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Ceramic media amended with metal oxide for the

capture of viruses in drinking water
a b
J. Brown & M.D. Sobsey
a
Department of Biological Sciences , University of Alabama , Box 870229, Tuscaloosa, AL,
35487–0299, USA
b
Department of Environmental Sciences and Engineering , University of North Carolina
School of Public Health , CB 7431 Rosenau Hall, Chapel Hill, NC, 27599–7431, USA
Published online: 19 Mar 2009.

To cite this article: J. Brown & M.D. Sobsey (2009) Ceramic media amended with metal oxide for the capture of viruses in
drinking water, Environmental Technology, 30:4, 379-391, DOI: 10.1080/09593330902753461

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 Environmental Technology
Vol. 30, No. 4, 1 April 2009, 379–391

Ceramic media amended with metal oxide for the capture of viruses in drinking water
J. Browna* and M.D. Sobseyb
a
Department of Biological Sciences, University of Alabama, Box 870229, Tuscaloosa, AL 35487–0299, USA;
b
Department of Environmental Sciences and Engineering, University of North Carolina School of Public Health, CB 7431
Rosenau Hall, Chapel Hill, NC, 27599–7431, USA
(Received 7 August 2008; Accepted 5 January 2009 )
Taylor and Francis Ltd

10.1080/09593330902753461

Ceramic materials that can adsorb and/or inactivate viruses in water may find widespread application in low-tech
drinking-water treatment technologies in developing countries, where porous ceramic filters and ceramic granular
media filters are increasingly promoted for that purpose. We examined the adsorption and subsequent inactivation of
Downloaded by [UGR-BTCA Gral Universitaria] at 08:33 05 March 2015

bacteriophages MS2 and ϕX-174 on five ceramic media in batch adsorption studies to determine media suitability
for use in a ceramic water filter application. The media examined were a kaolinitic ceramic medium and four
kaolinitic ceramic media amended with iron or aluminium oxides that had been incorporated into the kaolinitic clays
before firing. Batch adsorption tests indicate increased sorption and inactivation of surrogate viruses by media
amended with Fe and Al oxide, with FeOOH-amended ceramic inactivating all bacteriophages up to 8 log 10.
Unmodified ceramic was a poor adsorbent of bacteriophages at less than 1 log 10 adsorption-inactivation and high
recovery of sorbed phages. These studies suggest that contact with ceramic media, modified with electropositive Fe
or Al oxides, can reduce bacteriophages in waters to a greater extent than unmodified ceramic.
Keywords: ceramic filtration; bacteriophages; drinking water; sorption

Introduction meaning that they may be either positively or negatively

Waterborne viruses are important aetiologic agents of
diarrhoeal disease, contributing to the staggering world-
wide burden of water-related diseases [1]. With the
advent of better monitoring and analytical tools, viruses
are increasingly identified as aetiologic agents in
endemic diarrhoea and outbreaks of gastroenteritis in
developing countries [2] and developed countries [3–5].
The behaviour of waterborne viruses in contact with
ceramic surfaces may have public health importance
because ceramic media and porous ceramic filters are
widespread and increasingly used as drinking-water
treatment technologies, particularly in developing coun-
tries [6]. The application of these technologies is limited
by their ineffectiveness against viruses [6,7].
Viruses are colloid-sized amphoteric microbes with
a well-defined isoelectric point (IEP), specific to the
individual virus type and strain. The net charge of a
virus is largely dependent on the pH of the medium and
the surface chemistry of the virus, due to an increase in
the ionization of carboxyl groups (COOH → COO−)
and sulfhydril groups (SH → S−) and a decrease in
ionization of amine groups (NH3+ → NH2) at the
microbe surface as the pH is raised. Viruses have
isoelectric points typically in the range of 3–7 [8],
charged at the pH of natural waters (pH 4–9). However,
localized regions on the virus surface can vary widely in
charge as the surface is made up of multiple protein
subunits, each of which has its own unique hydrophilic
and hydrophobic regions as well as the ability to inter-
act with adjacent protein subunits of different chemical
composition and associated hydrophilic and hydropho-
bic properties to create new domains with unique
charged and hydrophobic regions [9]. At pH values
greater than the IEP, the virus carries a net negative
charge. Depending on the pH and surface charge of
other interacting particles, electrostatic attraction can
thus play an important role in virus fate and transport
[10,11].
Adsorption is a key process governing virus transport
in porous media, including in the subsurface environ-
ment [12,13], in media filtration [14], and in microfil-
tration [15–17]. Studies have shown that adsorption may
be virus-type and strain specific [12,18], varying by
virus isoelectric point (IEP) [10], virus morphology [8],
hydrophobicity [9,19,20] and aggregation [21]. Double
layer interactions have been proposed as the dominant
mechanism for virus adsorption in porous media [9].
Hydrophobic interactions [9,22], factors relating to

*Corresponding author. Email: joebrown@as.ua.edu

ISSN 0959-3330 print/ISSN 1479-487X online

© 2009 Taylor & Francis
DOI: 10.1080/09593330902753461
http://www.informaworld.com
 380 J. Brown and M.D. Sobsey
solution and surface chemistry [11,23–25], and other
interactions are also important in virus-media sorption
and inactivation [26].
There is a substantial body of literature on virus
attachment to various positively charged particles and
surfaces, especially metal oxides [9] and positively
charged media in the environment such as clays that are
rich in ferric oxides [12]. Murray and Laband [27]
found that the metal oxides α-SiO2, α-Fe2O3, α-Al2O3,
β-MnO2, and CuO were able to effectively capture
poliovirus from solution. Several studies have shown
that sand columns coated with metal oxide precipitates
are capable of reducing viruses in water significantly
more than those containing unmodified sand
[11,26,28–30]. These and other coatings which alter the
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surface charge of media (and filter elements) have
demonstrated the potential to increase reduction of
viruses in drinking water.
Attachment to some surfaces can protect viruses
from inactivation, especially those with high clay and
organic content [26,31–34]. Attachment to other
surfaces, however, appears to increase inactivation
[9,26,27,35]. Subsequent virus inactivation through
sorption to charged surfaces has been well documented
and is apparently strongly associated with the charge
differential, which leads to destructive chemical
changes to the virus capsid or nucleic acid once sorbed
to the surface [26,27,36]. If the charge differential is
great enough, viruses can be very effectively removed
and/or inactivated through sorption to charged media.
Viruses are composed of protein subunits that are
arranged into a unique, directed and energy-stable
pattern that is created when the viruses are formed
during replication. When external physical or chemical
stressors are applied to the virus, its structure can
become altered, leading to conformational changes and
loss of integrity that result in loss of infectivity or in
virus inactivation [37,38].
We hypothesize that ceramic media amended with
electropositive iron and aluminium oxides can increase
virus capture and subsequent inactivation. Batch
adsorption experiments were performed to examine the
extent of sorption and inactivation of two model
viruses to ceramic media, suspended in groundwater or
a surface water supplemented with primary wastewater
effluent (soluble portion, autoclaved). Media and water
type were chosen to represent a range of ceramic
media and water characteristics that may be directly
relevant to the processes of ceramic media filtration
and porous ceramic filtration technologies used for
drinking water treatment in developing countries.
Studies were also intended to aid in the identification
of virus, media and water characteristics affecting
virus sorption to ceramic media and subsequent
inactivation.
Materials and methods
In order to assess the ability of modified ceramic filter
surfaces to capture and inactivate viruses, samples of
ceramics modified with metal oxides were tested in
small batch adsorption studies. Ceramic samples were
crushed, sieved, and suspended in two challenge waters
spiked with 105–108 plaque-forming units (pfu) per
millilitre of bacteriophage MS2 or ϕX-174. The chal-
lenge waters consisted of a groundwater and a lake
water supplemented with 20% primary wastewater
effluent (soluble portion, autoclaved) to simulate a range
of source water qualities. The suspensions were mixed
long enough to attain sorption quasi-equilibrium at room
temperature (30 minutes) and then centrifuged to pellet
the ceramic particles and adsorbed phages. The super-
natant water was removed and assayed for unadsorbed
infectious phages, and the pellet was resuspended in a
beef extract (BE) eluant containing 2M NaNO3 at pH 5.5
to elute adsorbed phages. The eluate was also assayed
for phages to determine whether bacteriophages could
be recovered. Inactivation of adsorbed phages was
determined via direct plating of media suspensions to
examine adsorbed phage infectivity.
Preparation of ceramic media
Ceramic material preparation was modelled after meth-
ods used by Potters for Peace, an international NGO
that specializes in the manufacture of porous ceramic
water filters. Their model has been widely adapted and
used worldwide in the production of ceramic point-of-
use water treatment devices. Ceramic samples were
prepared from a low-fire, white, kaolinitic clay typical
of porous ceramic microfilter production, which was
obtained from Laguna Clay Company (Oviedo, FL).
Milled, high purity (>98%), natural (non-synthetic)
metal oxides were added to the base clay in a 1:6
oxide:dry clay ratio by weight and kiln-fired to cone
012 (840–880°C), the firing temperature most
commonly using for ceramic filter production in the
Potters for Peace factories. The 1:6 oxide:clay ratio was
used as the highest ratio allowable in the firing process
without negatively affecting the structural properties of
the ceramic in initial testing (data not shown). Using a
greater amount of metal oxide amendments produced a
brittle ceramic poorly suited for use as granular media
or porous ceramic filters. Metal oxide additives were:
α-FeOOH (goethite), Fe3O4 (magnetite), Fe2O3 (hema-
tite) and Al2O3 (alumina): common, naturally occurring
oxides of iron and aluminium [39]. Fired ceramic
samples were crushed using a mortar and pestle, dried
for 24 hours to a constant weight at 110 °C (±5 °C), and
sieved by method ASTM C136 [40] to a particle size
range of 75–250 µm (maximum diameter) for use in
batch tests.
 Environmental Technology
Incorporation of the mineral oxide into the clay
medium was selected as the preferred way to incorpo-
rate positively charged materials into ceramic media. It
was considered preferable to surface coating of clay
after firing. While there is much literature on oxide-
coated surfaces and media, these methods of amending
filters with positively charged materials may not work
well in some porous ceramic filtration applications
because of the regular abrasive scrubbing required to
maintain filter effectiveness. Also, chemical precipita-
tion of metal oxides as coatings on media (e.g. methods
used by Scott et al. [29]) can entail the use of toxic
chemicals and complex processes that may not be prac-
tical in typical ceramics workshops and ceramic filter
production facilities in developing countries.
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Zeta potential measurement of sorption media
The pH and zeta potential of all media were measured
as the key parameters possibly associated with adsorp-
tion characteristics. Zeta potential or electrophoretic
mobility of particle suspensions can be measured by
tracking particles through a charged field [41]. Briggs
cell microelectrophoresis was used on a fine particle
suspension (<1 µm diameter) of crushed ceramic media
samples. A Zeta-Meter (Zeta-Meter Inc, Staunton, VA)
instrument was used.
Suspensions were buffered with 0.001 M acetate at
pH 5.0, phosphate at pH 7.0 and borate at pH 9.0.
Sodium chloride (NaCl) was added as a swamping elec-
trolyte to a concentration of 0.05 M to ensure constant
ionic strength.
Challenge waters used for batch adsorption testing
Challenge water characteristics are summarized in
Table 1. Two model environmental waters were used as
suspending waters in batch adsorption tests to represent
Table 1. Challenge water characteristics.
Lake water + 20%
primary wastewater
Parameter Groundwater effluent (PE)
Temperature 22 °C ± 5 °C 22 °C ± 5 °C
Total dissolved 1000 ± 200 mg L−1 3000 mg L−1 ± 1000
solids
Free chlorine 0 mg L−1 0 mg L−1
pH 7.6 ± 0.5 6.8 ± 0.5
−1
Total organic <3 mg L 130 ± 10mg L−1
carbon
Turbidity <2 NTU 5 ± 2 NTU
UV absorbance at 0.005 0.05
254 nm
381
a range of water quality characteristics possibly influ-
encing virus attachment to ceramic media.
Challenge water CW1 was a well-characterized
groundwater from the fractured bedrock aquifer in the
Piedmont region of southern Orange County, approxi-
mately 6 km west of Chapel Hill, North Carolina, USA.
Water was collected from two private residential wells.
Challenge water CW2 was a high humic-content natural
surface water from Bay Tree Lake (80 km SE of
Fayetteville, North Carolina) spiked with 20% (volume/
volume) of well-characterized primary effluent waste-
water (soluble fraction, sterile) from the Orange Water
and Sewer Authority wastewater treatment facility,
Orange County North Carolina. Soluble organic matter
such as that found in eutrophic surface waters and
primary wastewater effluent has been shown to prevent
sorption of viruses and to desorb them from surfaces
[42,43] and was therefore used as a suitable ‘worst-
case’ challenge water for virus sorption.
Bacteriophages used in batch and filter challenge tests
Surrogates for waterborne human enteric viruses used
in batch tests and in filter challenge tests were MS2
and ϕX-174 coliphages. MS2 is a male-specific (F+),
single-stranded, non-enveloped coliphage with an
isoelectric point (IEP) of 3.9. It is often used in model-
ling human enteric viruses, because of its similarity to
poliovirus, other enteroviruses, noroviruses and hepati-
tis A virus in size (diameter = 24–25 nm), shape
(icosahedral) and nucleic acid (RNA) [8,44,45]. It is
also useful in laboratory applications because of its
ease of production, recovery, and enumeration; its
non-pathogenic nature; and the ease of attaining high
titres [46]. MS2 and other F+RNA viruses have been
shown to be conservative estimators of sorption
mechanisms when compared with mammalian viruses
[12,17,47].
The coliphage ϕX-174 is a somatic, spherical virus
(IEP = 6.6) with single-stranded DNA as the nucleic
acid. It is also useful as an indicator for human enteric
viruses in water, because of its similarity to human
parvoviruses, easy detection and correlation with
enteric viruses in water and wastewater [48].
Production method for virus stocks
The somatic and male-specific bacteriophages MS2 and
ϕX-174 were propagated to obtain high-titre stocks for
use in sorption experiments. Bacteriophages originally
obtained from laboratory stocks were twice purified on
E. coli C3000. Plaques were selected (‘picked’) from the
bacterial lawn and suspended in phosphate-buffered
saline (PBS). High titre stocks were produced through
confluent lysis on soft agar with PBS-suspended phages,
 382 J. Brown and M.D. Sobsey
log-phase host (E. coli F-amp for MS2, E. coli CN-13
for ϕX-174) and appropriate antibiotics and incubated at
37 °C for 24 hours. The lysate-agar mixture was
subjected to chloroform extraction. Chloroform was
added to the mixture in a 1:1 volume:volume ratio in
50 mL polypropylene centrifuge tubes, shaken vigor-
ously by hand for three minutes, and centrifuged for
20 minutes at 4 °C at 2500 rcf (relative centrifugal
force). Following centrifugation, the supernatant was
removed from individual centrifuge tubes and pooled.
Sterile glycerol was added to the supernatant in a
1:4 volume:volume ratio. Finally, aliquots of the stocks
were placed in 1 ml polypropylene microcentrifuge
tubes and stored at −80 °C. Phage stocks were assayed
to determine titre using plaque assay techniques as
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described by Adams [49] and more recently standardized
by the US EPA [50].
Plaque assay methods for test viruses
The double agar layer (DAL) assay was used to
enumerate phages in water samples and was performed
as described in EPA method 1602 [50]. Samples were
serially diluted in PBS. Bottom agar was prepared as
1.4–1.5 g Bacto-agar and 3 g of tryptic soya broth per
100 ml of sterile, reagent-grade water, autoclaved,
cooled to 42 °C, supplemented with appropriate antibi-
otics, and poured into sterile, 60 mm × 15 mm polysty-
rene Petri dishes. For tests using bacteriophage host E.
coli F-amp (ATCC#700891), the antibiotics used were
streptomycin/ampicillin prepared according to EPA
method 1602 [51]. For tests using bacteriophage host E.
coli CN-13 (ATCC#700609), the antibiotic used was
nalidixic acid prepared according to EPA method 1602
[50]. MS2 (a male-specific coliphage) infects E. coli F-
amp, and ϕX-174 (a somatic coliphage) infects E. coli
CN-13.
Top agar was prepared as 0.7–0.8 g Bacto-agar and
3 g of tryptic soya broth per 100 ml of sterile, reagent-
grade water, autoclaved, cooled to 42 °C, and supple-
mented with appropriate antibiotics. A series of 13 mm
× 100 mm sterile glass test tubes were filled with 7 mL
of top agar while maintaining constant temperature at
42 °C in a water bath. To each tube was added 0.1 mL
of log-phase host bacteria and 0.1 mL of sample (serial
dilutions, vortexed). The contents of each tube (host,
sample and top agar) were poured on to bottom agar
60 mm × 15 mm polystyrene plates. Top agar was
allowed to solidify at room temperature. All plates were
then inverted and incubated at 37 °C for 16–24 hours.
Two or more dilutions and replicates were used, along
with positive and negative controls.
Bacteriophages were enumerated on plates by count-
ing clear zones of lysis (plaques) on the bacterial lawn
and reported as pfu per 100 mL sample. Bacteriophages
were enumerated from plates with the most appropriate
dilutions (those with 20–300 plaques).
Adsorption testing protocol
Crushed ceramic samples (0.25 g dry weight) were
suspended in 1 ml of challenge water spiked with 105–
108 pfu mL−1 of bacteriophage MS2 or ϕX-174 in 2 ml
conical polypropylene micro-centrifuge tubes. Each
tube was sealed, placed horizontally and shaken along
the tube’s longitudinal axis to facilitate mixing and
contact at 100 rpm (i.e. 100 back-and-forth motions per
minute) for 30 minutes at 22 ± 2 °C to establish sorption
quasi-equilibrium. These conditions were pretested in
preliminary adsorption kinetics experiments to establish
that equilibrium was reached. Sorption was rapid in all
cases, with >95% of sorption occurring within five
minutes (data not shown). This contact time was also
intended to replicate the mean surface contact time of
water and associated contaminants in currently
produced porous ceramic filters. After contact, the
media-phage suspensions were centrifuged at 8000 × g
for 10 minutes at 22 ± 2 °C.
The supernatant was removed and assayed for
phages MS2 or ϕX-174 as previously described and
reported as pfu per 100ml sample. Log reduction values
(LRVs) were computed based on test phage titre in the
supernatant water after centrifugation versus spiked
challenge water (the log10 of the influent concentration
minus the log10 of the effluent concentration) as:
LRV ( Log10 reduction value ) = log10
( pre − contact titre ) − log10 ( post − contact titre )
Eluant
The crushed ceramic pellet from centrifugation was
resuspended at 5% w/v in a 3% solution of beef extract
and 2 M NaNO3 at pH 5.5, which was prepared with
reagent grade distilled water, autoclaved, stored at 4 °C,
and brought to room temperature before use. This eluant
has been used previously to effect virus desorption from
sediments and soils [12,51] and was selected for use in
these experiments based on initial work indicating
greater efficiency than other eluants for virus recovery
after adsorption (data not shown).
Bacteriophage recovery and infectivity assays
Elution
Elution of sorbed phages was performed by resuspend-
ing the pelletized phage-laden media in 1 ml of eluant.
The media with added eluant were sealed, placed
horizontally, and shaken along the tube’s longitudinal
axis for 60 minutes on a platform shaker at 100 rpm at
 Environmental Technology
22 ± 2 °C to facilitate mixing and contact. The suspen-
sion was centrifuged at 8000 × g for 10 minutes at 22 ±
2 °C, and the supernatant was removed for plaque assay
according to methods previously described. Virus assay
of the eluate was used to determine the extent of virus
inactivation (absence of infectivity) subsequent to
phage sorption.
Direct plating of sorbent suspension to determine
infectivity of sorbed phages
Direct plating of phage-laden crushed ceramic,
suspended in PBS and serially diluted in PBS, was used
to further determine the extent of inactivation. After
elution and recovery of the eluate for assay, the pellet-
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ized phage-laden sorbent was resuspended in 1 ml of
sterile PBS. Suspensions were vortexed, serially diluted
in PBS, and directly plated on prepared 150 mm × 15 mm
polystyrene host/trypticase soya agar (TSA) plates as in
the spot titre assay described previously. The infectivity
of the sorbed phages was verified by examining zones
of lysis on the plates, indicating infectious phage.
Because the particles may have had many viruses
attached, individual plaques may represent more than
one phage. Therefore, this method was considered to be
a presence/absence test for infectivity post-sorption.
Statistical analysis
Descriptive statistics were used to characterize the
results of experiments of batch adsorption experiments,
including arithmetic mean, median, standard deviation
and variance of log10 reduction of phages by phage type,
water type, and adsorption medium.
Parametric and non-parametric statistical tests were
used to compare results from different individual exper-
iments assessing the effect of media type, phage type
and water type on adsorption as measured by the log10
reduction in bacteriophages post-contact. Comparisons
were made initially using a two-sample mean compari-
son (t) test. Due to the small sample size of some exper-
iments (n < 10), the assumption of a normally
distributed range of values may not hold true, nor were
all tests performed an equal number of times. The
Welch t-test, a statistical test that computes the t-statis-
tic for variables with non-uniform variances, was used.
A non-parametric equivalent, the Mann–Whitney U-
test, was also used as a test of statistical significance
when comparing sample sets. In comparing log10 reduc-
tion values across parameters of media, challenge water
and phage type, ANOVA (parametric) and the Kruskal–
Wallis one-way ANOVA for non-parametric testing
were used.
Where both parametric and non-parametric tests
yielded a significant result (p < 0.05), the difference in
383
means was considered significant. Discrepancies
between parametric and non-parametric tests of signifi-
cance occurred in 14% of sample sets. Where signifi-
cance tests were in conflict, the Welch t-test was
preferred based on an assumed normal distribution of
the data (albeit with unequal variances). Both paramet-
ric and non-parametric ANOVA agreed when
performed in parallel.
Linear regression was performed to analyse possible
associations between zeta potential and adsorption of
phages. A Pearson correlation coefficient and R2 values
were used initially to characterize trends. An F-test for
correlation was applied to the data to indicate associa-
tions of statistical significance.
All tests were compared using a significance level of
α = 0.05. Statistical testing was performed in Stata
version 8.1 (Stata Corporation, College Station, TX).
Results
The results of batch sorption experiments on bacte-
riophage interaction with ceramic particles, modified
with metal oxide and suspended in test waters CW1
(groundwater) and CW2 (lake water with primary efflu-
ent), are summarized in Table 2. Viral adsorption to
ceramic media was computed as the difference between
virus concentrations in the supernatant of media suspen-
sions versus that in media-free controls. Reduction of
bacteriophages by inactivation or die-off not attribut-
able to sorption was considered negligible, due to the
stability of the microbes at room temperature over short
periods [12,18]. Virus adsorption to the interior surface
of the microcentrifuge tube was assumed to be negligi-
ble but was not measured. Meschke [12] reported less
than 5% adsorption of viruses to identical tubes used in
similar batch tests with soils.
Media type had the greatest effect on sorption in
both waters and for both bacteriophages (P < 0.0001).
Neither phage type nor challenge water showed consis-
tently greater adsorption as determined in parametric
and non-parametric ANOVA analysis. Consistently
greater bacteriophage sorption was observed in ceram-
ics modified with Fe2O3, FeOOH and Al2O3.
MS2 in CW1 was reduced an arithmetic mean 0.55
log10 when contacted with unmodified white base
ceramic (WBC), compared with 0.51 log10 on Fe3O4-
WBC (P = 0.5594), 4.6 log10 on Fe2O3-WBC (P <
0.0001), ≥8.0 log10 on FeOOH-WBC (P < 0.0001) and
1.9 log10 on Al2O3-WBC (P = 0.0035).
MS2 in CW2 was reduced an arithmetic mean 0.72
log10 when contacted with unmodified (WBC) ceramic,
compared with 0.63 log10 on Fe3O4-WBC (P = 0.6382),
5.6 log10 on Fe2O3-WBC (P < 0.0001), ≥8.0 log10 on
FeOOH-WBC (P < 0.0001) and 1.3 log10 on Al2O3-WBC
(P = 0.0113). A value of 0.34 log10 of MS2 sorption in
 Downloaded by [UGR-BTCA Gral Universitaria] at 08:33 05 March 2015

384

Table 2. Bacteriophage total log10 reductions values (LRV) and irreversible reduction (IrrLRV) over n trials.

Log10 total adsorption (LRV)b Log10 irreversible adsorption (IrrLRV)c

Mediuma Phage (water) Mean nd Median Std Dev Variance Mean n Median Std Dev Variance
WBC MS2 (CW1) 0.55 4 0.54 0.49 0.24 0.34 3 0.10 0.42 0.18
MS2 (CW2) 0.72 5 0.92 0.38 0.14 0.34 3 0.15 0.33 0.11
ϕX-174 (CW1) 0.59 3 0.68 0.24 0.059 0.35 3 0.15 0.32 0.11
ϕX-174 (CW2) 0.35 5 0.37 0.065 0.0042 0.19 3 0.10 0.15 0.023
Fe3O4-WBC MS2 (CW1) 0.51 7 0.53 0.090 0.0082 0.43 4 0.43 0.021 0.00043
MS2 (CW2) 0.63 7 0.24 0.48 0.23 0.12 2 0.12
ϕX-174 (CW1) 0.52 9 0.62 0.27 0.071 0.52 5 0.52 0.048 0.0023
ϕX-174 (CW2) 0.24 7 0.35 0.14 0.021 0.15 2 0.15
Fe2O3-WBC MS2 (CW1) 4.6 13 4.3 0.64 0.41 3.5 8 4.1 1.1 1.1
MS2 (CW2) 5.6 4 5.6 0.054 0.0030 3.5 2 3.5
ϕX-174 (CW1) 4.0 22 3.7 1.3 1.8 4.1 12 4.1 1.4 1.9
ϕX-174 (CW2) 4.5 9 4.6 1.9 3.4 2.8 3 2.9 0.21 0.044
FeOOH-WBC MS2 (CW1) ≥8.0e 6 * * * ≥8.0 6 * * *
MS2 (CW2) ≥8.0 6 * * * ≥8.0 6 * * *
ϕX-174 (CW1) ≥8.0 6 * * * ≥8.0 6 * * *
J. Brown and M.D. Sobsey

ϕX-174 (CW2) ≥8.0 6 * * * ≥8.0 6 * * *

Al2O3-WBC MS2 (CW1) 1.9 10 1.9 0.22 0.046 1.2 10 1.3 0.28 0.077
MS2 (CW2) 1.3 6 1.2 0.32 0.10 1.3 3 1.3 0.0032 0.0011
ϕX-174 (CW1) 2.5 13 2.7 0.77 0.60 2.3 8 2.6 1.0 0.98
ϕX-174 (CW2) 1.7 10 1.8 0.41 0.17 1.4 2 1.4 0.019 0.00039
a
Mass/volume ratio = 0.25 g sorbent/ml challenge water, particle size = 75–250 µm (maximum diameter).
b
Arithmetic mean LRV = log10 reduction value. LRV = log10[Ci/C] where Ci is the initial phage titre and C is the titre post-contact.
c
Arithmetic mean irreversible LRV = irreversible log10 reduction value, or the log10 reduction of phages that were not successfully eluted from the ceramic surface.
d
n = number of trials. Reported LRV and IrrLRV values are arithmetic means over n trials.
e
≥ denotes the case where no infective phages were recovered after contact, so the LRV and IrrLRV values potentially represent minima.
*Cannot be computed for these samples; all results are low-end estimates (no phages recovered post-contact).
 Environmental Technology 385

CW2 was not recovered from WBC, versus 0.12 log10
from Fe3O4-WBC, 3.5 log10 from Fe2O3-WBC, ≥8.0
log10 on FeOOH-WBC and 1.3 log10 from Al2O3-WBC.
Bacteriophage ϕX-174 in CW1 was reduced an
arithmetic mean 0.59 log10 when contacted with unmod-
ified (WBC) ceramic, compared with 0.52 log10 on
Fe3O4-WBC (P = 0.6561), 4.0 log10 on Fe2O3-WBC
(P < 0.0001), ≥8.0 log10 on FeOOH-WBC (P < 0.0001),
and 2.5 log10 on Al2O3-WBC (P < 0.0001). A value of
0.35 log10 of ϕX-174 sorption in CW1 was not recov-
ered from WBC, versus 0.52 log10 from Fe3O4-WBC,
4.1 log10 from Fe2O3-WBC, ≥8.0 log10 from FeOOH-
WBC and 2.3 log10 from Al2O3-WBC.
Bacteriophage ϕX-174 in CW2 was reduced an
arithmetic mean 0.35 log10 when contacted with unmod-
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Sorption of MS2 was significantly greater in CW2
for contact with Fe2O3-WBC (P < 0.0001) compared
with MS2 adsorption in CW1. No other significant
differences were detected for differential adsorption of
phages by challenge water type stratified by phage or
media type, nor overall as determined by parametric
(P = 0.7975) and non-parametric (P = 0.9146) ANOVA.
The extent of ϕX-174 adsorption was significantly
greater in CW1 (P = 0.0088) and CW2 (P = 0.0231)
when contacted with Al2O3-WBC. No other significant
differences between the extent of adsorption by either
phage were detected within media types or within
challenge water type, nor overall as determined by para-
metric (P = 0.3238) and non-parametric (P = 0.0762)
ANOVA.

ified (WBC) ceramic, compared with 0.24 log10 on
Fe3O4-WBC (P = 0.5641), 4.5 log10 on Fe2O3-WBC (P
< 0.0001), ≥8.0 log10 on FeOOH-WBC (P < 0.0001)
and 1.7 log10 on Al2O3-WBC (P < 0.0001). A value of
0.19 log10 of ϕX-174 sorption in CW2 was not recov-
ered from WBC, versus 0.15 log10 from Fe3O4-WBC,
2.8 log10 from Fe2O3-WBC, 8.0 log10 from FeOOH-
WBC, and 1.4 log10 from Al2O3-WBC.
Mean recoveries of viruses by media, virus type and
suspending water are given in Table 3. Bacteriophages
were recovered from ceramic media to differentiate
between adsorption that resulted in inactivation of
phages and adsorption that was chemically reversible.
Several eluants were initially tested for efficiency of
virus desorption of MS2 from ceramic media suspen-
sions. The eluants tested were beef extracts (1–10%) at

Table 3. Per cent recovery of bacteriophages from ceramic media by phage type and suspending water using 3% BE eluant.
Total phages adsorbed Total phages recovered Per cent Infectious phages detected
Mediuma Phage (water) (mean log10b) (mean log10) recoveryc in direct-plated media?d
WBC MS2 (CW1) 0.55 0.21 46% +
MS2 (CW2) 0.72 0.38 46% +
ϕX-174 (CW1) 0.59 0.24 45% +
ϕX-174 (CW2) 0.35 0.16 65% +
Fe3O4-WBC MS2 (CW1) 0.51 0.080 37% +
MS2 (CW2) 0.63 0.51 76% +
ϕX-174 (CW1) 0.52 <0.010 30% +
ϕX-174 (CW2) 0.24 0.09 71% +
Fe2O3-WBC MS2 (CW1) 4.6 1.1 <1% −
MS2 (CW2) 5.6 2.1 <1% −
ϕX-174 (CW1) 4.0 <0.010 <1% −
ϕX-174 (CW2) 4.5 1.7 <1% −
FeOOH-WBC MS2 (CW1) ≥8.0e <0.010 0% −
MS2 (CW2) ≥8.0 <0.010 0% −
ϕX-174 (CW1) ≥8.0 <0.010 0% −
ϕX-174 (CW2) ≥8.0 <0.010 0% −
Al2O3-WBC MS2 (CW1) 1.9 0.70 6% −
MS2 (CW2) 1.3 <0.010 5% −
ϕX-174 (CW1) 2.5 0.20 1% −
ϕX-174 (CW2) 1.7 0.30 4% −
a
Mass/volume ratio = 0.25g sorbent/ml challenge water, particle size = 75–250 µm (maximum diameter).
b
Arithmetic mean LRV = log10 reduction value. LRV = log10[Ci/C] where Ci is the initial phage titre and C is the titre post-contact.
c
Phages were recovered via 3%BE eluant at pH 5.5.
d
Inactivation was confirmed via direct plating of phage-laden media suspensions. A positive (+) result indicates that some infectious phages were
detected on media.
e
≥ denotes the case where no infective phages were recovered after contact, so the LRV and IrrLRV values potentially represent minima.
 386 J. Brown and M.D. Sobsey

a range of pHs and with the addition of NaNO3 or Table 4. Zeta potential measurements of modified and
KNO3. The eluant of 3% beef extract supplemented unmodified ceramic media at pH 5.0, 7.0, and 9.0.
with 2 M NaNO3 at pH 5.5 was the most efficient at Zeta potential (mV) IEPb
recovering sorbed viruses (data not shown) from clay
and ceramic materials. Other more efficient eluants may Ceramic media pHa pH 5 pH 7 pH 9 (pH)
exist but were not identified in this study. For this White base ceramic 5.0 19.2 −11.6 −24.0 6.5
reason, reversibility of adsorption was not considered (WBC)
absolute but specific to this eluant in this system. Fe3O4-WBC 5.0 27.0 −36.7 −48.9 6.0
Because inactivation of bacteriophages subsequent Fe2O3-WBC 5.6 36.5 −29.9 −44.6 6.2
to adsorption on to ceramic media was of primary inter- FeOOH-WBC 11.3 69.2 46.7 25.5 *
est in this study, a further step to determine infectivity Al2O3-WBC 5.7 54.1 −28.5 −49.1 6.7
of adsorbed bacteriophages was performed. Following a
assay of the eluant to determine phages released by Method for pH determination was McLean [52]; suspending water
was reagent-grade, deionized water.
elution, the ceramic media pellet with adsorbed phages b
Isoelectric point, the pH at which the surface charge is 0.
was resuspended in PBS, and dilutions of this suspen- *not observed
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sion were directly plated on a host-agar lawn as in the

DAL assay. This method for confirming bacteriophage
infectivity was not quantitative but rather a presence/
absence test for determining whether infectious phages
were retained on media after elution with 3% BE, as
individual zones of lysis, constituting plaque-forming
units, could generally not be attributed to individual
bacteriophages (infectious units).
The mean recovery of MS2 from WBC suspensions
was 46% in both suspending waters (CW1 and CW2).
The mean recovery of ϕX-174 from WBC suspensions
was 45% in CW1 and 65% in CW2. In both suspending
waters and in both virus types, infectious phages were
detected through direct plating of WBC ceramic media
suspensions, suggesting that some retained viruses were
still infectious post-sorption.
For Fe3O4-WBC, 37% and 76% of adsorbed MS2
were recovered through elution after contact with media
in suspending waters CW1 and CW2, respectively.
Recovery of ϕX-174 was 30% in CW1 and 71% in
CW2 after contact with Fe3O4-WBC. Infectious phages
were detected through direct plating of Fe3O4-WBC
ceramic media suspensions, suggesting that some
retained viruses were still infectious post-sorption in
both suspending waters and for both virus types.
Mean recoveries of MS2 and ϕX-174 bacterioph-
ages from Fe2O3-WBC suspensions were less than 1%
in both suspending waters (CW1 and CW2). Although
adsorbed phages were no longer infectious following
sorption.
For Al2O3-WBC, 6% and 5% of adsorbed MS2 was
recovered through elution after contact with media in
suspending waters CW1 and CW2, respectively. Recov-
eries of ϕX-174 were 1% in CW1 and 2% in CW2 after
contact with Al2O3-WBC. Infectious phages were not
detected through direct plating of Al2O3-WBC ceramic
media suspensions, suggesting that retained phages
were no longer infectious post-sorption in both
suspending waters and for both virus types.
The pH of the ceramic media was determined by meth-
ods articulated by McLean [52] (Table 4) and in suspen-
sions of test waters CW1 and CW2 (Table 5), using the
same mass:volume ratio of batch adsorption tests.
Electrophoretic mobility results obtained using the
Zeta-Meter instrument are shown in Table 4 and
Figure 1. The media examined are not pure minerals but
mixtures of high-purity metal oxides and base kaolinitic
clay subjected to a high-temperature (840–880 °C)
firing process to create ceramic. Because of potential
chemical changes to the media during the firing process
the ceramic samples were not well characterized and
cannot be compared with standard curves for the
electrophoretic mobility of media reported elsewhere.
Figure 1. Zeta potential measurement of base ceramic media and ceramic media amended with metal oxide as a function of pH.

significant numbers of phages were recovered from the Table 5. pH of ceramic media suspensions in waters CW1
media in log10 terms, the overall percentage recovery and CW2.
was low. Moreover, direct plating of Fe2O3-WBC pH of media pH of media
media suspensions did not detect any infectious MS2 or suspension (2.5 g to suspension (2.5 g to
ϕX-174 infectious units post-elution, suggesting that 10 ml) in 10 ml) in lake water
adsorbed phages were no longer infectious following Medium groundwater (CW1) + 20% PE (CW2)
sorption to Fe2O3-WBC. WBC 6.8 6.4
No MS2 or ϕX-174 bacteriophages were recovered Fe3O4-WBC 6.7 6.4
from FeOOH-WBC suspensions in either suspending Fe2O3-WBC 6.7 5.9
water (CW1 and CW2). Direct plating of FeOOH-WBC
FeOOH-WBC 10.3 11.8
media suspensions did not detect any infectious MS2 or
Al2O3-WBC 7.0 6.5
ϕX-174 infectious units post-elution, suggesting that
 Environmental Technology 387










  



 
 










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Figure 1. Zeta potential measurement of base ceramic media and ceramic media amended with metal oxide as a function of pH.
The pH of the media suspensions in both challenge
waters was measured and is shown in Table 5. With the
exception of the strongly basic FeOOH-WBC suspen-
sion, other media mixtures were slightly acid or neutral
(pH 5.9–7.0).
Figure 1 shows the pH-zeta potential relationship
for each of the media studied at all pH levels examined.
Statistical analysis of differences in the pH–zeta-poten-
tial relationship between media was accomplished via a
two-sample t test for individual comparisons and
ANOVA for association between media and zeta poten-
tial. FeOOH-WBC was significantly more electroposi-
tive over the pH range examined than other media (P =
0.0004) at α = 0.05. WBC, Fe3O4-WBC, Fe2O3-WBC
and Al2O3-WBC were not significantly more or less
electropositive than others over the pH range (pH 3.0–
11.0), although the media did exhibit some variability.
The relatively small differences between media in the
neutral pH range, although not statistically significant
by these methods, may have implications for phage
sorption to media. This is because media heterogeneity
and local charge variability at surface sites, rather than
net surface charges, may be more important to sorption
mechanisms.
Discussion
The results suggest that iron and aluminium oxide
amendments to ceramic media can enhance the sorption
and inactivation potential of media for bacteriophages
MS2 and ϕX-174. Total inactivation of test phages was
observed when phages were contacted with FeOOH-
amended ceramics, possibly due to pH effects of the
media suspension. Ceramics modified with Fe2O3 and
Al2O3 metal oxide exhibited increased sorption and
inactivation of phages in contact with them and Fe3O4-
modified ceramic and unmodified ceramic were not
effective at adsorbing or inactivating phages (< 1.0
log10). The order of adsorption capacity was FeOOH-
WBC > Fe2O3-WBC > Al2O3-WBC > Fe3O4-WBC =
WBC for both bacteriophages and in both suspending
waters. Direct plating of phage-laden media suspension
dilutions indicated that non-recoverable phages were no
longer infectious for Fe2O3-WBC, Al2O3-WBC and
FeOOH-WBC.
Adsorption characteristics of test phages MS2 and
ϕX-174 were not observed to be significantly different
across media types and test waters. No consistent, statis-
tically significant trend was observed between adsorp-
tion of phages and the suspending water. Also, the fact
that no phages were able to be recovered after contact
with FeOOH-WBC meant that the values reported are
functions solely of the pre-contact phage titre and may
represent low-end estimates of phage inactivation, and
so are reported as ‘greater than’ (≥) values. For this
reason, an examination of the differences between phage
attachment according to challenge water and phage are
not possible for experiments using FeOOH-WBC.
The chemistry of the fired ceramic is highly depen-
dent upon base clay composition, purity of the additives,
 388 J. Brown and M.D. Sobsey
firing temperature and conditions, and other factors.
Initial X-ray diffraction data for chemical composition
of the oxide-amended ceramic media indicate a substan-
tial reduction of oxide species in the firing process (data
not shown). Further chemical characterization of the
media content and nature is a next step and is beyond the
scope of this paper. Care should be taken in the attribution
of bacteriophage adsorption or inactivation characteris-
tics to individual oxide species used as ceramic amend-
ments. After firing, the ceramic media probably contain
a mixture of oxide species that may have formed from
the base clay material and additives.
Recovery of infectious bacteriophages through
elution varied from 0% to 76%. Recovery was in all
cases greater following contact with WBC (45–65%)
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and Fe3O4-WBC (30–76%). Less than 6% of infectious
phages were recovered from Al2O3-WBC (1–6%) and
less than 1% of phages were recovered from Fe2O3-
WBC. No viruses were able to be recovered following
contact with FeOOH-WBC.
Ceramic media modified with Fe2O3-WBC,
FeOOH-WBC and Al2O3-WBC were associated with
virus inactivation as determined by direct plating of
media suspensions. The order of inactivation capacity
was FeOOH-WBC > Fe2O3-WBC > Al2O3-WBC >
Fe3O4-WBC = WBC for both bacteriophages and in
both suspending waters.
The association between pH of the suspension and
bacteriophage inactivation is a potentially important
one, as the high pH of the FeOOH-WBC suspension
was of sufficiently high pH to result in phage inactiva-
tion chemically. The effect of pH on phage viability is
well documented. Battigelli and Sobsey [53] report a 4
log10 inactivation of MS2 over 360 minutes of contact
time at pH 11. Also, Ryan et al. [26] report inactivation
of bacteriophages MS2 and PRD1 in groundwater at pH
11.0 (k = 0.55 ± 0.17 d−1). This rate could explain total
inactivation of phages over the time period included in
the test. Although the extent of inactivation reported in
the present study is significantly greater (≥8 log10)
within a fraction of the contact time (60 minutes), the
pH effects on phage viability are important and
potentially explain the apparent inactivation of bacte-
riophages in batch tests using FeOOH-amended
ceramic media.
The electropositivity of media as indicated by zeta
potential is one factor potentially influencing the
attachment of charged colloid-sized species (e.g.
viruses) to surfaces. Other factors, such as ion strength
and composition, also play a role [11]. Based on zeta
potential measurements of the ceramic media by pH, at
pHs of the batch sorption studies the media are all net
negatively charged except for goethite, although this
would be near the isoelectric point (IEP) of the media,
and therefore potentially localized surface charge prop-
erties would be important in virus binding [26]. Also,
zeta potential measurements were taken on particles an
order of magnitude smaller (1 µm) than particles in
batch sorption tests. The balance of surface charges on
small particles may differ from that of the larger
media.
Theory suggests that inactivation is associated with
the strength of the charge differential between the nega-
tively charged virus surface and the electropositive
media surface [9,26]. A weak association was observed
between surface charge of media and inactivation in this
study, suggesting that other mechanisms may be
influencing sorption behaviour in ceramic media
suspensions.
These results are consistent with other studies that
have reported an increased reduction of bacteriophages
and viruses in contact with granular media amended
with metal oxide. In particular, sand media coated with
aluminium and ferric oxides have been shown to reduce
viruses, bacteria and protozoa to a greater extent than
unmodified media [30,54,55]. Granular activated
carbon treated with iron and aluminium oxides to
increase zeta potential has also been shown to effec-
tively reduce phages and viruses in water, to greater
than 2.0 log10 from less than 0.5 log10 in unmodified
filters [29]. Zhuang and Jin [11] report significant
reduction and inactivation of MS2 and ϕX-174 in sand
columns coated with aluminium oxide (up to 2.0 log10
over low pore volumes in phosphate-buffered saline),
and that ionic strength and composition play a major
role in the extent of sorption and inactivation of bacte-
riophages through electrostatic forces.
Although granular media filtration and microbial
transport through porous media have been the areas
of focus for the majority of studies relating to virus
sorption and inactivation through contact with
surfaces amended with metal oxides, virus sorption is
an important mechanism in other applications as
well. Viruses can be removed from water through
contact with positively charged filter and media
surfaces, a phenomenon that is well established in
the literature on virus detection, fate and transport
[10,12,26,56–59]. The fact that adsorbed viruses can
also be inactivated by some forms of this process
makes this property potentially useful for water treat-
ment applications. Advances in approaches to virus
sorption and inactivation by contact with positively
charged surfaces have suggested several applications
in water and wastewater treatment, including zerova-
lent iron-based treatment [60] and reaction with
quaternary ammonium silane polymers coated on to
granular medium surfaces [61].
Porous ceramic or crushed ceramic media filters
may need to be modified chemically in some way if they
are to provide an effective barrier against waterborne
 Environmental Technology
viruses to the extent required by protocols issued by
NSF-International (99.99% reduction) [62] and the US
EPA [63], or to reduce the disease risk of drinking water
to meet the health-based target of 10−6 Disability-
Adjusted Life Years (DALYs) per person per year as
recommended by the World Health Organization [64].
Initial testing of locally produced ceramic filters and
ceramic granular media filters has shown bacteriophage
reductions in drinking waters from 0% to 99% [65–67].
Modified filter surfaces may provide a more effective
barrier against viruses in water, however, if they medi-
ate, promote or facilitate virus sorption and/or inactiva-
tion. Many ceramic water filters in operation today use
chemical augmentation, such as silver preparations
impregnated into the filter [68], to enhance the virus
Downloaded by [UGR-BTCA Gral Universitaria] at 08:33 05 March 2015
reduction capacities of the filter. The extent of effective-
ness against viruses and the mechanisms governing the
anti-viral effects of these additives are not well charac-
terized, however. Metal oxide additives that are posi-
tively charged or have other virus retention or
inactivation properties may be an option to consider in
existing ceramic microfiltration. The microbiological
effectiveness of small-scale granular media filters such
as ceramic grog filters, crushed brick filters and sand
filters may also be improved through the incorporation
of low-cost ceramic media enhanced with positively
charged metal oxides. Media amended with metal oxide
may increase the effectiveness of these filters against
viruses.
This work has been the first to examine the incorpo-
ration of Fe and Al oxides into ceramic media (before
firing) to enhance the reduction capacity of viruses
through sorption processes. This method of applying
metal oxides to media that can be used in granular
media or low-cost microfiltration technologies (e.g.
ceramic filters) may be advantageous for use in devel-
oping countries, where raw mineral oxides are widely
available and used in the manufacture of ceramics.
Acknowledgements
The authors wish to thank Thomas Clasen, Otto Simmons,
Dave Love, Douglas Wait, Christine Stauber and Sharon
Nappier for their kind assistance.
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