagik (1975) have also observed that enteric viruses adsorbed to either Montmorillonite clay or other naturally occurrmng solids displayed an elevated titer. These authors proposed that the clay particles may function in allowing the virus to establish better proximity to the cells. Table 19 also shows that the infectivity of adsorbed T2 phage was lower than that displayed by polioviruso(!Jr~tfillS2pphgge and much more variable (57.8% and 93.5% infective). Puck and Sagik (1953) have shown that T2 phage, after its attachment to a cationic resin, was split into its DNA and protein fractions and therefore, could not be recovered in active form. It appears that upon adsorbing to this surface, the T2 phage ejac-ulates its DNA much as it does ordinarily at the surface of its host cell. It is postulated that the reduced infectivity of T2 phage may have resulted from the ejaculation of its DN~ upon adsorbing to magnetite. Furthermore, the variability found in infectivity may be due to the way in which the phage is adsorbed. If the phage is adsorbed by the tail to the magnetite, then ejaculation and inactivation may occur. However, if adsorption occurs on the phage head, then inactivation does not occur. Finally, it can be said that viruses adsorbed to magnetite ate infective. Moore et al. (1975) have studied the association of poliovirus and phages-r2, T7 and f2 with clays and suspended solids. They found that all the viruses tested, with the excep-tion of f2 phage, were infective in the adsorbed state. In our study, we found that the tailless RNA phage, MS2,dGl.;i;~:played a similar behavior as poliovirus type 1 and these two viruses were 100% infective in the adsorbed state. In view of these findings, it should be clear that the safe treatment and disposal of virus-laden magnetite is critical in controlling the spread of disease.

Elution of Poliovirus from Magnetite

Prior to initiating experiments on the concentration of poliovirus by magnetic filtration, it was important to deter-mine which solution would be best for the elution of poliovirus from magnetite. Table 20 shows the eluents used and the percent

Table 19. Infectivity of bacteriophage T2, bacteriophage MS2 and Poliovirus Type I (Sabin) adsorbed to magnetite

Virus a Direct Plate Direct Plate @ T = 0 min @ T = 20 min

(pfu/ml) (pfu/ml)

T2 phage 1190 700 790 740

MS2 phage 717 800

% tm:lfectivit~b of Adsorbed

Virus

57.8 93.5

>100.0

Supernatant (pfu/ml)

30 20

o

% Virus Adsorbedc

97.5 97.5

100.0

Poliovirus 1200 1335 >100.0 13 98.9

a

b

c

The bacteriophage T2, bacteriophage MS2 and Poliovirus were each shaken (~ 170 oscillations/ min) for 20 min in the presence of 500 ppm of magnetite and 1610 ppm of CaC1 2. Samples were removed and assayed by direct plating (no dilutions made) immediately after addition of the virus (T = 0 min) and after the adsorption period (T = 20 min). Following the adsorption period, the magnetite was pelleted by placing the mixtures next to a magnet and then the supernatants were assayed for virus. Controls without magnetite were also studied and they showed no significant inactivation of the three viruses during the 20 minutes of shaking.

The % infectivity of adsorbed virus was determined using the following equation:

~ tti£ectivit' = Direct Plate @ T = 20 (Efu/ml) - SUEernatant (Efu/ml) X 100 Y Direct Plate @ T = 0 (pfu/ml) - Supernatant (pfu/ml) ~)

The % virus adsorbed is based on the percentage of the direct plate at T = 0 min.

I CJ1 -1>0 I

-55-

Table 20. Elution of Poliovirus Type I (Sabin) from the magne-tite surface

Eluentb Used

Distilled Water

1610 ppm CaC1 2

Tris Buffer, pH = 9

3%, isotonic Beef Extract, isotonic buffered, pH = 9

10%, isotonic Fetal Calf Serum, isotonic buffered, pH = 9

Eagles Minimal Essential Medium (MEM) + 5% Calf Serum + 0.03 M Hepes Buffer, pH = 7.0

Glycine Buffer, 0.05 M, pH = 11.3

Dome Water,d Tris buffered, pH = 9

% RecoveryC of Adsorbed Poliovirusa

7.1

l.8

9.9

3.2

59.lB

39.5C

8.8

a The Poliovirus was shaken (rv 200 oscillations/min) for 20 min in the presence of 500 ppm of magnetite and 1610 ppm of CaC12. Aliquots were then removed and the magnetite was pelleted by

cpiLacing the mixture next to a magnet. Supernatants were assayed and showed that 99% of the initial Poliovirus concentration (1.0 x 104 pfu/ml) was adsorbed to magnetite.

b

c

d

Elution was undertaken as follows: the magnetite pellets were resuspended in the indicated eluents for 1 hour at 40 C (except for glycine buffer which was allowed only 1 min of contact time), were then pelleted again and finally, the supernatants were assayed for viruses.

The % recovery values displaying different superscript capital letters are significantly different at the 0.01 level for a two-tailed test. If no superscript capital letter appears, then a test of significance was not performed.

The dome water was sampled at a cypress dome located north of Gainesville, Florida.

-56-

recoveries obtained. The composition and source of the eluents used appear in the Appendix. The 1610 ppm CaC12 solution was the medium in which adsorption initially took place and. as expected, the re.c(i)weTyof adsorbed poliovirus using this solution as eluent was very low (108%). The 'best eiluent was mnti!$llltonic~" 10% solution of fetal calf serum, Tris buffered, pH = 9 and it enabled the desorption of 77.9% of poliovirus from the magnetite surface (Table 20). These results are not surprising since Rao et al. (1968) also used a similar solution to effectively elute e~teroviruses from an iron oxide. However, the low recovery obtained with an isotonic, 3% solution of beef extract, Tris buffered at pH 9 (3.2% recovery) was not expected. This solution has been shown to be an efficient eluent when desorbing viruses from an iron oxide·;surface (Pearson and Metcalf, 1974; Rao et al., 1968). Rao et al. (1968) has stated that not all lots of beef-extract have~he-same virus eluting capacity. Thus,it appears that the lot of beef extract used in this study probably had a low virus eluting capacity. Generally, the best eluents are proteinaceous materials adjusted to an alkaline pH (8-9). In many cases, good eluents also effectively interfere with the initial adsorption of viruses to surfaces. Dome water, which had previously been shown to interfere with the adsorption of poliovirus to magnetite, was studied to determine its efficiency in eluting poliovirus from magnetite. Table 20 shows that dome water, Tris buffered, pH = 9 could only elute 8.8% of adsorbed poliovirus. Therefore, in this case, a good interfering substance (dome water) was shown not to be a good eluent. Experiments with glycine buffer, 0.05M, pH = 11.3 were performed allowing only 1 minute of contact time (see Table 20). This was done in order to avoid the inactivation of the virus due to the high pH. In fact, when 1 hour of contact time was allowed, no infectivity could be detected in the eluate as compared to 39.5% recovery of adsorbed poliovirus when the contact time was 1 minute. From the results presented, it was decided to use the 10%, isotonic fetal calf serum, Tris buffered, pH = 9 solution as the eluent in the concentration experiment described in the next section.

Concentration of Poliovirus by Magnetic Filtration

Viruses occur in natural water bodies in very low concentra-tions and, therefore, their detection is dependent upon their adequate concentration. Various virus adsorbents have been used to effectively concentrate viruses and these have been discussed in the literature review section. Since magnetite can 3dsa~~~ under ~ppropriate conditions, over 99% of poliovirus, we have carried out a