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Adsorption of viruses on magnetic particles—I. Adsorption of MS2 bacteriophage and the effect of cations, clay and polyelectrolyte
~t:;tcr Rc~. Vol, I-. No. 8, pp, 9.,t3-94S. 1953 Printed in Great Britain. MI rights reserved
~X)43-135,/.'33 080943-06503.00 0 Cop.,.right D 1953 Pergamon Press Ltd
A D S O R P T I O N O F VIRUSES ON MAGNETIC PARTICLES I ADSORPTION OF MS2 BACTERIOPHAGE AND THE OF CATIONS, CLAY AND POLYELECTROLYTE
EFFECT
J. G. ATnERTOY and S. S. BELL Department of Microbiology. University of Queensland. St Lucia 4067. Brisbane. Australia (Receired October 1982)
Abstract--Washed samples of finely-divided magnetite are found, after alkali treatment, to adsorb MS2 bacteriophage very efficiently, from suspensions in 100 mg 1- ~ NaCI at pH 6. Suspended material, such as clay. components of sewage effluent and ions interfere with virus adsorption. but this interference can be largely overcome by the addition of suitable polyelectrolyte in tow concentration.
INTRODUCTION
the adsorption of poliovirus to magnetite (Bitton et
The usefulness of magnetite in the treatment of raw water has been studied by the C S I R O Division of Chemical Technology (CSIRO), South Melbourne. It has been found that magnetite, after chemical treatment, is capable of removing a large proportion of the dissolved and st.spended organic matter, as well as clay and other colloidal material, from previously untreated w a t e r / K o l a r i k et al., 1977). A study of the efficiency of removal of viruses from water has also been undertaken (Atherton & Bell, 1979), In this study, bacteriophage MS2 of Escherichia colt was used as a model for the behaviour of the humanpathogenic enteric viruses. Because of their small particle size (22-28 nm) and the protein nature of their surface, the enteric viruses will behave as charged colloidal particles in aqueous suspension, their net charge being dependent on their ionic environment. Finely-divided particles of magnetite behave similarly (Kolarik et al., 1980; Amirhor & Englebrecht, 1975). If the isoelectric pH (iep) of the virt, s differs from that of magnetite, pH conditions can be chosen such that the virus particles will be electrostatically bound to those of magnetite. At pH 10. where both magnetite and the MS2 virus particles carry negative charges, the virus, previously adsorbed to magnetite, might be expected to be released into the supernatant fluid, thus allowing the magnetite to be recycled after washing. Previous studies on the adsorptive capacity of iron oxide toward viruses include: the removal of influenza virus by haematite (Warren et al., 1966), the use of magnetic iron oxide-packed columns to concentrate viruses (Pearson & Metcalf, 1974) or to remove enteroviruses from water (Rao et al., 1968), removal of b a c t e r i o p h a g e T7 by magnetic filtration (Bitton & Mitchell, 1974) and more recently, factors affecting 943
al., 1976).
The present studies were undertaken to assess the removal of viruses which might take place in the Sirofloc water-treatment process employing magnetite, developed by the CSIRO. The effect, on the adsorptive process, of cations, pH, time, suspended and dissolved organic matter and the presence of polyelectrolytes, was also studied,
MATERIALS AND METHODS
MS2 bacteriophage and its host bacterium Escherichia colt C-3000 (a prototrophic strain of KI21 were kindly supplied by Professor J. Pittard. University of Melbourne. The host bacterium was inoct, lated into a solid culture medium containing 10g tryptone. 10g NaCI. 5g yeast extract and 15 g agar I-t of distilled water. For MS2 bacteriophage propagation a similar medium, without agar ("broth") but with 10g Na,HP~.I2H,O and 3.8g KH.,PO.~ I- ~. buffered at pH 9 was used (Sargeant. 1970). Stocks of MS2 were prepared by the layer agar method [Adams. 1959). Monodisperse phage preparations were prepared by collecting the freeze-thawed agar from the propagation plates, homogenising at low speed in a Waring blender, centrifuging at 10.000 g to separate the agar fragments, then filtering the phage-containing supernatant through a 0.2.ttm membrane filter with a prefilter stack, in a Sartorius pressure filtration cylinder. The filtrate was checked by electron microscopy and the titre determined by counting plaques (Adams. 1959). The stock MS2 preparation had a titre of 9.3 x l0 t° pfu ml- ~ and was stored in 1 m[ amounts in sealed plastic ampoules at -50-C. All inorganic chemicals used were Univar Analytical Reagent grade [Ajax Chemicals. Sydney) and bacteriological media were supplied by Difco Laboratories. Detroit. Michigan. Magnetite. supplied as a finely crushed powder of 1-5/tm particle size consisted of 66.3?o Fe_,O3 and 27.6°o FeO, and had a specific surface area of 0.6 m'- g- t. It was prepared for use in the following manner: First the magnetite was mechanically stirred for 10 rain each. with four changes of distilled water, then with four
944
J.G. ATHERTON and S. S. BELL
changes of 0. l N NaOH. the distilled water washing repeated and then similar treatment with four changes of 0.01 M HCI. In each case. the volume of the washing solution ~as 4 times that of the original magnetite suspension. This complete process of water-alkali-water-acid wash was repeated eight times. The "'8-cycled'" magnetite was then stored wet. in a wide-mouthed airtight polythene bottle. [mmediately before use the magnetite was again wash-cycled once, and demagnetised by exposure to 50 Hz alternating magnetic field produced by a coil carrying 240 V. 0.8 A. which surrounded the flask. This material is referred to as 'conditioned" magnetite. For use in experinaents, wash-c>cled magnetite was kept in a wide-bore burette in the unmagnetised state. The magnetite particles were allowed to settle for 12 h, and formed a "slurry" which could be run into reaction flasks in 1 ml amounts. Stable suspensions of clay in salt solutions were prepared from mixed (unstable) suspensions by sonication, in 4--15s bursts at 6 3 W c m -3. Suspensions containing 0.12 g I- 1 correspond to a turbidity of 90 NTU, 0.06 g I- t to 42 NTU. The clay used was a milled sample of kaolinite with a particle size of 1-2 #m. These clay particles have an isoelectric pH of 2.5. Polyelectrolyte AS101 was supplied by Catoleum (Australia). It is a strongly basic, highly charged cationic molecule of moderate chain length. Three different salt solutions were used in the study. I. 100 mg I- ~ NaCI, 2. 400 mg 1- t NaCI. and 3. 100mgI - t NaC1, 30mgl - t Ca-'-, 20mg1-1 Mg(500 mg I- 1 TDS). Sewage effluent was obtained, after secondary treatment, from the Brisbane City Council, Oxley Ck. Wastewater Treatment Plant. After autoclaving at 121~'C for 15 rain, it had a pH of 9, TDS 800-1000 mgl-~ and a turbidity of 62 NTU.
under the following conditions: exposure to pH over the range 4.8-11, NaCI concentrations 0-8500 mg 1- 1 and autoclaved sewage effluent at pH 9.
Adsorption of 3,1S2 bacterioptzage to magnetite Shaking speed. An adsorption experiment was performed in 100 mg 1-~ NaCI at pH 4. with rotational speeds from 100 to 300 rev m i n - ~, at an amplitude of 20 mm. Maximal adsorption ,,,,as obtained at speeds of 260 rev m i n - t or higher. All further experiments were performed at a shaking speed of 260 rev m i n - ~. pH. The effect of pH of the solution of 100 mg 1- t NaCI used a suspending medium for magnetite was tested over the range 3-11. using magnetite which had been washed and pH-cycled once only ("unconditioned" magnetite). Virus adsorption was much diminished above pH 5 (Figs I and 2). Multiple wash-cycling of magnetite. The virusadsorptive characteristics of magnetite which had been washed and pH-cycled only once, was compared to that of magnetite which had been so treated 8 times ("conditioned" magnetite). Virus adsorption is much improved in the latter and occurs up to pH 7
0
6
O0
Stability of MS2 bacteriophage Sufficient volume of monodisperse suspension of MS2 bacteriophage was added to a 500 ml stoppered conical flask containing 100ml of sterile aqueous suspending medium under test, to give a virus titre of approx. 10~' pfuml- ~ and a sample taken for counting initial titre. Rotational shaking on a "'New Brunswick" gyratory shaker at 260revmin-t at an amplitude of 20mm was then begun. Samples were taken at 5 rain intervals up to 30 rain. diluted in broth and plated for plaque-counting.
13-
0 0
0
o"uncond~tioned"mognetite o"conditioned'" magnetite
IE
pH Fig. 1
100
80
8
60
/-,0
o '~JncondRioned" magnetite
20
n "'conditioned" rnognetite
RESULTS
Stability of MS2 bacteriophage No significant reduction in titre of MS2 bacteriophage could be demonstrated over a period of 30 rain
0 I"1 [] 0 0 ClyD
• virus control
Adsorption oJ"MS2 bacteriophage to magnetite Two flasks, each containing I I0ml of suspending medium (salts solutions or sewage effluent) were used for each pH to be tested. One flask of each pair had I ml of wash-cycled magnetite slurry added to it. and both flasks autoclaved at 115 C for 15 rain. When cool each flask was adjusted to the desired pH with sterile 0.01 M HCI or NaOH by pipetting off I0 ml for use as an external aliquot for calculation of the acid or alkali necessary to adjust the pH of the remaining 100 ml. Virus was then added to give a titre of approx. 10~' pfu ml-t. a sample taken for virus titration and rotational shaking at 260 rev min-t at an amplitude of 20 mm, commenced. After 20min, shaking was stopped, the magnetite sedimented by magnetisation over a permanent magnet of 0.1T and samples taken for titration of unadsorbed virus.
~
7
pH Figs 1 and 2. Effect of pH on the adsorption of MS2 to "'conditioned" and "unconditioned" magnetite.
Adsorption of viruses on magnetic particles--I
945
lOO
go
"8
2
a~
~70
1
/ 2
3 [magl
t. No. of regeneration cyctes of magnetite
(Ioglo rng F )
Fig. 3. Effect of magnetite concentration on adsorption of
MS2. (Figs 1 and 2). All further experiments were performed using "conditioned" magnetite. Magnetite concentration. A series of tared flasks was set up, containing magnetite concentrations from 0.0156 to 1.0% in 100 mg 1- z NaCI at pH 4. Virus was added and the flasks shaken at 260rev rain -t for 20 rain. Samples of the supernatant were taken for virus titration, after sedimentation of the magnetite over a magnet. All flasks were then taken to dryness in a 56°C oven, reweighed and the magnetite content calculated. Figure 3 shows that virus adsorption was satisfactory at concentrations of 2000 mg 1- ~ (0.2%) or higher. Kinetics. Samples for virus titration were taken at intervals after the addition of MS2 bacteriophage to a suspension of 1% magnetite in 100 m g l - t NaCI at pH 4. Adsorption was seen to be rapid, being 99.6% completed after 3 min. Variable cirus input. 1% magnetite in 100mg1-1 NaCI at pH 4 was exposed to virus titres ranging from 10'~ to l0 s pfu ml-~. Figure 4 shows that the data conforms to the Freundlich isotherm with a
Fig. 5. Effect of regeneration pH cycle on MS2 bacteriophage-adsorptive capacity of magnetite. slope equal to 1.017 and a y-intercept equal to -3.98. The correlation coefficient r 2 = 0.99. Multiple stages of titus adsorption. The virus adsorbing capacity of a sample of magnetite exposed to virus after each cycle of pH change and washing, was compared with that of "fresh" magnetite (not previously exposed to virus) and controls. In the test flask, each time after virus samples had been removed for titration at the completion of the 20 rain shaking period the magnetite was wash-cycled, demagnetised and resuspended in 100 mg 1-1 NaCI at pH 4, before another virus dose was added to test the adsorptive capacity. Figure 5 shows that the virus adsorptive capacity of the recycled magnetite showed no diminution after 11 cycles.
Treatment of cirus-containing fluids by two stages of magnetite adsorption Test and control flasks were set up with 100 mg lNaCi. at pH4. MS2 bacteriophage approx. 10 9 pfu ml-1 was added to each flask. After shaking, the supernatant (containing approx. 105 pfu ml-~ of
~-.
4
I ~
input titre
-&
oe3
i 2 o -//
c
5
6
7
8
Input[virus] (togl0Pfu ml -t)
\ , l°°2t.tabe, \
0
~
No. of cycles of magnetite treahnent
Fig. 6. Amount
Fig. 4. Effect of input virus concentration on total virus adsorbed. w.R. 17 8--H
~
of virus adsorbed in successive stages of
magnetite treatment of MS2 suspension in 100mgl -~ NaCI at pH 4.
J. G. ATHERTONand S. S. BELL
946
5! input titre
input titre E
2 o
i fl
3
o.~
0.I05
o
o.o2
Potyetectrotyte (mg t-')
ob~
o.b6
0.08 '
oho
012 '
0.14 ' 016
Clay [g [-')
Fig. 7. Effect of polyelectrolyte concentration on MS2 adsorption at pH 4.
Fig. 9. Effect of clay concentration on MS2 adsorption to magnetite at pH 4.
unadsorbed virus) was added to a fresh flask containing 1 g magnetite. This flask was then shaken and sampled and the procedure repeated. Figure 6 shows the relationship between virus titre and number of stages of treatment with magnetite. Virus content was removed by a factor of 10"t in each successive adsorption stage.
Effect of clay on cirus adsorption
Effect of polyelectrolyte A8101 on rirus adsorption Flasks with 1~o magnetite were set up in 100 mg 1- t at pH 4 and polyelectrolyte concentrations between 0 and 8 mg 1- t added. MS2 bacteriophage was added to each flask to give a titre of 106 pfu ml-z. The flasks were then shaken and sampled for unadsorbed virus. Concentrations of polyelectrolyte as low as 0.5 mg lproved to be inhibitory to virus adsorption. Figure 7 shows the results when this experiment was repeated using lower concentrations of polyelectrolyte. Virus adsorption was improved at very low polyelectrolyte concentrations (0.0125-0.025 mg 1- t). The inhibitory effect of polyelectrolyte at a concentration of 0.45 mg 1- = observed at pH 4 may be overcome by raising the pH to 7, above which satisfactory adsorption by magnetite was obtained (Fig. 8).
6
Olr~
•
A series of flasks was prepared, each containing 15/o magnetite in 100 mg 1-t NaCI at pH 4. Dilutions of clay from 0.02 to 0.16 mg 1- t were added, then virus, and the flasks shaken and finally sampled for unadsorbed virus. Figure 9 shows that clay at concentrations of 0.15 mg I- t (approx. 100 NTU) interferes to the extent of diminishing the amount of virus adsorbed six-fold. Subsequent experiments were performed using 0.15 mg 1- ~ clay and varying amounts of polyelectrolyte A8101 (Fig. 101. Added polyelectrolyte significantly counteracted the efli~ct of clay.
Effect of cations on virus adsorption Two different suspending solutions were used, One contained 400mg I-~ NaC1, the other contained cations similar to those in sewage effluent, viz. 100mgl-~ NaCI, 3 0 m g l - ~ Ca z + and 2 0 m g l - I Mg-' +. The total dissolved solids (TDS) of the latter solution was 500 mg 1- t. Polyelectrolyte in concentrations up to 2.0 mg lwas added to the magnetite suspensions after they had been autoclaved. Other steps in the experiments
• input titre o ctay 0.15 g t-'and polyeIectrolyte [] magnetite alone
•
= 5 'K /. o______o
3
• poIyetectrotyte o
2
without mognetite polyelectrotyte with 1% magnetite
o \
\
.d
O4
\o
'
;
;
;
pI-I
Fig. 8. Effect of p H on M S 2 adsorption at a polyelectrolyte concentration of 0.45 m g I- '.
0
I 0.01
i
o.'o2 oio3 o.'0~ dos 0106<'< 0.15
Po|yeIect rol.yte (mg [-') Fig. 10. Effect of polyelectrolyte on MS2 adsorption at standard clay concentration of 0.15 g 1- ~.
.Adsorption of viruses on magnetic particles- I
7~-
o
947
input titre 5
°°
3
~
e°
a=
o
o g ti,ew oe
\
bt
o m~netite w~ poiyel.ect ol.yt
"3"
o.;
o
21o
zr
0
--.//I
"/3
I
4
t
t
5
6
Potyetectrotyte (rag I-')
Fig. 11. Effect of polyelectrolyte concentration on MS2 adsorption to magnetite in 500 mg 1-t "I'DS solution, including 30mgl -t Ca z~, 20mgl -t Mg-'" and 100mgl -t Na z - ions at pH 4. (synthetic sewage effluent). were carried out in the manner previously described. A comparison of the adsorptive behaviour of magnetite in the presence of different concentrations of salts and polyelectrolyte is shown in Fig. 11. High salt, without polye[ectrolyte, reduced adsorption, but polyelectrolyte within the range of 0.2-0.5 mg I- ~ improved adsorption when added to these solutions, as with 100 mg l- ~ NaCI solutions. Higher polyelectrolyte concentrations were detrimental to adsorption.
Effect of ,sewa,qe eJfluent When sewage effluent was used as a suspending medium for magnetite, virus adsorption was inhibited at pH values between 6 and 8, and was much reduced at p H 4 compared with that in 1 0 0 m g l - t NaCI (Fig. 12). If the effluent was treated by one cycle of exposure to magnetite, the turbidity was reduced from 62 to 38 NTU. The virus adsorptive capacity of magnetite suspended in magnetite-treated effluent was extended over the range pH3--8. Further improvement was effected by the addition of polyelectrolyte at 0.45 mg I- ~ to the system (Fig; 13).
: 7 pH
I 8
= 10
I 9
@i 11
Fig. 13. Effect of pH and polyelectrolyte at standard concentration of 0.45 mg l- ~ on MS2 adsorption to magnetite in sewage effluent previously treated by one cycle of exposure to magnetite, at pH 4.
Adsorption of t'iruses fi'ont sewage effluent by successice stages of magnetite treatment Treatment of virus suspensions in sewage effluent at pH 4 with magnetite, resulted in little removal of virus in the first stage, whether polyelectro[yte was present or not. When polyelectro[yte was present at 0.45 rag l-~ and a second magnetite treatment was carried out, virus removals of the order of 103-10 "~ were obtained. A third magnetite treatment reduced the virus content to below detectable levels (Fig. 14). DISCUSSION
The results of this study indicate that magnetite is an efficient adsorbent for the small RNA-containing bacterial virus MS2, of Escherichia coll. MS2 was chosen as a convenient virus for study because it is easily prepared in high titre in purified form, the ease, speed and accuracy with which viable virus can be detected and quantified (titrated) and because of its similarity in size, structur~ and chemical composition 10
6 3
%
%
5
o
\ \
\
~wi~
magnetite
\
2
~.
( not detectabte )
\
N \
-¢/~ . . . . . . . . . . . 3
/-
5
6
7
~ 8
9
10
11
pH
Fig. 12. Effect of pH on MS2 adsorption to magnetite in sewage effluent.
I
2
3
No. of cycles of magnetite treatment
Fig. 14. Amount of MS2 bacteriophage removed in successive stages of magnetite treatment of MS2 suspension in sewage effluent, with 0.45 mg 1-1 polyelectrolyte added,
948
J.G. ATHERTONand S. S. BFLL +
b
Nett
,
suttee - -' // / , 3 chcrge
\
\ ,'
t:crzteriog~hoge--,\ L
,~
5~
6,
\
7i
8i
pH range of ~J odsorphon---.-~
Fig. 15. Effect of pH on net surface charge of MS2 bacteriophage and magnetite particles. to the human-pathogenic enteroviruses, such as poliovirus, which may be found in sewage. The magnetite used in the study was treated by successive cycles of acid and alkali washing, a process designed to improve its efficiency of removal of colour, turbidity and other impurities from water in the Sirofloc process (Kolarik et al., 1977). The effect of this chemical treatment on magnetite was investigated by Kolarik et al. (1980) using microelectrophoresis. Both acid and alkali treatment raise the isoelectric pH (iep) of the magnetite particles and reduce the silica content of the magnetite. The standard 8-cycle washing stabilizes the iep of magnetite at pH 7.5 + 0.5. Thus the surface charge on MS2 bacteriophage with an iep of 3.9 (Overby et al., 1966) and that on magnetite particles would be expected to be of opposite sign between pH 3.9 and 6.5 (Fig. 15). Consequently virus adsorption would be expected to be maximal between those limits and to diminish above pH 7.5. This behaviour was observed. Virus adsorption to magnetite was rapid and conformed to the Freundlich isotherm when the virus concentration varied from 1.0 x 104 to 6.4 x 107 pfu ml-~. No evidence of saturation of adsorption sites on the magnetic particles was observed, even with the highest virus concentrations. The polyelectrolyte used in the study (Catoleum ASI01) is a basic cationic molecule of moderate chain length. At low concentrations, in the absence of divalent cations, the polyelectrolyte might be expected to adsorb to the negatively-charged virus particles, and if able to bridge between virus particles, will facilitate adsorption to magnetite. Higher concentrations of polyelectrolyte, by reversing the virus negative charges, might be expected to stabilize the dispersion of virus particles and to inhibit their adsorption to the positively charged magnetite. The effects might not be expected to be so evident at higher ionic strength because of charge-shielding by the additional ions. This accords with observations. The presence of divalent cations leads to a reduction in the thickness of the electrical double-layer of charged particles. Optimum cationic polyelectrolyte concentrations promoting adsorption to magnetite are thus higher.
In suspending media of undefined composition, such as sewage effluent containing many organic compounds, particle interaction is complex. Improved adsorption of virus to magnetite in the presence of relatively high concentrations of polyelectrolyte is explicable because of the competing binding of polyelectrolyte to clay which aids removal of this and other interfering substances which otherwise reduce virus-magnetite adsorption. In general the role of polyelectrolyte should thus be to bind to clay and other interfering substances, reducing their charges. while raising the charge on magnetite, with consequently improved virus adsorption. Viruses from different Families differ widely in size, structure and degree of complexity. The limited data available indicates that they differ in their isoelectric pH values. As a consequence, the optimal conditions for adsorption to magnetite and the methods of application of magnetite adsorption for removal of viruses from water, might be expected to differ from virus to virus. Acknowled¢,Iements--This work has been supported by a grant-in-aid from the Commonwealth Scientific and Industrial Research Organisation (Chemical Technology Division). The authors are grateful to Dr Brian Bolto and Dr David Dixon for much information and for consultation during the progress of the work. Thanks are expressed to colleagues in the Department of Microbiology for helpful discussion and criticism, and also for technical assistance. REFERENCES Adams M. H. (1959) Bacteriophages. Interscience, New York. Amirhor P. & Englebrecht R. S. (1975) Interaction of virus and polyelectrolyte in wastewater effluent. Prog. Wat. Technol. 7, 677-686. Atherton J. G. & Bell S. S. (1979) Use of magnetite for removal of viruses from water and wastewater. Proc. 8th Fed. Cont,. A W W A 19-39. Bitton G. & Mitchell R. (1974) The removal of Escherichia coli bacteriophage T7 by magnetic filtration. Water Res. 8, 549-551. Bitton G., Pancorbo O. & Gifford G. (1976) Factors affecting the adsorption of poliovirus to magnetite in water and wastewater. Water Res. 10, 973-980. Kolarik L. O., Priestley A. J. & Weiss D. E. (1977) The Sirofloc process for turbidity and colour removal. Proe. 7th Fed. Cone. A W W A 143. Kolarik L. O., Dixon D., Freeman P. A.. Furlong D. N. & Healy T. W. (1980) Transactions of the American lnstimte of Minin¢l and Metallurgical Engineering, Confi, rence, Las Vegas.
Fine Particle
Overby L. R., Barlow G. H., Doi R. H., Jacob M. & Spiegelman S. (1966) Comparison of two serotogically distinct ribonucleic acid bacteriophages. J. Bact. 91, 442-448. Pearson F. & Metcalf T. G. (1974) The use of magnetic iron oxide for recovery of virus from water. Completion Report--Project No. A-014-1. Under grant No. 14-31-001-3029. Water Resources Research Center, University of New Hampshire, Durham. Rao V. Ch., Sullivan R.. Read R. B. & Clarke N. A. {1968} A simple method for concentrating and detecting viruses in water. J. Am. Wat. Wks Ass. 60, 1288-1294. Sargeant K. (1970} Large-scale bacteriophage production. Adv. appl. Microbiol. 13, 121-137. Warren J.. Neal A. and Rennels D. (1966) Adsorption of myxoviruses on magnetic iron oxides. Proc. Soc. exp. Biol. Med. 12, 1250-1253.
~X)43-135,/.'33 080943-06503.00 0 Cop.,.right D 1953 Pergamon Press Ltd
A D S O R P T I O N O F VIRUSES ON MAGNETIC PARTICLES I ADSORPTION OF MS2 BACTERIOPHAGE AND THE OF CATIONS, CLAY AND POLYELECTROLYTE
EFFECT
J. G. ATnERTOY and S. S. BELL Department of Microbiology. University of Queensland. St Lucia 4067. Brisbane. Australia (Receired October 1982)
Abstract--Washed samples of finely-divided magnetite are found, after alkali treatment, to adsorb MS2 bacteriophage very efficiently, from suspensions in 100 mg 1- ~ NaCI at pH 6. Suspended material, such as clay. components of sewage effluent and ions interfere with virus adsorption. but this interference can be largely overcome by the addition of suitable polyelectrolyte in tow concentration.
INTRODUCTION
the adsorption of poliovirus to magnetite (Bitton et
The usefulness of magnetite in the treatment of raw water has been studied by the C S I R O Division of Chemical Technology (CSIRO), South Melbourne. It has been found that magnetite, after chemical treatment, is capable of removing a large proportion of the dissolved and st.spended organic matter, as well as clay and other colloidal material, from previously untreated w a t e r / K o l a r i k et al., 1977). A study of the efficiency of removal of viruses from water has also been undertaken (Atherton & Bell, 1979), In this study, bacteriophage MS2 of Escherichia colt was used as a model for the behaviour of the humanpathogenic enteric viruses. Because of their small particle size (22-28 nm) and the protein nature of their surface, the enteric viruses will behave as charged colloidal particles in aqueous suspension, their net charge being dependent on their ionic environment. Finely-divided particles of magnetite behave similarly (Kolarik et al., 1980; Amirhor & Englebrecht, 1975). If the isoelectric pH (iep) of the virt, s differs from that of magnetite, pH conditions can be chosen such that the virus particles will be electrostatically bound to those of magnetite. At pH 10. where both magnetite and the MS2 virus particles carry negative charges, the virus, previously adsorbed to magnetite, might be expected to be released into the supernatant fluid, thus allowing the magnetite to be recycled after washing. Previous studies on the adsorptive capacity of iron oxide toward viruses include: the removal of influenza virus by haematite (Warren et al., 1966), the use of magnetic iron oxide-packed columns to concentrate viruses (Pearson & Metcalf, 1974) or to remove enteroviruses from water (Rao et al., 1968), removal of b a c t e r i o p h a g e T7 by magnetic filtration (Bitton & Mitchell, 1974) and more recently, factors affecting 943
al., 1976).
The present studies were undertaken to assess the removal of viruses which might take place in the Sirofloc water-treatment process employing magnetite, developed by the CSIRO. The effect, on the adsorptive process, of cations, pH, time, suspended and dissolved organic matter and the presence of polyelectrolytes, was also studied,
MATERIALS AND METHODS
MS2 bacteriophage and its host bacterium Escherichia colt C-3000 (a prototrophic strain of KI21 were kindly supplied by Professor J. Pittard. University of Melbourne. The host bacterium was inoct, lated into a solid culture medium containing 10g tryptone. 10g NaCI. 5g yeast extract and 15 g agar I-t of distilled water. For MS2 bacteriophage propagation a similar medium, without agar ("broth") but with 10g Na,HP~.I2H,O and 3.8g KH.,PO.~ I- ~. buffered at pH 9 was used (Sargeant. 1970). Stocks of MS2 were prepared by the layer agar method [Adams. 1959). Monodisperse phage preparations were prepared by collecting the freeze-thawed agar from the propagation plates, homogenising at low speed in a Waring blender, centrifuging at 10.000 g to separate the agar fragments, then filtering the phage-containing supernatant through a 0.2.ttm membrane filter with a prefilter stack, in a Sartorius pressure filtration cylinder. The filtrate was checked by electron microscopy and the titre determined by counting plaques (Adams. 1959). The stock MS2 preparation had a titre of 9.3 x l0 t° pfu ml- ~ and was stored in 1 m[ amounts in sealed plastic ampoules at -50-C. All inorganic chemicals used were Univar Analytical Reagent grade [Ajax Chemicals. Sydney) and bacteriological media were supplied by Difco Laboratories. Detroit. Michigan. Magnetite. supplied as a finely crushed powder of 1-5/tm particle size consisted of 66.3?o Fe_,O3 and 27.6°o FeO, and had a specific surface area of 0.6 m'- g- t. It was prepared for use in the following manner: First the magnetite was mechanically stirred for 10 rain each. with four changes of distilled water, then with four
944
J.G. ATHERTON and S. S. BELL
changes of 0. l N NaOH. the distilled water washing repeated and then similar treatment with four changes of 0.01 M HCI. In each case. the volume of the washing solution ~as 4 times that of the original magnetite suspension. This complete process of water-alkali-water-acid wash was repeated eight times. The "'8-cycled'" magnetite was then stored wet. in a wide-mouthed airtight polythene bottle. [mmediately before use the magnetite was again wash-cycled once, and demagnetised by exposure to 50 Hz alternating magnetic field produced by a coil carrying 240 V. 0.8 A. which surrounded the flask. This material is referred to as 'conditioned" magnetite. For use in experinaents, wash-c>cled magnetite was kept in a wide-bore burette in the unmagnetised state. The magnetite particles were allowed to settle for 12 h, and formed a "slurry" which could be run into reaction flasks in 1 ml amounts. Stable suspensions of clay in salt solutions were prepared from mixed (unstable) suspensions by sonication, in 4--15s bursts at 6 3 W c m -3. Suspensions containing 0.12 g I- 1 correspond to a turbidity of 90 NTU, 0.06 g I- t to 42 NTU. The clay used was a milled sample of kaolinite with a particle size of 1-2 #m. These clay particles have an isoelectric pH of 2.5. Polyelectrolyte AS101 was supplied by Catoleum (Australia). It is a strongly basic, highly charged cationic molecule of moderate chain length. Three different salt solutions were used in the study. I. 100 mg I- ~ NaCI, 2. 400 mg 1- t NaCI. and 3. 100mgI - t NaC1, 30mgl - t Ca-'-, 20mg1-1 Mg(500 mg I- 1 TDS). Sewage effluent was obtained, after secondary treatment, from the Brisbane City Council, Oxley Ck. Wastewater Treatment Plant. After autoclaving at 121~'C for 15 rain, it had a pH of 9, TDS 800-1000 mgl-~ and a turbidity of 62 NTU.
under the following conditions: exposure to pH over the range 4.8-11, NaCI concentrations 0-8500 mg 1- 1 and autoclaved sewage effluent at pH 9.
Adsorption of 3,1S2 bacterioptzage to magnetite Shaking speed. An adsorption experiment was performed in 100 mg 1-~ NaCI at pH 4. with rotational speeds from 100 to 300 rev m i n - ~, at an amplitude of 20 mm. Maximal adsorption ,,,,as obtained at speeds of 260 rev m i n - t or higher. All further experiments were performed at a shaking speed of 260 rev m i n - ~. pH. The effect of pH of the solution of 100 mg 1- t NaCI used a suspending medium for magnetite was tested over the range 3-11. using magnetite which had been washed and pH-cycled once only ("unconditioned" magnetite). Virus adsorption was much diminished above pH 5 (Figs I and 2). Multiple wash-cycling of magnetite. The virusadsorptive characteristics of magnetite which had been washed and pH-cycled only once, was compared to that of magnetite which had been so treated 8 times ("conditioned" magnetite). Virus adsorption is much improved in the latter and occurs up to pH 7
0
6
O0
Stability of MS2 bacteriophage Sufficient volume of monodisperse suspension of MS2 bacteriophage was added to a 500 ml stoppered conical flask containing 100ml of sterile aqueous suspending medium under test, to give a virus titre of approx. 10~' pfuml- ~ and a sample taken for counting initial titre. Rotational shaking on a "'New Brunswick" gyratory shaker at 260revmin-t at an amplitude of 20mm was then begun. Samples were taken at 5 rain intervals up to 30 rain. diluted in broth and plated for plaque-counting.
13-
0 0
0
o"uncond~tioned"mognetite o"conditioned'" magnetite
IE
pH Fig. 1
100
80
8
60
/-,0
o '~JncondRioned" magnetite
20
n "'conditioned" rnognetite
RESULTS
Stability of MS2 bacteriophage No significant reduction in titre of MS2 bacteriophage could be demonstrated over a period of 30 rain
0 I"1 [] 0 0 ClyD
• virus control
Adsorption oJ"MS2 bacteriophage to magnetite Two flasks, each containing I I0ml of suspending medium (salts solutions or sewage effluent) were used for each pH to be tested. One flask of each pair had I ml of wash-cycled magnetite slurry added to it. and both flasks autoclaved at 115 C for 15 rain. When cool each flask was adjusted to the desired pH with sterile 0.01 M HCI or NaOH by pipetting off I0 ml for use as an external aliquot for calculation of the acid or alkali necessary to adjust the pH of the remaining 100 ml. Virus was then added to give a titre of approx. 10~' pfu ml-t. a sample taken for virus titration and rotational shaking at 260 rev min-t at an amplitude of 20 mm, commenced. After 20min, shaking was stopped, the magnetite sedimented by magnetisation over a permanent magnet of 0.1T and samples taken for titration of unadsorbed virus.
~
7
pH Figs 1 and 2. Effect of pH on the adsorption of MS2 to "'conditioned" and "unconditioned" magnetite.
Adsorption of viruses on magnetic particles--I
945
lOO
go
"8
2
a~
~70
1
/ 2
3 [magl
t. No. of regeneration cyctes of magnetite
(Ioglo rng F )
Fig. 3. Effect of magnetite concentration on adsorption of
MS2. (Figs 1 and 2). All further experiments were performed using "conditioned" magnetite. Magnetite concentration. A series of tared flasks was set up, containing magnetite concentrations from 0.0156 to 1.0% in 100 mg 1- z NaCI at pH 4. Virus was added and the flasks shaken at 260rev rain -t for 20 rain. Samples of the supernatant were taken for virus titration, after sedimentation of the magnetite over a magnet. All flasks were then taken to dryness in a 56°C oven, reweighed and the magnetite content calculated. Figure 3 shows that virus adsorption was satisfactory at concentrations of 2000 mg 1- ~ (0.2%) or higher. Kinetics. Samples for virus titration were taken at intervals after the addition of MS2 bacteriophage to a suspension of 1% magnetite in 100 m g l - t NaCI at pH 4. Adsorption was seen to be rapid, being 99.6% completed after 3 min. Variable cirus input. 1% magnetite in 100mg1-1 NaCI at pH 4 was exposed to virus titres ranging from 10'~ to l0 s pfu ml-~. Figure 4 shows that the data conforms to the Freundlich isotherm with a
Fig. 5. Effect of regeneration pH cycle on MS2 bacteriophage-adsorptive capacity of magnetite. slope equal to 1.017 and a y-intercept equal to -3.98. The correlation coefficient r 2 = 0.99. Multiple stages of titus adsorption. The virus adsorbing capacity of a sample of magnetite exposed to virus after each cycle of pH change and washing, was compared with that of "fresh" magnetite (not previously exposed to virus) and controls. In the test flask, each time after virus samples had been removed for titration at the completion of the 20 rain shaking period the magnetite was wash-cycled, demagnetised and resuspended in 100 mg 1-1 NaCI at pH 4, before another virus dose was added to test the adsorptive capacity. Figure 5 shows that the virus adsorptive capacity of the recycled magnetite showed no diminution after 11 cycles.
Treatment of cirus-containing fluids by two stages of magnetite adsorption Test and control flasks were set up with 100 mg lNaCi. at pH4. MS2 bacteriophage approx. 10 9 pfu ml-1 was added to each flask. After shaking, the supernatant (containing approx. 105 pfu ml-~ of
~-.
4
I ~
input titre
-&
oe3
i 2 o -//
c
5
6
7
8
Input[virus] (togl0Pfu ml -t)
\ , l°°2t.tabe, \
0
~
No. of cycles of magnetite treahnent
Fig. 6. Amount
Fig. 4. Effect of input virus concentration on total virus adsorbed. w.R. 17 8--H
~
of virus adsorbed in successive stages of
magnetite treatment of MS2 suspension in 100mgl -~ NaCI at pH 4.
J. G. ATHERTONand S. S. BELL
946
5! input titre
input titre E
2 o
i fl
3
o.~
0.I05
o
o.o2
Potyetectrotyte (mg t-')
ob~
o.b6
0.08 '
oho
012 '
0.14 ' 016
Clay [g [-')
Fig. 7. Effect of polyelectrolyte concentration on MS2 adsorption at pH 4.
Fig. 9. Effect of clay concentration on MS2 adsorption to magnetite at pH 4.
unadsorbed virus) was added to a fresh flask containing 1 g magnetite. This flask was then shaken and sampled and the procedure repeated. Figure 6 shows the relationship between virus titre and number of stages of treatment with magnetite. Virus content was removed by a factor of 10"t in each successive adsorption stage.
Effect of clay on cirus adsorption
Effect of polyelectrolyte A8101 on rirus adsorption Flasks with 1~o magnetite were set up in 100 mg 1- t at pH 4 and polyelectrolyte concentrations between 0 and 8 mg 1- t added. MS2 bacteriophage was added to each flask to give a titre of 106 pfu ml-z. The flasks were then shaken and sampled for unadsorbed virus. Concentrations of polyelectrolyte as low as 0.5 mg lproved to be inhibitory to virus adsorption. Figure 7 shows the results when this experiment was repeated using lower concentrations of polyelectrolyte. Virus adsorption was improved at very low polyelectrolyte concentrations (0.0125-0.025 mg 1- t). The inhibitory effect of polyelectrolyte at a concentration of 0.45 mg 1- = observed at pH 4 may be overcome by raising the pH to 7, above which satisfactory adsorption by magnetite was obtained (Fig. 8).
6
Olr~
•
A series of flasks was prepared, each containing 15/o magnetite in 100 mg 1-t NaCI at pH 4. Dilutions of clay from 0.02 to 0.16 mg 1- t were added, then virus, and the flasks shaken and finally sampled for unadsorbed virus. Figure 9 shows that clay at concentrations of 0.15 mg I- t (approx. 100 NTU) interferes to the extent of diminishing the amount of virus adsorbed six-fold. Subsequent experiments were performed using 0.15 mg 1- ~ clay and varying amounts of polyelectrolyte A8101 (Fig. 101. Added polyelectrolyte significantly counteracted the efli~ct of clay.
Effect of cations on virus adsorption Two different suspending solutions were used, One contained 400mg I-~ NaC1, the other contained cations similar to those in sewage effluent, viz. 100mgl-~ NaCI, 3 0 m g l - ~ Ca z + and 2 0 m g l - I Mg-' +. The total dissolved solids (TDS) of the latter solution was 500 mg 1- t. Polyelectrolyte in concentrations up to 2.0 mg lwas added to the magnetite suspensions after they had been autoclaved. Other steps in the experiments
• input titre o ctay 0.15 g t-'and polyeIectrolyte [] magnetite alone
•
= 5 'K /. o______o
3
• poIyetectrotyte o
2
without mognetite polyelectrotyte with 1% magnetite
o \
\
.d
O4
\o
'
;
;
;
pI-I
Fig. 8. Effect of p H on M S 2 adsorption at a polyelectrolyte concentration of 0.45 m g I- '.
0
I 0.01
i
o.'o2 oio3 o.'0~ dos 0106<'< 0.15
Po|yeIect rol.yte (mg [-') Fig. 10. Effect of polyelectrolyte on MS2 adsorption at standard clay concentration of 0.15 g 1- ~.
.Adsorption of viruses on magnetic particles- I
7~-
o
947
input titre 5
°°
3
~
e°
a=
o
o g ti,ew oe
\
bt
o m~netite w~ poiyel.ect ol.yt
"3"
o.;
o
21o
zr
0
--.//I
"/3
I
4
t
t
5
6
Potyetectrotyte (rag I-')
Fig. 11. Effect of polyelectrolyte concentration on MS2 adsorption to magnetite in 500 mg 1-t "I'DS solution, including 30mgl -t Ca z~, 20mgl -t Mg-'" and 100mgl -t Na z - ions at pH 4. (synthetic sewage effluent). were carried out in the manner previously described. A comparison of the adsorptive behaviour of magnetite in the presence of different concentrations of salts and polyelectrolyte is shown in Fig. 11. High salt, without polye[ectrolyte, reduced adsorption, but polyelectrolyte within the range of 0.2-0.5 mg I- ~ improved adsorption when added to these solutions, as with 100 mg l- ~ NaCI solutions. Higher polyelectrolyte concentrations were detrimental to adsorption.
Effect of ,sewa,qe eJfluent When sewage effluent was used as a suspending medium for magnetite, virus adsorption was inhibited at pH values between 6 and 8, and was much reduced at p H 4 compared with that in 1 0 0 m g l - t NaCI (Fig. 12). If the effluent was treated by one cycle of exposure to magnetite, the turbidity was reduced from 62 to 38 NTU. The virus adsorptive capacity of magnetite suspended in magnetite-treated effluent was extended over the range pH3--8. Further improvement was effected by the addition of polyelectrolyte at 0.45 mg I- ~ to the system (Fig; 13).
: 7 pH
I 8
= 10
I 9
@i 11
Fig. 13. Effect of pH and polyelectrolyte at standard concentration of 0.45 mg l- ~ on MS2 adsorption to magnetite in sewage effluent previously treated by one cycle of exposure to magnetite, at pH 4.
Adsorption of t'iruses fi'ont sewage effluent by successice stages of magnetite treatment Treatment of virus suspensions in sewage effluent at pH 4 with magnetite, resulted in little removal of virus in the first stage, whether polyelectro[yte was present or not. When polyelectro[yte was present at 0.45 rag l-~ and a second magnetite treatment was carried out, virus removals of the order of 103-10 "~ were obtained. A third magnetite treatment reduced the virus content to below detectable levels (Fig. 14). DISCUSSION
The results of this study indicate that magnetite is an efficient adsorbent for the small RNA-containing bacterial virus MS2, of Escherichia coll. MS2 was chosen as a convenient virus for study because it is easily prepared in high titre in purified form, the ease, speed and accuracy with which viable virus can be detected and quantified (titrated) and because of its similarity in size, structur~ and chemical composition 10
6 3
%
%
5
o
\ \
\
~wi~
magnetite
\
2
~.
( not detectabte )
\
N \
-¢/~ . . . . . . . . . . . 3
/-
5
6
7
~ 8
9
10
11
pH
Fig. 12. Effect of pH on MS2 adsorption to magnetite in sewage effluent.
I
2
3
No. of cycles of magnetite treatment
Fig. 14. Amount of MS2 bacteriophage removed in successive stages of magnetite treatment of MS2 suspension in sewage effluent, with 0.45 mg 1-1 polyelectrolyte added,
948
J.G. ATHERTONand S. S. BFLL +
b
Nett
,
suttee - -' // / , 3 chcrge
\
\ ,'
t:crzteriog~hoge--,\ L
,~
5~
6,
\
7i
8i
pH range of ~J odsorphon---.-~
Fig. 15. Effect of pH on net surface charge of MS2 bacteriophage and magnetite particles. to the human-pathogenic enteroviruses, such as poliovirus, which may be found in sewage. The magnetite used in the study was treated by successive cycles of acid and alkali washing, a process designed to improve its efficiency of removal of colour, turbidity and other impurities from water in the Sirofloc process (Kolarik et al., 1977). The effect of this chemical treatment on magnetite was investigated by Kolarik et al. (1980) using microelectrophoresis. Both acid and alkali treatment raise the isoelectric pH (iep) of the magnetite particles and reduce the silica content of the magnetite. The standard 8-cycle washing stabilizes the iep of magnetite at pH 7.5 + 0.5. Thus the surface charge on MS2 bacteriophage with an iep of 3.9 (Overby et al., 1966) and that on magnetite particles would be expected to be of opposite sign between pH 3.9 and 6.5 (Fig. 15). Consequently virus adsorption would be expected to be maximal between those limits and to diminish above pH 7.5. This behaviour was observed. Virus adsorption to magnetite was rapid and conformed to the Freundlich isotherm when the virus concentration varied from 1.0 x 104 to 6.4 x 107 pfu ml-~. No evidence of saturation of adsorption sites on the magnetic particles was observed, even with the highest virus concentrations. The polyelectrolyte used in the study (Catoleum ASI01) is a basic cationic molecule of moderate chain length. At low concentrations, in the absence of divalent cations, the polyelectrolyte might be expected to adsorb to the negatively-charged virus particles, and if able to bridge between virus particles, will facilitate adsorption to magnetite. Higher concentrations of polyelectrolyte, by reversing the virus negative charges, might be expected to stabilize the dispersion of virus particles and to inhibit their adsorption to the positively charged magnetite. The effects might not be expected to be so evident at higher ionic strength because of charge-shielding by the additional ions. This accords with observations. The presence of divalent cations leads to a reduction in the thickness of the electrical double-layer of charged particles. Optimum cationic polyelectrolyte concentrations promoting adsorption to magnetite are thus higher.
In suspending media of undefined composition, such as sewage effluent containing many organic compounds, particle interaction is complex. Improved adsorption of virus to magnetite in the presence of relatively high concentrations of polyelectrolyte is explicable because of the competing binding of polyelectrolyte to clay which aids removal of this and other interfering substances which otherwise reduce virus-magnetite adsorption. In general the role of polyelectrolyte should thus be to bind to clay and other interfering substances, reducing their charges. while raising the charge on magnetite, with consequently improved virus adsorption. Viruses from different Families differ widely in size, structure and degree of complexity. The limited data available indicates that they differ in their isoelectric pH values. As a consequence, the optimal conditions for adsorption to magnetite and the methods of application of magnetite adsorption for removal of viruses from water, might be expected to differ from virus to virus. Acknowled¢,Iements--This work has been supported by a grant-in-aid from the Commonwealth Scientific and Industrial Research Organisation (Chemical Technology Division). The authors are grateful to Dr Brian Bolto and Dr David Dixon for much information and for consultation during the progress of the work. Thanks are expressed to colleagues in the Department of Microbiology for helpful discussion and criticism, and also for technical assistance. REFERENCES Adams M. H. (1959) Bacteriophages. Interscience, New York. Amirhor P. & Englebrecht R. S. (1975) Interaction of virus and polyelectrolyte in wastewater effluent. Prog. Wat. Technol. 7, 677-686. Atherton J. G. & Bell S. S. (1979) Use of magnetite for removal of viruses from water and wastewater. Proc. 8th Fed. Cont,. A W W A 19-39. Bitton G. & Mitchell R. (1974) The removal of Escherichia coli bacteriophage T7 by magnetic filtration. Water Res. 8, 549-551. Bitton G., Pancorbo O. & Gifford G. (1976) Factors affecting the adsorption of poliovirus to magnetite in water and wastewater. Water Res. 10, 973-980. Kolarik L. O., Priestley A. J. & Weiss D. E. (1977) The Sirofloc process for turbidity and colour removal. Proe. 7th Fed. Cone. A W W A 143. Kolarik L. O., Dixon D., Freeman P. A.. Furlong D. N. & Healy T. W. (1980) Transactions of the American lnstimte of Minin¢l and Metallurgical Engineering, Confi, rence, Las Vegas.
Fine Particle
Overby L. R., Barlow G. H., Doi R. H., Jacob M. & Spiegelman S. (1966) Comparison of two serotogically distinct ribonucleic acid bacteriophages. J. Bact. 91, 442-448. Pearson F. & Metcalf T. G. (1974) The use of magnetic iron oxide for recovery of virus from water. Completion Report--Project No. A-014-1. Under grant No. 14-31-001-3029. Water Resources Research Center, University of New Hampshire, Durham. Rao V. Ch., Sullivan R.. Read R. B. & Clarke N. A. {1968} A simple method for concentrating and detecting viruses in water. J. Am. Wat. Wks Ass. 60, 1288-1294. Sargeant K. (1970} Large-scale bacteriophage production. Adv. appl. Microbiol. 13, 121-137. Warren J.. Neal A. and Rennels D. (1966) Adsorption of myxoviruses on magnetic iron oxides. Proc. Soc. exp. Biol. Med. 12, 1250-1253.