ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 21 (2004) 1–10
www.elsevier.nl/locate/jnlabr/yfmic

The efficacy of sand-immobilized organoclays as filtration bed

materials for bacteria
P. Herrera, R. Burghardt, H.J. Huebner, T.D. Phillips*
Veterinary Anatomy & Public Health, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4458, USA
Received 6 January 2003; accepted 12 May 2003

Abstract

Previous research has shown that montmorillonite clays exchanged with cationic surfactants including cetylpyridinium (CP),
hexadecyltrimethylammonium (HDTMA), and cetyldimethylethylammonium (CDEA) were effective in reducing Salmonella
enteritidis colony counts in bulk antibacterial assays. In order to increase the porosity (and hydraulic conductivity) of these materials
for use in filtration beds, organoclays were tightly adhered and immobilized onto the surface of sand. Bulk antibacterial assays
performed with 10 mg of the sand-immobilized organoclay (S/IOC) showed that the composite materials retained their antibacterial
activity after processing. Reductions in plate colony counts ranged from 16.5% for sand-immobilized CP-exchanged sodium
montmorillonite to 83.9% for immobilized CDEA-exchanged low pH montmorillonite. The sand/adhesive matrix and sand-
immobilized activated charcoal failed to produce any significant decreases. Pilot studies with columns containing 1.0 g of S/IOC
showed that maximal filtration efficiency was obtained at a filtration rate of 1.0 ml/min. Initial column studies with Salmonella
suspensions at a concentration of 3.0
108 cfu/ml, produced reductions in colony plate counts varying from 28.5% for immobilized
CP-exchanged calcium montmorillonite to 59.5% for immobilized CDEA-exchanged low pH montmorillonite. The sand/adhesive
matrix and immobilized activated charcoal yielded much lower reductions (7.6% and 10.4%, respectively). Studies with Escherichia
coli suspensions (3.0
107 cfu/ml) indicated reductions varying from 59.3% for immobilized CP-exchanged calcium montmorillonite
to 99.9% for immobilized CDEA-exchanged low pH montmorillonite. These initial results suggest that S/IOC can be effective in
reducing microbial numbers in wastewater following some modifications.
r 2003 Elsevier Ltd. All rights reserved.

Keywords: Montmorillonite clay; Organoclay; Wastewater reconditioning; Escherichia coli; Salmonella enteritidis; Water treatment; Filtration bed

1. Introduction One area of interest involves the reconditioning and

Water is used in many steps of carcass production in
the poultry industry, including washing carcasses and
equipment, scalding baths to facilitate hair and feather
removal, and chilling baths to quickly cool the carcasses
in preparation for refrigeration (Hubbert and Hagstad,
1986). As potable water is becoming increasingly scarce,
the cost of obtaining usable water and disposing of
waste water represents a substantial economic expendi-
ture. Interest in developing methods of water deconta-
mination and conservation are becoming increasingly
important (Hamza et al., 1978; Chang and Sheldon,
1989).
*Corresponding author. Tel.: +1-979-846-6414; fax: +1-979-862-
4929.
E-mail address: tphillips@cvm.tamu.edu (T.D. Phillips).
recycling of water. Initial studies involving the recondi-
tioning of water used in carcass processing have focused
on filtration, flocculation, or flotation to remove
particulate matter followed by chemical disinfection
(Palumbo et al., 1997). A major concern of meat
processors who utilize recycled water is the removal of
pathogenic bacteria, especially Escherichia coli and
Salmonella, to prevent cross-contamination of carcasses.
The Food Safety and Inspection Service (1998) (FSIS)
allows the use of recycled water when reconditioning
produces at least a 60% reduction in total micro-
organisms and when visible light transmission of the
treated water is at least 60% of fresh water (CFR, 1998).
At this level of treatment, 1.75 gallons (6.62 l) of the
reconditioned water can be used to replace 1 gallon
(3.79 l) of fresh water. Greater reductions in bacterial
counts and increases in light transmission allow the

0740-0020/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0740-0020(03)00050-9
 ARTICLE IN PRESS
2 P. Herrera et al. / Food Microbiology 21 (2004) 1–10
replacement of fresh water with smaller quantities of
reconditioned water. At 90% reduction of bacterial
counts and 80% light transmission, 1.25 gallons (4.73 l)
of reconditioned water can be used to replace 1 gallon
(3.79 l) of fresh water.
The use of montmorillonite clays for the adsorption,
filtration and sequestration of natural and anthropo-
genic contaminants is longstanding. Their high surface
area, high cationic exchange capacity, and the ability to
‘‘swell’’ in aqueous environments (which enhances
access to interlaminar surfaces) make them ideal
adsorbents for multiple applications (Browne et al.,
1980; Friebele et al., 1980). However, certain traits of
these clays make them less than ideal in removing
bacteria from water. The swelling of montmorillonites
under aqueous conditions decreases their hydrodynamic
conductivity thus limiting their use as filtration media.
For this reason, montmorillonites have been used as
barrier materials to prevent the movement of water-
borne contaminants (Allen-King et al., 1995). Also,
under physiologic conditions the surfaces of many
aluminosilicate minerals as well as bacteria are nega-
tively charged (Stotzky, 1989). This represents a large
energy barrier to the adsorption of bacteria to the clay
particle surface.
The surface chemistry of montmorillonite clays can be
altered by exchanging the predominant interlaminar
cations with organic materials that are positively
charged (Koh and Dixon, 2001). For example, the
exchange of cations within the gallery of montmorillo-
nite clays with cationic surfactants having long alkyl
chains results in the production of hydrophobic
organoclays. As the concentration of organic cations
exchanged onto the clay increases, hydrophobic inter-
actions between the alkyl chains attract more organic
cations to the clay surface. The further addition of
organic cations in excess of the exchange capacity of the
clay, results in an organoclay with a positive (instead of
negative) surface charge (Xu et al., 1997). These
organoclays have proven useful in the adsorption of
aromatic and halogenated hydrocarbons as well as other
organic contaminants that the parent (hydrophilic) clays
would not normally sequester.
Quaternary ammonium compounds (QAC) are a class
of organic cations sharing a common structure: a
positively charged ammonium moiety and one or more
hydrophobic alkyl chains of varying length (Merianos,
1991). The combination of positive charge and hydro-
phobic alkyl chains gives the compounds wetting and
cleansing properties. These compounds are also prized
for their antimicrobial activity. Cetylpyridinium (CP) is
one of the most common and widely used QAC. It is the
active ingredient in the mouthwash Cepacolr and is also
used as a topical antiseptic and a preservative in
pharmaceutical preparations. Recently, a patent was
granted for the use of CP as a spray on poultry and beef
carcasses to prevent foodborne microbial contamination
(Compadre et al., 1999).
Previous research in this laboratory has shown that
exchanging montmorillonite clays with CP increases
their ability to sorb and remove bacteria from aqueous
suspension in bulk adsorption studies (Herrera et al.,
2000). Other compounds that were found to be effective
are: hexadecyltrimethyl-ammonium (HDTMA) and
cetyldimethylethylammonium (CDEA). Initial attempts
to use organoclays in filtration columns failed because
the flow of the aqueous filtrate decreased over time, in
spite of increasing head pressure applied to the column.
To address this problem, water permeable clay-based
composites have been constructed for the remediation of
groundwater contaminated with chlorophenols and
polyaromatic hydrocabons (Ake et al., 2001). Based on
this work, bacteriocidal organoclays were immobilized
onto the surface of sand (as a solid support) to produce
a porous composite media for the filtration and
remediation of bacteria. The major goals of this study
were to characterize the filtration efficacy and anti-
bacterial activity of these sand-immobilized clays
(S/IOCs). Charcoal-based composites were compared
with S/IOCs to delineate any differences.
2. Methods
2.1. Materials
Salmonella enteritidis phage type 13A was obtained
from the National Veterinary Services Laboratory in
Ames, Iowa. Escherichia coli TH008 was provided by
Dr. K.C. Donnelly, College of Veterinary Medicine,
Texas A&M University, College Station, Texas. Two
clays used in these studies, STx-1 (a calcium montmor-
illonite) and SWy-2 (a sodium montmorillonite), were
obtained from the Source Clay Minerals Repository at
the University of Missouri (Columbia, Missouri).
Additional clays tested included low pH montmorillo-
nite (LpHM) from Engelhard Corporation (Jackson,
Mississippi) and acid-activated montmorillonite
(AAM) from Kaiser Aluminum & Chemical Corpora-
tion (Pleasanton, California). Activated charcoal (AC),
Norit A was purchased from Aldrich Chemical
Company (Milwaukee, Wisconsin). The QACs used
included: cetylpyridinium chloride (CP) and cetyldi-
methylethylammonium bromide (CDEA) from ICN
Biomedical (Costa Mesa, California) and hexadecyltri-
methylammonium chloride (HDTMA) from Fluka
(Ronkonkoma, New York). The commercially prepared
mucilage adhesive used in these experiments was
obtained from American Glue Company (Taylor,
Michigan). White quartz sand (–50 +70 mesh) was
purchased from Sigma Chemical Company (St. Louis,
Missouri).
 ARTICLE IN PRESS
P. Herrera et al. / Food Microbiology 21 (2004) 1–10
2.2. Preparation of organoclays
Parent clays were ground and sieved to a uniform
particle size of o45 mm. The clays were then washed in
deionized water at a ratio of 5 g clay:500 ml water for 24 h
under agitation. The resulting clay suspensions were
centrifuged and the wash water discarded. All clays were
rehydrated with 500 ml water to which the cationic
surfactant was added at an amount in excess of 1.5 times
the cation exchange capacity of the clays (based on
90 cmolec/kg). The resulting mixture was exchanged under
agitation for 24 h. The exchanged clays were centrifuged,
the supernatant discarded, and the clay washed for 24 h
with 500 ml deionized water. After centrifugation and
removal of the wash, the material was dried at 50 C for
24 h, then ground and sieved to a size of o45 mm.
2.3. Preparation of sand-immobilized organoclay
(S/IOC)
Sand (10 g) was placed into a glass mortar. The
bonding adhesive was diluted 1:1 with deionized water;
750 ml of this adhesive was then added to the sand. This
mixture was stirred with a pestle until the sand was
uniformly wetted by the adhesive. Organoclay (1 g) was
added to the sand/adhesive mixture and mixed until the
clay was incorporated. The resulting S/IOC composite
was transferred into a glass Petri dish (100
15 mm) and
placed in an oven at 50 C for 12 h. Once dry, the S/IOC
composite was ground and sieved to a uniform particle
size (between 212 mm and 300 mm). The S/IOC compo-
site was transferred into a glass Petri dish and washed
under gentle agitation for 5 min with three 20 ml
aliquots of deionized water. After washing, the final
product was dried in an oven at 50 C for 12 h.
2.4. Nomenclature of the organoclays
The four clays used in these experiments included:
STx-1, SWy-2, LpHM, and AAM. The three QACs used
were: CP, CDEA, and HDTMA. To denote which
compound was exchanged onto which clay, the abbre-
viation for the QAC was separated from the clay with an
asterisk (i.e., CP-exchanged AAM is designated
CPAAM). If a material was immobilized onto sand,
it is preceded by the abbreviation ‘‘S/’’. Thus immobi-
lized CP-exchanged AAM appears as S/CPAAM.
When discussing immobilized organoclays in general,
they are designated as S/IOC. Sand immobilized
activated charcoal and the sand/adhesive matrix are
denoted as S/AC and SA, respectively.
2.5. Visible light microscopy
Samples of sand-immobilized AC and HDTMALpHM
(100 mg) were dispersed on glass microscope slides for
3
microscopic analysis. The images were taken with a
10
objective and brightfield illumination using
a Zeiss Axioplan 2 Microscope (Carl Zeiss, Inc.,
Thornwood, New Jersey) interfaced with a Hamamatsu
3 CCD color camera (Hamamatsu Corporation,
Bridgewater, New Jersey) using Adobe Photoshop 5.0
(Adobe Systems, Seattle, Washington) image capture
software.
2.6. Filtration column preparation
An inert porous frit was placed into a disposable
borosilicate glass tube (14.5 cm
0.5 cm i.d.). Sand
(200 mg) was then added to the tube. The filtration
material to be studied (1 g) was layered onto the sand.
This was topped with another 200 mg of sand.
2.7. Preparation of standardized bacterial suspensions
Methods used in the preparation and plating of the
standardized bacterial suspensions were modifications
of procedures from a previously published study (Hargis
et al., 1995). Sterile Trypticase Soy Broth (60 ml) was
inoculated with either S. enteritidis or E. coli and
incubated overnight at 37 C. The culture was centri-
fuged at 5000 rpm for 10 min. The media was removed
and the pellet resuspended in 30 ml of a sterile 0.9%
saline solution. The bacteria were then centrifuged
again. This washing step was repeated two more times.
Based on the results of standardized curves, the resulting
bacterial suspensions were carefully diluted until visible
light absorbance (l ¼ 625 nm) was equal to a value of
0.40 for S. enteritidis or 0.35 for E. coli when measured
by a Beckman DU-65 spectrophotometer. This pro-
duced roughly 200 ml of a S. enteritidis suspension with
a concentration of approximately 3.0
108 colony
forming units (cfu) per ml or an E. coli suspension with
approximately 4.0
107 cfu/ml. To produce suspensions
with varying bacterial concentrations, serial 10-fold
dilutions with sterile 0.9% saline were performed.
2.8. Bulk antibacterial assays
Sand-immobilized organoclay (10 mg) or non-immo-
bilized organoclay (1 mg) were weighed out into a set of
four test tubes. A set of four tubes with no clay materials
served as controls. Two ml of the S. enteritidis
suspension with a concentration of approximately
3.0
108 cfu/ml was then added to the tubes. The tubes
were agitated for 1 h at 600 rpm at 25 C. The tubes were
then centrifuged for 10 min at 500 rpm. This speed was
determined in pilot studies to remove the clay particles
from suspension without significantly affecting the
bacterial counts (data not shown). The supernatants
were removed from the clay pellets. Aliquots (10 ml) were
diluted by two serial hundred-fold dilutions. These were
 ARTICLE IN PRESS
4 P. Herrera et al. / Food Microbiology 21 (2004) 1–10
further diluted fivefold and 50 ml aliquots were plated on
modified Brilliant Green Agar containing 20 mg nali-
dixic acid and 25 mg novobiocin per liter of prepared
agar. The plates were incubated at 37 C for 24 h, after
which the number of colonies on each plate was
counted. Reductions in bacterial counts were measured
by comparing the experimental plate counts to the
controls. Significant differences between the individual
treatments and controls were determined by the
Student’s t-test ðP ¼ 0:05Þ:
2.9. Filtration column studies
The bacterial suspension with the desired initial
concentration was transferred into a sterilized filtration
flask. Ten ml of the suspension was retained for later use
as the control. Sterile nalgene tubing was immersed in
the bacterial suspension and passed through a rubber
stopper in the mouth of the flask, connecting the flask to
the column. The column was mounted on a Retriever IV
fraction collector set to collect 1 ml of filtrate per tube.
The flask was connected via the side connector to a
regulated source of compressed nitrogen, which was
used to continuously feed the bacterial suspension to the
test columns (Fig. 1). Unless otherwise specified, the
flow rate of the bacterial suspension through the column
was 1 ml/min. Every sixth tube on the fraction collector
was retained for sampling. The control suspension was
diluted by serial 10-fold dilutions with sterile 0.9%
saline to obtain a bacterial concentration of approxi-
mately 3.0
104 cfu/ml. The same dilution factor was
applied to the samples. The resulting dilutions were
further diluted fivefold. An aliquot (50 ml) of this final
dilution was plated on modified brilliant green agar.
After incubation at 37 C for 24 h, the percent reductions
in plate counts were calculated by comparing the
experimental plates to the controls. Bacterial reduction
Fig. 1. Diagram of sand-immobilized organoclay filtration column
setup.
in the samples was also measured by visible light
transmittance at a wavelength of 625 nm. Significant
differences between the individual treatments
and controls were determined by the Student’s t-test
ðP ¼ 0:05Þ:
2.10. S/IOC column breakthrough studies
The procedures for the breakthrough studies followed
those of the filtration column studies with the following
modifications. Filtration columns were prepared using
0.5 g of the sand-immobilized organoclays. A standar-
dized Salmonella suspension (3.0
107 cfu/ml) was sent
through the columns at a flow rate of 1 ml/min for 3 h.
Collection of samples and plating were performed in the
same fashion as the filtration column studies. Percent
reductions in plate counts were plotted against filtrate
volume to produce filtration curves. The linear portions
of the curves were formula fitted using TableCurve 2D
(Jandel Scientific Software) and the 50% breakthrough
volume was calculated. By multiplying the breakthrough
volume by the bacterial suspension concentration, a
50% breakthrough bacterial load was calculated for the
S/IOC column.
3. Results
The technique used to adhere organoclays and AC
onto the surface of sand produced composites displaying
a heterogeneous distribution of adsorbent. Upon visible
light microscopic analysis, it was noted that not all sand
particles were covered with organoclay or activated
charcoal (Fig. 2). As seen in the figure, the immobilized
materials appear as the darker areas on the translucent
particles of sand. In both photographs the immobilized
sorbent materials appear as the darker areas on the
translucent sand particles. The distribution of the
sorbent materials on the sand/adhesive matrix (SA)
was not uniform. Generally, bonded sorbent materials
were observed on the surface, in crevices or as aggreg-
ates at the edges of sand grains. The amount of organo-
clays immobilized in the finished batches of S/IOCs varied
between 0.26 and 0.79 g/10 g of sand.
In order to determine whether sand immobilization
affected the ability of the organoclays to reduce
bacterial counts, non-immobilized organoclays (1 mg)
and S/IOCs (10 mg) were compared using bulk anti-
bacterial assays. The amounts of the materials used in
this study contained approximately the same amount of
organoclay, assuming complete adherence of the orga-
noclays to the sand. The sand/adhesive matrix (SA) was
included to determine the antibacterial potential of the
matrix material upon which all other adsorbent materi-
als were based (data not shown). Non-immobilized and
sand-immobilized AC were included, given charcoal’s
 ARTICLE IN PRESS
P. Herrera et al. / Food Microbiology 21 (2004) 1–10 5

Fig. 2. Light micrographs (100
) of sand-immobilized activated charcoal (left) and HDTMALpHM (right). In both photographs, the
immobilized sorbent materials appear as the darker areas on the translucent sand particles.

Immobilized Non-Immobilized 3.00E+04 3.00E+06

3.00E+05 3.00E+07
120
3.00E+08
100
120
Percent Reduction

80
Percent Reduction (CFU/Plate)
100
60

80
40

20 60

0 40
AC

CP*SWy-2

CP*AAM

CP*STx-1

HDTMA*LpHM

CDEA*LpHM

20

0
Fig. 3. Bulk binding studies performed with Salmonella enteritidis 0 20 40 60 80 100 120
(3.0
108 cfu/ml) and non-immobilized or sand-immobilized organo- Eluate Volume (ml)
clays. 1 mg of non-immobilized organoclay and 10 mg of the
corresponding S/IOC were used since they would contain equivalent Fig. 4. Pilot column studies performed with immobilized S/CPAAM
amounts of organoclays (assuming complete efficiency of the at a flow rate of 1.0 ml/min and varying Salmonella enteritidis filtrate
immobilization process). concentrations. Filtration efficiency was shown to be concentration
dependent.

ability to bind a wide variety of organic compounds. exhibited greater reductions in plate counts (59.1% and
The organoclays tested included: CP-exchanged AAM 83.9%, respectively).
(CPAAM), CP-exchanged STx-1 (CPSTx-1), CP- To determine the optimal conditions under which
exchanged SWy-2 (CPSWy-2), HDTMA-exchanged filtration studies should be conducted, pilot studies were
LpHM (HDTMALpHM), and CDEA-exchanged run with columns packed with S/CPAAM at a filtrate
LpHM (CDEALpHM). The study was performed flow rate of 1 ml/min, and S. enteritidis suspensions with
with an initial S. enteritidis concentration of concentrations ranging from 3.0
104 to 3.0
108 cfu/
3.0
108 cfu/ml. Reductions in bacterial counts were ml. It was determined that filtration efficiency was
measured by colony plate counts (Fig. 3). Results show dependent on bacterial concentration (Fig. 4). In the
that non-immobilized AC and organoclays produced studies where bacterial concentrations ranged from
near complete reductions in bacterial counts. S/AC and 3.0
104 to 3.0
106 cfu/ml, the columns produced
SA failed to produce any detectable reduction under the near total reduction in plate counts over the time
conditions tested. The sand-immobilized organoclays period tested. With a S. enteritidis concentration of
exhibited fair to good antibacterial activity. The 3.0
107 cfu/ml, reductions in plate counts were initially
CP-exchanged organoclay composites (i.e. S/CPSWy- very high. However, filtration efficiency decreased
2, S/CPAAM, and S/CPSTx-1) produced 16.6%, sharply over time. At a concentration of 3.0
108 cfu/
22.4%, and 24.1% reductions in colony plate counts, ml, percent reduction in plate counts remained relatively
respectively. S/HDTMALpHM and S/CDEALpHM stable and averaged 39.2%. Given the results of the
 ARTICLE IN PRESS
6 P. Herrera et al. / Food Microbiology 21 (2004) 1–10

80 80

Parent Clay Parent Clay

Percent Light Transmission (@625 nm)

Exchanged Clay
Exchanged Clay
Percent Reduction (Colonies/Plate)

60

70

40

60
20

0
SA

AC

CP*STx-1

HDTMA* LpHM

CP*AAM

CP*SWy-2

CDEA*LpHM
50

SA

AC

CP*STx-1

HDTMA* LpHM

CP*AAM

CP*SWy-2

CDEA*LpHM
Fig. 5. Column studies performed with Salmonella enteritidis
Fig. 6. Column studies performed with Salmonella enteritidis
(3.0
108 cfu/ml) and columns filled with sand-immobilized parent
(3.0
108 cfu/ml) and columns filled with sand-immobilized parent
and exchanged clays. Reductions in bacterial loads were measured by
and exchanged clays. Reductions in concentration of bacteria in
plate counts. The exchanged clay columns exhibited a greater ability to
suspension were measured by changes in visible light transmission. In
reduce bacteria counts than the parent clay columns. However, none of
general, the majority of the columns was able to produce filtrates with
the columns produced 60% reduction in plate counts.
60% light transmission.

S/CPAAM column studies, a bacterial concentration 100

of 3.0
108 cfu/ml was used for steady-state filtration

Percent Reduction (CFU/Plate)

studies and 3.0
107 cfu/ml was used as a starting point 80

for breakout filtration studies.

60
The column studies were repeated with a standardized
Salmonella suspension (3.0
108 cfu/ml), a flow rate of 40
1 ml/min, and columns filled with immobilized parent
and exchanged clays. Reductions of bacteria counts 20
were measured by the standardized plating method and
by changes in visible light transmission (l ¼ 625 nm). 0
SA

S/AC

S/CP*STx-1

S/CP*AAM

S/HDTMA*LpH M

S/CP*SWy-2

S/CDEA*LpHM
Fig. 5 shows the results of the filtration column studies
as measured by colony plate counts. SA and S/AC
columns produced 7.6% and 10.4% reductions in plate
counts, respectively. The parent clay columns produced
less of a reduction in plate counts as compared to either Fig. 7. S/IOC column studies performed with Escherichia coli
the SA or S/AC columns. Reductions measured by plate (4.0
107 cfu/ml). Reductions in the concentration of bacteria in
counts ranged from 1.7% (S/STx-1) to 5.9% (S/SWy-2). suspension was measured by colony plate counts. Except for the S/
CPSTx-1 column, all the exchanged clay columns produced 60%
The immobilized organoclay columns produced reduc- reductions in plate counts.
tions significantly greater than either the SA, S/AC, or
parent clay columns. Percent reductions measured by
colony counts ranged from 28.5% (S/CPSTx-1) to
59.5% (S/CDEALpHM). produced stable filtration curves. All other aspects of
Fig. 6 shows the results of the filtration column these studies were the same as the Salmonella column
studies as measured by visible light transmission. In studies. Both the SA and S/AC columns exhibited fair
general, most of the immobilized materials tested ability to reduce bacterial loads as measured by plate
produced filtrate with light transmission greater counts, 17.2% and 16.1%, respectively (Fig. 7). The
than 60%. The notable exceptions being, SA (53.3%), sand-immobilized organoclay columns produced reduc-
S/SWy-2 (55.8%), S/CPSTx-1 (57.0%), and tions ranging from 59.3% (S/CPSTx-1) to 99.9%
S/HDTMALpHM (55.7%). (S/CDEALpHM). Fig. 8 shows the results of the E.
The column studies were repeated with immobilized coli column studies as measured by visible light
organoclay columns and a standardized E. coli suspen- transmission. All the columns tested, except SA
sion. Pilot studies performed with E. coli suspensions (53.3%), produced filtrates with visible light transmis-
showed that an initial concentration of 4.0
107 cfu/ml sion that was at least 60% of fresh water.

90
Percent Light Transmission (@ 625 nm)
80
70
60
50
SA
S/AC
Fig. 8. S/IOC column studies performed with Escherichia coli
(4.0
107 cfu/ml). Reductions in the concentration of bacteria in
suspension was measured by visible light transmission (at 625 nm). All
S/IOC columns produced filtrates with 60% visible light transmission.
100
Percent Reduction (CFU/Plate)
80
60
40 S/CP*SWy-2
S/CP*AAM
20
S/HDTMA*LpHM
S/CP*STx-1
0 incomplete immobilization of the organoclay on the
0 30
Fig. 9. S/IOC column breakthrough studies performed with Salmo-
nella enteritidis (3.0
107 cfu/ml). Percent reductions in the bacterial
plate counts were plotted against filtrate volume. The plots were
formula fitted in TableCurve 2D and the 50% breakthrough volume
was calculated. The calculated 50% breakthrough bacterial loads
ranged from 3.6
109 cfu (S/CPSWy-2) to 8.1
109 (S/CPSTx-1).
Salmonella breakthrough studies were performed with
an initial concentration of 3.0
107 cfu/ml. Percent
reductions in the bacterial plate counts were plotted
against the filtrate volume (Fig. 9). From the resulting
filtration curves, the volume at which 50% of the initial
bacterial concentration broke through was determined.
The calculated 50% breakthrough bacterial loads for the
sand-immobilized organoclays were: S/CPSWy-2
(3.6
109 cfu), S/CPAAM (7.1
109 cfu), S/HDTMA
LpHM (7.4
109 cfu), and S/CPSTx-1 (8.1
109 cfu).
4. Discussion
We found that organoclays and AC are incompletely
immobilized and unevenly dispersed on the surface of
sand particles using the experimental methods described
ARTICLE IN PRESS
P. Herrera et al. / Food Microbiology 21 (2004) 1–10 7
in this study (Fig. 2). The amount of organoclay
immobilized to the sand matrix differed between
batches. A partial cause for this variation was due to
sieving and washing steps designed to remove non-
bonded clay particles, which was necessary to prevent
blockage of the S/IOC columns and maintain the flow.
Although a rank order in the antibacterial activities of
the various immobilized materials were observed in
these studies, it would be premature to assign these
differences directly to the activity of the various
surfactant/clay combinations. The difference in activity
S/CP*STx-1
S/CP*AAM
S/HDTMA*LpHM
S/CP*SWy-2
S/CDEA*LpHM
may be related to variations in the amount of
organoclay immobilized in the various batches. Before
developing indices for comparing the antibacterial
activities of S/IOC materials, it will be necessary to
standardize the immobilization process and adjust for
variations in organoclay content in the final composite
products.
Previous studies showed that CP-exchanged mon-
tmorillonites exhibited antibacterial activity under bulk
binding conditions (Herrera et al., 2000). In this study, it
was determined that HDTMA and CDEA exchanged
LpHM were also effective in reducing bacterial loads.
Near total reductions in bacterial plate counts were
produced with 1 mg of the non-immobilized organoclays
(Fig. 3). Significantly lower reductions were produced
with 10 mg of the S/IOCs. Since the amounts of
available organoclays should be comparable in both
cases, part of this difference can be attributed to the
60 90 120 150 180
sand matrix. It was also noted that non-immobilized AC
Eluate Volume (ml)
produced near complete reductions similar to the
organoclays, but S/AC failed to produce any detectable
reduction.
The initial column experiments suggested that filtra-
tion efficiency of the S/IOC columns was dependent on
the concentration of suspended bacteria (Fig. 4). When
Salmonella concentrations were less than 3.0
106 cfu/
ml, the columns produced near total reduction in plate
counts. The filtration efficiency of the S/IOC columns
decreased sharply with increasing bacterial concentra-
tion. These findings suggest that the S/IOC columns
would be better suited to applications where the
remediation of low-to-moderate bacterial loads is
required.
Filtration column studies were then performed using
columns of immobilized parent and exchanged clays and
a standardized Salmonella suspension (3.0
108 cfu/ml).
The filtration efficiencies produced by the SA column
were less than 8% when measured by plate counts. This
suggests that about 8% of the reduction produced by
the S/IOC columns may be attributed to filtration by the
sand/adhesive matrix. In comparison, the S/AC column
produced filtration efficiencies similar to the SA column,
but significantly less than all the immobilized organo-
clays tested. The filtration efficiencies of the parent clays
 ARTICLE IN PRESS
8 P. Herrera et al. / Food Microbiology 21 (2004) 1–10
columns were much less than those of the SA and S/AC
columns. All the immobilized organoclay columns tested
exhibited fair to good ability to reduce bacterial counts
as measured by plate counts (Fig. 5). However, none of
the S/IOC columns tested produced the 60% reduction
in bacterial loads needed to comply with FSIS regula-
tions. In contrast, the majority of S/IOC columns tested
produced the necessary change in visible light transmis-
sion required for regulatory compliance (Fig. 6).
It was further noted in these column studies that
reductions as measured by plate counts were greater
than the reductions measured by visible light absorbance
(data not shown). These findings are consistent with
those of previous studies performed in this laboratory
(Herrera et al., 2000). In establishing standard curves for
the standardized bacterial suspensions, it was found that
visible light absorbance values were highly correlated to
the concentration of bacteria in suspension. Therefore if
reductions in bacterial concentration were solely due to
the removal of cells from suspension, then the changes in
absorbance values should closely mirror the changes in
plate counts. The greater reductions observed in the
plate counts suggest that the antibacterial activity of the
S/IOC columns is not solely due to the filtration of
bacterial cells. The findings suggest that the columns
also act to render some of the bacterial cells remaining in
suspension unculturable.
The E. coli column studies were performed with
immobilized exchanged clay columns and a standar-
dized suspension (3.0
107 cfu/ml). With the exception
of S/CPSTx-1, the S/IOC columns produced the 60%
reduction in bacterial plate counts necessary for
compliance with FSIS regulations (Fig. 7). All of the
S/IOC columns met the regulatory requirements for
changes in visible light transmittance (Fig. 8). The
filtration efficiencies produced by the SA and the S/AC
columns were approximately 16–17% as measured by
plate colony counts. While it is possible that E. coli was
more efficiently removed by the S/IOC columns as
compared to Salmonella, the differences in the filtration
efficiencies may be attributed to the filtration system’s
dependence on bacterial concentration (i.e. the standar-
dized E. coli suspension was one-tenth as concentrated
as the Salmonella suspension).
The breakthrough studies performed with Salmonella
suggested that the S/IOC columns have a large capacity
for bacterial loads (Fig. 9). For 0.5 g of S/IOC, the
calculated 50% breakthrough bacterial loads ranged
from 3.6
109 cfu (S/CPSWy-2) to 8.1
109 (S/
CPSTx-1).
The filtration of bacteria in coarse sandy soils involves
two mechanisms: straining and adsorption (Huysman
and Verstraete, 1993). Straining is the capture of
particles in the interstitial space between matrix
particles; whereas, adsorption involves the capture of
bacteria on the surface of the adsorbent particles by
physiochemical forces. Hydrophobic and electostatic
interactions, van der Waals forces, and cation bridging
have been implicated as the major forces associated with
the attachment of bacteria to soil particles (Truesdail
et al., 1998; Sposito, 1984). Bacteria have strain-specific
differences in surface hydrophobicity and electrostatic
charge and thus vary in the surfaces to which they
are attracted (Dickson and Koohmaraie, 1989; Van
Loosdrecht et al., 1987). In sandy soils, straining is the
primary mechanism for the capture of bacteria
(Peterson and Ward, 1989). In soils with clay and
or organic materials adsorption becomes important
(McDowell-Boyer et al., 1986).
Activated charcoal is an excellent material to bind a
wide variety of compounds due to its porosity, surface
area, and organophilic nature (Hassler, 1974). AC is
able to absorb small molecules in great amounts due to
its porosity; the majority of the pores in AC are less than
25 nm wide. Salmonella and E. coli are rods with
dimensions of approximately 100
600 nm (Brenner,
1984). Thus, absorption of bacteria by AC is not a
major mechanism. Activated charcoals are hydrophobic
materials and thus preferentially adsorb nonpolar,
nonionized compounds (Hassler, 1974). However, due
the formation of oxygenated compounds on the
charcoal’s surface during the activation process, AC
can also bind polar and electrostatically charged
compounds in lesser amounts.
Montmorillonite clays suspended in aqueous solution
at physiological pHs are negatively charged (Nzengung
et al., 1996). Most bacterial cells under physiological
conditions also have negatively charged surfaces
(Stotzky, 1989). Thus bacteria and clays in suspension
should mutually repel each other. This may explain why
the immobilized parent clays had lower antibacterial
activity than the SA matrix. Exchanging montmorillo-
nites with cationic surfactants produces materials that
are more hydrophobic (Nzengung et al., 1996). Con-
tinuing the exchange process beyond a montmorilloni-
te’s cation exchange capacity, results in the material
undergoing a charge reversal and becoming positively
charged (Xu et al., 1997).
The following mechanisms may account for the
results of these column studies. The sand/adhesive
matrix, which is the basis of the S/IOC materials,
removes bacteria from suspension primarily by strain-
ing. AC should adsorb bacteria primarily by hydro-
phobic interactions. However, the fact that immobilized
AC failed to filter bacteria, beyond what could be
attributed to straining, suggests that partitioning effects
are not major mechanisms for the removal of bacteria
from suspension. Previous studies in our laboratory
determined that the two strains of bacteria used were
highly hydrophilic. It has been noted that as bacteria
become more hydrophilic, electrostatic surface charges
become a more important influence in the mechanism of
 ARTICLE IN PRESS
P. Herrera et al. / Food Microbiology 21 (2004) 1–10
adsorption (Van Loosdrecht et al., 1987). The finding
that the positively charged organoclays were more
effective in adsorbing bacteria than the negatively
charged parent clays, suggest that electrostatic attrac-
tion may be a primary mechanism. The reductions in
colony counts by the S/IOCs is further enhanced by the
antimicrobial activity of the cationic surfactants. This
may explain the finding that filtration was not the only
mechanism involved in the reduction of bacterial plate
counts.
The results of these studies suggest that the use of S/
IOC columns, with minor modification, could be used to
remove bacteria from waste water in order to comply
with FSIS regulations. The antibacterial activity of the
organoclay sorbent could be enhanced by increasing the
amount of cationic surfactant exchanged into the gallery
of the parent clay. Immobilized materials can be made
more efficient by standardizing the immobilization
process to produce S/IOCs with a more uniform
coating. Our results suggest that the antibacterial
efficacy of the S/IOC columns are dependent on the
concentration of bacteria suspended in the filtrate, thus
the columns would be best suited for applications with
low-to-moderate bacterial loads. Poultry processing
wastewater contains a variety of materials, other than
microbes, including suspended solids in the form of
feather dust, grease, blood, and fecal material, and
dissolved solids in the form of proteins and salts (Chang
et al., 1989). S/IOC columns should be used be used as
one of the last steps in water reconditioning and
polishing, following other methods that decrease the
concentration of dissolved and suspended contaminants.
Further studies are warranted to optimize these materi-
als for a variety of applications.
Acknowledgements
Supported by NIH P42 ES04917, USDA 9703230,
and TAES H6215.
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