Vol. 57, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1991, p. 2502-25060099-2240/91/092502-05$02.00/0Copyright C) 1991, American Society for Microbiology

Use of Modified Diatomaceous Earth for Removal and Recoveryof Viruses in Watert

S. R. FARRAH,'* D. R. PRESTON,'t G. A. TORANZOS,1§ M. GIRARD,' G. A. ERDOS,1AND V. VASUHDIVAN2

Department of Microbiology and Cell Science' and Department of Material Scienceand Engineering,2 University of Florida, Gainesville, Florida 32611-0100

Received 17 April 1991/Accepted 1 July 1991

Diatomaceous earth was modified by in situ precipitation of metallic hydroxides. Modification decreased thenegative charge on the diatomaceous earth and increased its ability to adsorb viruses in water. Electrostaticinteractions were more important than hydrophobic interactions in virus adsorption to modified diatomaceousearth. Filters containing diatomaceous earth modified by in situ precipitation of a combination of ferricchloride and aluminum chloride adsorbed greater than 80% of enteroviruses (poliovirus 1, echovirus 5, andcoxsackievirus B5) and coliphage MS2 present in tap water at ambient pH (7.8 to 8.3), even after filtration of100 liters of tap water. Viruses adsorbed to the filters could be recovered by mixing the modified diatomaceousearth with 3% beef extract plus 1 M NaCI (pH 9).

Early workers observed that poliovirus and influenza viruscould be concentrated by adsorption to metallic precipitatessuch as calcium phosphate (5, 16, 22). Wallis and Melnick(26) found that several viruses, including reoviruses andenteroviruses, could be efficiently adsorbed to aluminumhydroxide and other metallic precipitates.The ability of aluminum hydroxide and other metallic

precipitates to efficiently adsorb viruses has led to their usein methods for the recovery of viruses from water. Alumi-num hydroxide flocs have been used as the virus adsorbentin the first or second stages of virus concentration proce-dures (6, 28). Preformed magnesium hydroxide flocs havebeen used to further concentrate viruses eluted from micro-porous filters (24). Metallic ions have also been found toincrease the amount of floc formed by organic compounds atlow pH. The addition of ferric ions to filter eluates has beenshown to increase flocculation of organic compounds con-centrated by the filters along with viruses or of beef extractused as the eluent for microporous filters (13, 20). In bothcases, the flocs were found to efficiently adsorb viruses.

Since metallic hydroxides have been found to efficientlyadsorb viruses in water, attempts have been made to com-bine them with filters to produce flowthrough systems thatare capable of both processing large volumes of water andefficiently removing viruses from water. Seeley and Prim-rose (17) coated microporous filters with aluminum hydrox-ide. Clogging of these filters produced a slow filtration ratewith distilled water and precluded their use with surfacewater. Wallis and Melnick (27) combined ferric hydroxideflocs with 10-in. (ca. 25-cm) fiberglass filters. This combina-tion of a filter with ferric hydroxide floc was reported toadsorb all of the poliovirus in 3.8 liters of water. Brown et al.(1, 2) found that coating diatomaceous earth with aluminumhydroxide flocs increased its ability to adsorb viruses in

* Corresponding author.t Journal paper no. R-01642 from the Florida Agriculture Exper-

iment Station, Gainesville.t Present address: U.S. Patent & Trademark Office, A&T Unit

187, Washington, D.C. 20231.§ Present address: Department of Biology, Faculty of Natural

Science, University of Puerto Rico, Rio Piedras, Puerto Rico.

water. Two general problems have been found in attempts tocombine aluminum hydroxide flocs with filters, including (i)clogging of the filters (8, 12, 17) and (ii) poor adhesion of thefloc to the filters (2, 8, 23).

In previous studies (8, 23) we found that microporous anddepth filters can be modified by in situ precipitation ofindividual metallic salts or combinations of two metallicsalts. The modified filters were capable of adsorbing virusesin water with little or no decrease in the flow rate of waterthrough the filters. This procedure has been found to becapable of modifying diatomaceous earth (7) and sand (9)and increasing their ability to adsorb viruses in water. In thispaper, data on virus adsorption to diatomaceous earth mod-ified by precipitation of several metallic salts are presentedand the use of filters made with modified diatomaceous earthto remove and recover viruses from water is described.

MATERIALS AND METHODS

Modification of diatomaceous earth. Diatomaceous earth(grade 1; Sigma Chemical Co., St. Louis, Mo.) was mixedwith one of the solutions listed in Table 1 (4 ml of solutionper g of diatomaceous earth) for 30 min on a rotating shaker.The diatomaceous earth was collected by centrifugation for5 min at 500 x g and allowed to dry at 35°C. The driedmaterials were mixed with 3 N ammonium hydroxide (4ml/g) for 5 min and centrifuged as described above. Themodified materials were dried at 35°C and stored at roomtemperature until used.Measurement of zeta potential. The zeta potentials of

diatomaceous earth samples in 3 mM potassium phosphatebuffer were determined by using a Lazer Zee model 501 zetameter (Penkem, Inc., Bedford Hills, N.Y.). Values weredetermined at pH 5, 7, and 8.

Virus adsorption studies. Batch studies of virus adsorptionwere done by mixing 0.5 g of diatomaceous earth and 20 mlof solution with added virus on a rotating shaker for 5 min.The samples were centrifuged as described above, and thesupernatant samples were removed and sampled for viruses.The diatomaceous earth was then mixed with 20 ml of 3%beef extract plus 1 M NaCl (pH 9) for 5 min and centrifugedas described above. All solutions were buffered with 0.02 M

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TABLE 1. Percentage of virus adsorption and correlationbetween zeta potential and virus adsorption

by modified diatomaceous eartha

% Virus adsorption in:

Treatment of Zeta Buffer Trickling filterdiatomaceous potential effluent

earthb (mV) Indige-MS2 P1 nous P1

phage

None -71 1 2 47 7 0 0 45± 11Rinsed with ammo- -66 4 4 63 + 11 0 0 36 14nium hydroxide

1 M AICl3 -22 >99 99 2 >99 98 31 M CaCl2 -55 4 4 68 4 3 5 30 31 M MgCJ2 -21 86 6 >99 46 3 83± 141 M FeCl3 -48 98 4 99 + 2 92 6 93 51 M FeCl3 + 1 M -37 92 10 >99 97 5 >99

AlCl31MFeCl3+1M -12 >99 >99 71±10 82±11CaC12

1 M FeCl3 + 1 M -24 >99 >99 58 22 78 3MgCl2

rc 0.857 0.873 0.679 0.726r2c 0.734 0.762 0.462 0.527

a Adsorption of viruses in buffer (pH 8.0) and that in trickling filter effluent(pH 7.8) were compared with the zeta potential of diatomaceous earth at pH8.0. Samples (100 ml) with approximately 105 PFU of poliovirus 1 (P1) orcoliphage MS2 per ml or 102 PFU of indigenous phages per ml were passedthrough 0.5 g of diatomaceous earth in 25-mm holders. Values represent themean + standard deviation of triplicate determinations.

b Diatomaceous earth was modified by using the indicated salt solutionswith in situ precipitation as described in the text. Samples of untreateddiatomaceous earth and diatomaceous earth rinsed with 3 M ammoniumhydroxide served as control samples.

c Correlation between zeta potential and percent adsorption.

glycine plus 0.05 M imidazole. The solutions used are shownin Table 5.

Suspensions of diatomaceous earth were made in deion-ized water and passed through AP20 fiberglass filters (Milli-pore Corp., Milldale, Conn.) in 25- or 47-mm holders byexerting positive pressure with 50-ml syringes. Approxi-mately 0.5 g could be collected on 25-mm filters, and 1.5 gcould be collected on 47-mm filters. In some cases, diato-maceous earth was applied to 47-mm filters in two separateholders which were then connected in tandem. In thesecases, it was convenient to connect a Millipore high-pressure316 filter with a threaded female exit port to a MilliporeSwinnex filter with a threaded male input port. Samples (100ml) of buffer or unchlorinated effluent with indigenous phageor added -coliphage MS2 and poliovirus 1 were passedthrough diatomaceous earth in 25-mm filters. Samples (1liter) of dechlorinated tap water with added viruses werepassed through 47-mm filters containing diatomaceous earthafter passage through the filters of 10 or 100 liters of tapwater without viruses.

Virus recovery. Volumes (20 liters) of dechlorinated tapwater were seeded with virus and passed through one or two47-mm filters with AP20 filters and modified diatomaceousearth. After passage of the water, the diatomaceous earthand AP20 filter were removed and mixed with 40 ml of 3%beef extract plus 1 M NaCl (pH 9) for 5 min. After mixing,the samples were centrifuged at 500 x g for 5 min. Thesupernatant fractions were removed, neutralized, and as-sayed for viruses.

FIG. 1. Scanning electron micrograph of diatomaceous eartheither untreated (a) or modified by the in situ precipitation of ferricchloride (b).

Chemicals. The following chemicals were obtained fromSigma: aluminum chloride, calcium chloride, magnesiumchloride, ferric chloride, sodium chloride, sodium citrate,Tween 80, and Nonidet P-40. Beef extract was obtained fromDifco, Detroit, Mich.

Viruses and viral assays. Enteroviruses (poliovirus 1, cox-sackievirus B5, and echovirus 5) were grown in BGM cells.Cell culture harvests were treated with Freon and centri-fuged at 100,000 x g for 1 h. The pellets were suspended inbuffer (0.02 M glycine plus 0.05 M imidazole [pH 7]),centrifuged at 10,000 x g, filtered through 0.2-p.m-pore-sizefilters, and frozen until used. Enteroviruses were assayed asPFU on BGM cells by using an agar overlay. Virus produc-tion and assays were similar to previously described proce-dures (19). Bacterial phages in trickling filter effluent andphage MS2 were analyzed as PFU by using Escherichia coliC-3000 (ATCC 15597) as the host.

Adsorption kinetics and thermodynamic parameters. Thekinetics of virus adsorption were studied at 4, 25, and 37°C.The energy of activation (Ea), enthalpy of activation (AH*),Gibb's free energy of activation (AG*), and entropy ofactivation (AS*) were calculated as previously described(14).

Statistical analyses. Mean values, standard d-viations, andcorrelations were determined by using a calculator (TexasInstruments Model TI 60).

RESULTS

Clear pores in fragments of diatoms are clearly visible in ascanning electron micrograph of untreated diatomaceousearth (Fig. la). Modification of diatomaceous earth by in situprecipitation of metallic salts led to coating of the particlesand incorporation of precipitate within the pores (Fig. lb).Modification greatly increased the concentration of metalson the diatomaceous earth (7).

Modification with different salts led to an increase in thezeta potential of the diatomaceous earth (Table 1). Thisincrease was associated with increased adsorption of coli-phage MS2, coliphages in unchlorinated trickling filter ef-fluent, and poliovirus 1 by filters containing diatomaceousearth. The correlation between zeta potential and virusadsorption was higher for viruses in buffer (r> 0.850) thanfor viruses in trickling filter effluent (r > 0.670). Rinsing

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TABLE 2. Adsorption of viruses in tap water to filterscontaining diatomaceous earth'

Salt solution % Virus adsorption afterVirusb used to modify passage of:

diatomaceous earth 10 liters 100 liters

MS2 None 18 ± 8c 23 ± 31 M FeCl3 + 1 M MgCl2 66 ± 4 46 ± 71 M FeCl3 + 1 M AICl3 86 ± 6 84 6

CB5 None 22 ± 5 6 41 M FeCl3 + 1 M MgCl2 80 ± 8 89± 101 M FeCl3 + 1 M AlC13 96 2 87 5

E5 None 3 2 <11 M FeC13 + 1 M MgCl2 65 5 58 41 M FeCl3 + 1 M AlCl3 93 6 80 2

P1 None 42 1 28 91 M FeCl3 + 1 M MgCl2 87 3 86 41 M FeCl3 + 1 M AICl3 96 3 90 7

"One liter of dechlorinated tap water with 103 to 104 PFU of added virusesper ml was passed through approximately 1 g of diatomaceous earth in a

47-mm holder after passage of 10 or 100 liters of tap water. Viruses in theinitial samples and filter effluents were measured to determine the percentageof virus adsorption. The pH of the tap water was between 7.8 and 8.3.

b Abbreviations: MS2, coliphage MS2; CB5, coxsackievirus B5; E5, echo-virus 5; P1, poliovirus 1.

c Values represent the mean and range of duplicate determinations.

diatomaceous earth with ammonium hydroxide did not pro-duce an appreciable change in either its zeta potential or itsability to adsorb viruses. The ability of filters containingdiatomaceous earth to adsorb viruses after filtration of tapwater is shown in Table 2. After passage of 100 liters of tapwater, filters containing untreated diatomaceous earth ad-sorbed less than 30% of the viruses tested. Filters containingdiatomaceous earth modified by treatment with ferric chlo-ride and aluminum chloride adsorbed greater than 80% ofcoliphage MS2 and the three enteroviruses tested. Filterscontaining diatomaceous earth modified with ferric chlorideand magnesium chloride adsorbed considerably more of allof the viruses tested than filters with unmodified diatoma-ceous earth but adsorbed less echovirus 5 and coliphageMS2 than filters containing diatomaceous earth modifiedwith ferric chloride and aluminum chloride.

Initial studies on recovery of viruses from water by usinga single 47-mm filter containing modified diatomaceous earthgave variable and often low recoveries. By placing two filterscontaining diatomaceous earth modified with ferric chlorideand magnesium chloride in tandem, the recovery of viruses

added to tap water was improved (Table 3). However, thevariation in the percentage of virus recovered was still high;the standard deviations were equal to or higher than themean values for three of four of the tested viruses. Lessvariation and higher mean values were obtained by using twofilters containing diatomaceous earth modified with ferricchloride and aluminum chloride.The thermodynamic parameters of coliphage MS2 adsorp-

tion to diatomaceous earth modified with ferric chloride andmagnesium chloride were determined and are compared withthe values obtained in another study (14). As shown in Table4, the mean values for MS2 adsorption previously obtained(14) are similar to the values obtained in this study.

Detergents had little effect on virus adsorption by modifieddiatomaceous earth (Table 5). Salts had different effects on

virus adsorption to modified diatomaceous earth. Solutionsof sodium thiocyanate or sodium chloride did not have a

noticeable effect on virus adsorption. Sodium fluoride de-

TABLE 3. Recovery of viruses from tap water'

Salt solution used to modify Vir Sb No. of %Recvrdiatomaceous earth rus trials ecovery

1 M FeC13 + 1 M MgC12 MS2 5 37 ± 10P1 9 18 ± 21E5 3 26 ± 36CB5 4 15 ± 15

1 M FeC13 + 1 M AIC13 MS2 6 53 ± 12P1 4 49 ± 15E5 3 48 ± 10CB5 3 29 ± 5

"Twenty liters of dechlorinated tap water (pH 7.8 to 8.5) with the indicatedviruses were passed through two 47-mm filter holders in tandem containingapproximately 3 g of diatomaceous earth. The amount of virus added wasvaried between 103 and 106 PFU. Adsorbed viruses were recovered by using40 ml of 3% beef extract plus 1 M NaCI (pH 9). Values represent the mean ±standard deviation for the indicated number of trials.

b For abbreviations, see Table 2, footnote b.

creased adsorption by approximately 40%, and solutions ofsodium citrate reduced adsorption to less than 5%. Greaterthan 80% of the adsorbed virus could be recovered by using3% beef extract plus 1 M NaCl, indicating that adsorptionrather than inactivation was occurring (data not shown).

DISCUSSION

The ability of metallic precipitates to adsorb viruses inwater has led to efforts to use such precipitates in flow-through systems. In general, two problems have been en-countered in using insoluble metallic hydroxide flocs inconjunction with filter supports, namely, poor adhesion ofthe precipitate to the filter material and clogging of the filter.These problems have been found in our studies and havebeen reported by other workers. Seeley and Primrose (17)found that the aluminum hydroxide-coated microporousfilters rapidly clogged when filtering clean water samples anddid not attempt to use such filters with natural water sam-ples. Moeglich (12) discussed the problem with clogging offilters combined with metallic hydroxides and suggested anelectrolytic procedure for coating solids. Wallis and Melnick(27) reported that fiberglass filters modified with precipitatedferric hydroxide adsorbed poliovirus in water. However,these workers removed viruses from only 1 gal (3.8 liters) ofwater. The reason for the use of only a small volume of waterwith filters that have been shown capable of recoveringviruses from 100-gal samples of water (25) was not given. Itis likely that the precipitate was removed from the filter aslarger volumes of water were filtered. Brown et al. (1, 2)used aluminum hydroxide-coated diatomaceous earth toremove viruses from water. These authors reported that"poor adhesion of the alumina" limited the use of suchfilters for recovery of viruses from water. We have foundferric hydroxide or aluminum hydroxide flocs are poorlyretained by microporous filters, depth filters, diatomaceousearth, and sand, but that such filters can be modified by usingin situ precipitation of metallic salts (7-9, 23). Modificationof solids by this procedure in this and previous studies hasbeen found to permit stable modification of the solids and togreatly increase their ability to adsorb viruses in water.Modification did not greatly change the flow rate of waterthrough the filters.The finding that the charge on diatomaceous earth is

related to its ability to adsorb viruses is consistent with

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TABLE 4. Thermodynamic parameters' of virus adsorption to solids

Solid E AH* AG* bSdopin(kcallmol) (kcal/mol) (kcal/mol) (cal/mol) (°K) S Adsorption

Diatomaceous earth modified with 1 M 5.1 4.5 21.5 -57.1 90FeCl3 + 1 M MgCl2

Nitrocellulose, clay, and Seitz S1 filtersc 5.1 + 3.9 4.3 + 3.7 21.3 + 0.7 -56.4 + 14.8 88 + 8

a Values were calculated for 25°C as described in Materials and Methods.b Percentage of virus adsorption after 30 min at 25°C.' Mean values for MS2 adsorption to the three indicated solids reported by Preston and Farrah (14).

previous reports (15, 21). More efficient virus adsorption tomicroporous filters at pH values near neutrality has beenfound for filters with a positive or slightly negative zetapotential than for filters with a more negative charge (15, 21).The lower correlation between virus adsorption and zetapotential with trickling filter effluent than between virusadsorption and zeta potential with buffer suggests that com-ponents of the trickling filter effluent influenced virus adsorp-tion.Other procedures are available for increasing the charge

on diatomaceous earth and microporous filters. Cationicpolymers have been used to modify diatomaceous earth (1,2) and microporous filters (15). This method of modificationincreased the zeta potential and virus adsorption by themodified materials. Although modification with cationicpolymers may prove useful for modifying filters used inrecovering viruses in water, it might not be suitable formodifying materials used in filtering water for human con-sumption. The possible leaching of polymer or monomerunits might reduce public acceptance or invite regulatorycontrol of their use. Several of the metals used in this study,such as iron and magnesium, are not considered toxic unlesslarge amounts are consumed (3). There may be some con-cern with solubilization of aluminum. However, the use ofaluminum precipitates has been an accepted component ofwater treatment procedures for years (10). Also, the alumi-num and ferric precipitates used in this study are relativelyinsoluble. The solubility constants for aluminum hydroxideand ferric hydroxide are 5 x 10-33 and 6 x 10-38, respec-tively (11).

TABLE 5. Influence of salts and detergents on adsorption ofcoliphage MS2 to diatomaceous eartha

Salt used to modify Solution used for % Virusdiatomaceous earth virus adsorption adsorption

None Buffer <11 M FeCl3 + 1 M AICl3 Buffer 99 1

0.1% Tween 80 98 ± 20.1 M sodium thiocyanate 98 ± 10.1 M sodium chloride 97 + 20.1 M sodium fluoride 62 ± 80.1 M sodium citrate <1

1 M FeCI3 + 1 M MgCl2 Buffer 98 20.1% Nonidet P-40 99 ± 10.1 M sodium citrate 4 + 2

a A 20-ml volume of buffer (0.02 M glycine plus 0.2 M imidazole [pH 7])alone or with the indicated salts or detergents was seeded with approximately105 PFU of virus per ml and mixed with 0.5 g of diatomaceous earth on arotating shaker for 5 min. The samples were centrifuged at 2,000 x g for 5 min,and the supernatant fractions were examined. Virus adsorbed to the diatoma-ceous earth was recovered by mixing with 20 ml of 3% beef extract plus 1 MNaCI and mixing and centrifuging as described above. Values represent themean + standard deviation for three determinations.

The reasons for the variability in recovery of viruses fromwater by using filters containing modified diatomaceousearth have not been definitely established. Some of thevariability likely results from the fact than the filters arehandmade before use. Variations in packing and displace-ment of filter material directly in the center of the filter by theflow of water have been observed. These sources of varia-tion were reduced by using two filters in tandem. Additionalstudies are required to determine if the use of modifieddiatomaceous earth offers any advantages over previouslydescribed virus concentration procedures (5, 6, 17, 18, 20,21).The mean values of thermodynamic parameters reported

in a previous study are similar to the values obtained in thiswork (Table 4). This suggests that similar mechanisms ofvirus adsorption were operating in all of the cases studied.The finding that solutions of detergents had no significant

effect on virus adsorption to modified diatomaceous earthwas surprising. In previous studies, solutions of detergentshave been found to reduce virus adsorption to microporousand depth filters (18, 25). These results indicate that hydro-phobic interactions play at most a minimal role in adsorptionof virus to modified diatomaceous earth. The fact that achaotropic salt (sodium thiocyanate) does not affect virusadsorption is consistent with the fact that hydrophobicinteractions are not a major factor in virus adsorption tomodified diatomaceous earth. In a previous study, chaotro-pic salts were found to interfere with virus adsorption tomicroporous filters (4). Only salts with a relatively highcharge density (sodium fluoride and sodium citrate) inter-fered with virus adsorption to modified diatomaceous earth.This suggests that electrostatic interactions are the primarymechanism of virus adsorption to the modified solids studiedin this work (18).

In summary, we have found that modification of diatoma-ceous earth by in situ precipitation of metallic salts increasesits zeta potential and its ability to adsorb viruses in water.Filters made with such modified diatomaceous earth mightprove useful in the following: (i) recovering viruses fromwater, (ii) removing viruses from small volumes of water foremergency needs or for travelers, and (iii) large-scale treat-ment of water, especially recycled wastewater, where anadded barrier for virus transmission might be desired.

ACKNOWLEDGMENT

This work was supported by the Center for Natural Resources,Institute of Food and Agricultural Sciences, University of Florida.

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