U.S. patent application number 11/966621 was filed with the patent office on 2008-05-01 for method of water purification.
This patent application is currently assigned to SYDNEY WATER CORPORATION. Invention is credited to Heriberto Alejandro Bustamante, Marilyn E. KARAMAN, Richard Mark Pashley, Sivaraj Shanker.
Application Number | 20080099403 11/966621 |
Document ID | / |
Family ID | 3806690 |
Filed Date | 2008-05-01 |
United States Patent
Application |
20080099403 |
Kind Code |
A1 |
KARAMAN; Marilyn E. ; et
al. |
May 1, 2008 |
METHOD OF WATER PURIFICATION
Abstract
The invention provides a method for the removal of biological
species, such as Cryptosporidium, from water using aluminum based
media which contains surface Al--OH groups.
Inventors: |
KARAMAN; Marilyn E.;
(Aranda, AU) ; Shanker; Sivaraj; (Willoughby,
AU) ; Bustamante; Heriberto Alejandro; (Sylvania,
AU) ; Pashley; Richard Mark; (Aranda, AU) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP;INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W.
SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Assignee: |
SYDNEY WATER CORPORATION
SYDNEY
AU
|
Family ID: |
3806690 |
Appl. No.: |
11/966621 |
Filed: |
December 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09646347 |
Jan 4, 2001 |
7332088 |
|
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PCT/AU99/00173 |
Mar 18, 1999 |
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11966621 |
Dec 28, 2007 |
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Current U.S.
Class: |
210/661 ;
210/669; 210/691 |
Current CPC
Class: |
C02F 1/281 20130101;
B01J 20/08 20130101 |
Class at
Publication: |
210/661 ;
210/669; 210/691 |
International
Class: |
B01D 15/04 20060101
B01D015/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 18, 1998 |
AU |
PP2428 |
Claims
1-26. (canceled)
27. A method for the removal of Cryptosporidium from water
comprising the steps of contacting the water with a medium
comprising particulate alumina having surface Al--OH groups
occurring at an average rate of 1 hydroxyl group per 0.25 nm to 1
hydroxyl group per 0.18 nm.sup.2 for a time from 5 seconds to 1
hour so as to effect at least a 1 log reduction of Cryptosporidium
present in the water by adsorption thereof onto said alumina.
28. The method according to claim 27, wherein the water has been
pre-treated prior to contact with the medium.
29. The method according to claim 27, wherein the medium is
contained in discrete filter beds, each filter bed comprising
particles of alumina in different size ranges.
30. The method according to claim 27, wherein the medium is
contained in a cartridge.
31. The method according to claim 28, wherein the medium contained
in a filter bed is used as a polishing filter.
32. The method according to claim 27, wherein the medium is
contained in a water permeable bag which is immersed in water to
effect reduction of Cryptosporidium.
33. The method according to claim 30, wherein the water is brought
into contact with the medium under gravity flow.
34. The method according to claim 27, comprising alumina having a
particle size of about 0.5 mm to 1.5 mm.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for the
purification of water. More particularly, the present invention
relates to the removal of microbiological contaminants from
water.
BACKGROUND ART
[0002] The presence of microbial pathogens in water bodies, such as
rivers, dams, seawater and swimming pools, where human contact is
likely to occur, or, in water intended for human or animal contact
and/or consumption, is a potential hazard with the potential to
result in illness, disability or even death where these pathogens
are inadvertently ingested by humans or animals, Accordingly, there
exists a variety of methods for their removal so as to render
contaminated water safe for human contact and/or consumption.
[0003] Known methods of removing pathogens from contaminated water
include mechanical filtration, i.e. physical exclusion based on the
size of the microbial pollutants, chemical treatment such as
chlorination and ozonation and electrolysis which generates
oxidants fatal to the pathogens.
[0004] Cryptosporidium can survive up to six months in a moist
environment and have been known to contaminate public swimming
pools. Several outbreaks of cryptosporidiosis due to contaminated
swimming pools have been reported. the contamination is usually due
to faecal accidents in the pool and the spread of infection amongst
pool users can be rapid. This is partly due to the ineffectiveness
of current disinfection procedures. As swimming pools do not
normally monitor for Cryptosporidium, awareness of a problem is
invariably through incriminating epidemiological evidence. In many
cases, cryptosporidiosis manifests as infectious diarrhoea with
risks of complication in the immunocompromised/immunosuppressed
population, for example the very young, the very old, transplant
recipients and those undergoing immunotherapy.
[0005] In the case of raw water, Cryptosporidium oocysts may be
removed by conventional water treatment processes during the
production of potable water. These processes involve coagulation
with coagulants such as ferric chloride or alum followed by
addition of polyelectrolytes as coagulant aids and in some cases
high molecular weight polymeric organic filter aids. The coagulated
material is removed by either sedimentation or filtration through
sand filters.
[0006] Water treatment processes are not completely reliable for
the removal of Cryptosporidium oocysts and in many cases oocysts
breakthrough the plant into the reticulation system. Accordingly
conventional disinfectants, such as chlorine or ozone, are added to
the filtered water prior to reticulation as a precaution in order
to destroy some common water borne pathogenic microorganisms that
may have broken through the water treatment plant. However,
Cryptosporidium oocysts are unaffected by these disinfectants.
[0007] In addition, it is always possible that viable
microorganisms may be introduced between the water treatment plant
and the domestic user. This may occur by sewage infiltration.
[0008] Therefore Cryptosporidium oocysts that enter into the
reticulation system pose extremely serious public health concerns
since no cure exists for cryptosporidiosis.
[0009] In addition to potable water, water in swimming pools, spa
pools and other recreational waters may contain, Cryptosporidium
mainly through faecal contamination introduced by pool users as
well as potentially through the potable water used in the swimming
pools.
[0010] Normally in swimming pools and spas, the water is
continuously filtered through sand filters to remove particulate
material and disinfected by chlorine addition followed by
recirculation. In some instances low concentrations of inorganic
coagulants are added to optimise solids removal. However, if
Cryptosporidium contamination occurs, removal by filtration or
coagulation/filtration through sand filters may not be completely
effective. Detection of oocysts results in pool closure for the
treatment of the water, for example, by superchlorination at a
level of 3-5 mg/L. The efficiency of superchlorination as treatment
for deactivating Cryptosporidium is not guaranteed. The use of
other stronger disinfectants such as ozone, chlorine oxide or
mixtures of strong oxidants has also been tested with mixed
success.
[0011] Thus whilst there are a number of processes well recognised
for the treatment of water sources to produce potable water or to
treat recreational waters, a significant problem remains in the
ability of those processes to produce water, particularly potable
water, that complies with strict regulatory requirements in
relation to pathogenic microorganism content. Regrettably,
conventional water treatment processes have proven unreliable for
their removal from water sources.
[0012] The present inventors have recognised the critical
importance of providing a means by which microorganisms,
particularly pathogenic microorganisms, that may remain after
conventional water treatment processes may be removed prior to the
distribution of the potable water to the end users.
DISCLOSURE OF INVENTION
[0013] The present inventors have now discovered that aluminium
based media possessing surface Al--OH groups provides a means for
the removal of pathogens present in water.
[0014] Accordingly, in a first aspect the present invention
provides a method for the removal of biological species from water
comprising the step of contacting the water with an aluminium based
medium which contains surface Al--OH groups for a time and under
conditions such that a proportion of the biological species are
adsorbed onto said medium and removed from the water.
[0015] In a second aspect, the invention contemplates the use of an
aluminium based medium which contains surface Al--OH groups in the
removal of biological species from water.
[0016] The present inventors believe that for the first time, this
invention provides a practical way to remove dangerous water borne
pathogens, such as Cryptosporidium, using a process which is
readily adaptable to existing water treatment processes.
[0017] Many pathogens possess active surface groups, such as
carboxylate and phosphate groups associated with cellular
glycoproteins, which are available for interaction, for example, by
chemical or electrostatic means, when contacted with active surface
groups of an external medium. One particular pathogen is
Cryptosporidium, which the present inventors have found possesses a
pk.sub.a value of 2.5 suggesting the presence of negatively charged
groups (such as carboxylate or phosphate groups) on the organism's
surface. Additional studies indicated that maximum negative surface
potential of around -27 mV was achieved at a pH greater than 5.7 in
aqueous solution.
[0018] Close contact between surfaces can result in the formation
of chemical bonds between surface sites on the approaching
surfaces. This is called chemisorption and typically occurs between
carboxylate, phosphate and wide range of metal cations such as
aluminium, calcium, iron etc. in natural systems. The precise
nature of these chemical interactions is often complex but may
involve ligand bonding to carboxylate and phosphate groups on the
surface of micro-organisms. In addition, van der Waals forces
generally act to pull colloids together into strong adhesive
contact.
[0019] Thus, as used herein, the terms "adsorb" and "adsorption"
may refer to either electrostatic adsorption or chemisorption.
[0020] The biological species for removal by the method of the
invention include human or animal pathogens such as protozoa
exemplified by Cryptosporidium and Giardia, bacteria exemplified by
Pseudomonas, Escherichia coli, and Vibria cholerae, viruses,
exemplified by poliovirus 1 and coliphage MS-2 and algae.
[0021] A preferred aluminium based medium for use in the invention
is alumina (Al.sub.2O.sub.3) which is hydrated at the surface so as
to form surface Al--OH groups. This material presents a chemically
active substrate for the direct adsorption of suitable biological
species. Surprisingly, the present inventors have found that
surface hydrated alumina has the ability to strongly bind
microorganisms especially protozoa such as Cryptosporidium and
Giardia. It is, however, critically important that the alumina is
in the appropriately hydrated form.
[0022] The alumina may be presented in any number of physical forms
such as powders, granules, crystalline solids, or compressed discs
or wafers and may exist in the amorphous state or as
.alpha.-Al.sub.2O.sub.3 or .gamma.-Al.sub.2O.sub.3.
[0023] Particulate alumina, such as powdered and granulated forms,
provide an increased surface area per volume and are suitable for
packaging into cartridges which can be used alone or in conjunction
with other filtration systems. Powdered and granular alumina is
readily available in different diameter size ranges for example,
from about 15 mm down to about 50 microns (0.05 mm). The size of
the particulate alumina used may be varied depending on the
application. By way of example only, one particulate size range
contemplated by the invention is from about 5 mm to about 1 mm for
example, about 3-2 mm. Another particulate size range is from about
1.5 mm to about 0.5 mm. Yet another particulate size range
contemplated by the present invention is from about 0.5 mm to about
0.05 mm, for example 0.3 mm to about 0.1 mm.
[0024] Depending on the application generally the particle sizes
will be between 500 microns (0.5 mm) to 13 mm. The most suitable
size range will be selected in terms of effective size and
uniformity coefficient.
[0025] In the case of municipal water treatment, usually larger
particles size, typically greater than 1 mm would be preferred in
order to achieve appropriate water throughputs. However, pilot
plant testing may be carried out to establish the optimum
relationship between the thickness of the alumina bed and the
particle size to ensure maximum removal whilst maintaining high
water throughputs.
[0026] Similarly in the case of water treatment for industrial
purposes, such as in the preparation of water for use in the
manufacture of food and pharmaceuticals, relatively large volumes
of water will be treated. Accordingly, a similar approach to
municipal water will usually be adopted. It must however, be
realised that the use of filter cartridges containing the hydrated
alumina may be desirable in some manufacturing facilities.
[0027] In both municipal and private swimming pools applications it
may be appropriate to use finer particles, say between 0.5-2 mm to
maximise collision and capture of biological species by the
particles.
[0028] In the purification of domestic water it would be
appropriate to use smallest particle sizes to both minimise the
size of the filter device and to achieve maximum surface area
whilst ensuring that pressure drops across the filter cartridge
containing the alumina are minimised.
[0029] It is within the scope of this invention to utilise the
hydrated alumina as part of a mixed filter bed. In this form, the
hydrated alumina is generally disposed on the downstream side of
the inflowing water. In this way, the water will preferably have
been conventionally treated prior to contacting the hydrated
alumina. The person skilled in the art will appreciate that the
mixed filter bed may include discrete beds of hydrated alumina of
different particle size ranges.
[0030] It is also important to appreciate that in some
applications, it may be permissible to utilise beds of hydrated
alumina that are fed under gravity.
[0031] In order to maximise the adsorptive capacity of the hydrated
alumina for biological species, preferably the alumina bed will be
used as polishing filter. Thus, in some embodiments of the present
invention it is envisaged that the hydrated alumina will be used as
a separate polishing "monofilter" after the conventional filters
that remove the flocs from the flocculated raw water, In this
configuration it is easier to take the filter off-line when it is
exhausted in order to chemically regenerate the alumina. It must be
recognised that there may be some applications where the alumina
may be used with little or no pretreatment of the inflowing
water.
[0032] Prior to contacting the water with the hydrated alumina, in
the case of the treatment of municipal water, both turbidity and
colour are usually removed by the addition of suitable inorganic
coagulants and organic polyelectrolytes. If the municipal water is
hard, preferably the water will be softened by lime softening,
lime-soda ash softening or excess--lime treatment.
[0033] Furthermore, the hydrated alumina may be used for the
treatment of the supernatant of the backwash water in the
preparation of municipal water, thus ensuring that biological
species such as Cryptosporidium is removed. Backwash water is
generated in water treatment plants by reversing the water flow
through a filter in order to remove the material trapped. The
backwash water is normally decanted and the supernatant may be
recycled to the head of works.
[0034] In a domestic water situation, the water will have already
have been treated by the normal processes as described above.
However it is always possible that viable micro-organisms may
remain in the water supply or may be introduced between the water
treatment plant and the domestic user. This may occur by for
instance sewage infiltration to the reticulation system.
[0035] Whilst swimming pool water is not classified as potable
water it is important that its microorganism contents is kept
within standard limits. This is particularly important in the case
of public swimming pools and spas. In order to maintain water
quality swimming pool water is desirably subjected to filtration
and disinfection. As chlorine is inefficient as a disinfectant
against Cryptosporidium it is important to be able to remove it
from the swimming pool water as the water is being filtered prior
to recirculation.
[0036] An advantage of the present invention is that it may be
readily utilised as an adjunct to existing water treatment
facilities. As mentioned above, in most applications, the hydrated
alumina bed will be used as a final polishing filter. This permits
an existing water treatment facility to be upgraded by retrofitting
an additional stage after the current water treatment stages.
[0037] The aluminium based medium, preferably hydrated alumina, may
be packed into a suitable, high flow rate filtration cartridge and
may, for example, be used as the final stage in a swimming pool
pumping-filtration unit. Alternatively, such cartridges may be used
directly in conjunction with a domestic water reticulation system.
In this form, the cartridge may be fitted to tap(s) from which
drinking water is to be obtained or to the inflow from the
municipal water supply.
[0038] In a domestic situation, it may also be appropriate to use a
bed of hydrated alumina contained within a gravity fed cartridge.
In this situation, water is simply fed under gravity through a
cartridge that is open to receive the water at one end and at the
other end, allows the water to drain into a receiving vessel.
Alternatively, the hydrated alumina may be contained in a water
permeable bag. In this situation, the bag containing the hydrated
alumina is immersed in a vessel of water to be treated for a
suitable contact period.
[0039] For the majority of applications, the contact time between
the aluminium based medium and the water to be treated will be
minimal. Typically contact times of between about 5 seconds and 1
hour will be sufficient to achieve normal removal. The contact time
is, however, dependent on a variety of factors applicable to each
use situation such as the extent of the contamination, the
available surface area of alumina for contact with the water, i.e.
particle size and volume of alumina, the surface density of
hydroxyl groups and the flow rate of water over or through the
alumina. The person skilled in the art will appreciate that a
suitable contact time may be established through appropriate
testing and evaluation.
[0040] The surface density of Al--OH groups on the surface of the
aluminium based media occurs ideally at an average rate greater
than about 1 hydroxy group per 10 nm.sup.2 of surface (1 hydroxy
group per 10 nm.sup.2), preferably greater than about 1 hydroxy per
group 5 nm.sup.2, 1 hydroxy per group 3 nm.sup.2 especially 1
hydroxy per group 2 nm.sup.2. Most preferably, the density of the
surface hydroxy groups occurs at an average rate greater than about
1 hydroxy per group 1 nm.sup.2, especially greater than about 1
hydroxy per group 0.75 nm.sup.2 or about 1 hydroxy per group 0.5
nm.sup.2. When the Al.sub.2O.sub.3 surface is essentially fully
hydrated, thereby providing a maximized surface area available for
adsorption of the biological species to be separated, the average
rate of surface Al--OH groups per nm.sup.2 of surface area, is
about 1 hydroxy per group 0.18 nm.sup.2 to about 1 hydroxy per
group 0.25 nm.sup.2. In general terms, fully hydrated alumina is
most effective for the removal of biological species.
[0041] Because of the nature of the alumina surface, activated
alumina (dehydrated alumina) still contains some hydroxylated sites
for example less than about 1 hydroxy group per 10 nm.sup.2.
However, this material is ineffective in removal of Cryptosporidium
from contaminated water. The introduction of surface Al--OH groups
onto activated alumina is thermodynamically favoured and can be
achieved by hydrating methods known to those skilled in the art,
for example activated alumina may be soaked with water for a
prolonged time. A second method involves treatment with sodium
hydroxide (NaOH), where the upper alumina surface is dissolved thus
allowing other hydroxyl groups to be formed. In a third method, the
activated alumina may be treated by exposure to ultraviolet light
in the presence of water vapour. This process produces ozone which
breaks the Al--O--Al bond allowing the formation of Al--OH. In a
fourth method activated alumina is treated with, the peroxide
produces a hydroxyl radical which attacks the Al--O--Al bond
allowing the formation of Al--OH. These methods may be controlled
to introduce the desired frequency of Al--OH groups over the
surface area. By way of example, only the alumina surface may be
hydroxylated by treatment of the alumina in 1.times.10.sup.-2M NaOH
or in 30% w/v/H.sub.2O.sub.2 for more than one hour or treatment
with ozone in the presence of water vapour.
[0042] In general terms, operation of the invention will result in
at least a 1 log reduction in the biological species present in the
water. In the context of this specification, a log reduction refers
to a 10 fold reduction. For example, if there were 1000
microorganisms per ml in a water sample, a 1 log reduction would
result in 100 microorganisms remaining. A 2 log reduction would
result in 10 microorganisms remaining. Preferably there will be a 2
log reduction, desirably a 3 log reduction, most preferably a 4 log
reduction. It is especially preferred that the invention operates
to such that there is at least a 5 log reduction, particularly a 6
log reduction. Removal of the proportion of the biological species
may be achieved in one treatment or, optionally the process of
contacting the contaminated water with the Al--OH surface may be
repeated to provide the desired level of removal of the biological
species from the water.
[0043] As the following Examples describe, Cryptosporidium oocysts
are found to strongly adsorb onto alumina surfaces containing
surface Al--OH groups. The results indicate that once adsorption
onto the alumina takes place, the oocysts are strongly resistant to
desorption, even in solutions at higher pH values, where the
surfaces will be more negatively charged. Such an independence of
pH is a process safeguard because the oocysts will not be released
on the water due to small pH variations.
[0044] It appears that alumina is a specific substrate for
Cryptosporidium oocyst adsorption. The adsorbed oocyst layer
appears to be stable and resistant to desorption with pH and EDTA
treatment. Without limiting the invention by theory,
Cryptosporidium adsorption may be due to a specific chemisorption
between the alumina surface and either the carboxylate or phosphate
groups or some other groups present on the oocyst surface.
MODES FOR CARRYING OUT THE INVENTION
[0045] The invention will now be described with reference to the
following non-limiting Examples and Figures. In these examples,
removal of Cryptosporidium from water is described. It will of
course be appreciated by persons skilled in the art that other
biological species, particularly microbiological pathogens may be
removed from water using this invention.
BRIEF DESCRIPTION OF DRAWINGS
[0046] FIG. 1 depicts an electron micrograph of a Cryptosporidium
oocyst.
[0047] FIG. 2 is a graphical representation of the change in zeta
potential of Cryptosporidium oocysts in 10.sup.-3M NaCl as a
function of pH.
[0048] FIG. 3 is a photograph of Cryptosporidium oocysts adsorbed
onto polished alumina wafers.
[0049] FIG. 4 schematically depicts a size comparison between
Cryptosporidium oocysts and individual alumina or silica
spheres.
[0050] FIG. 5 schematically depicts the retention of
Cryptosporidium oocysts onto packed and unpacked columns of silica
and alumina.
[0051] FIG. 6 is a photograph of the optically smooth surface of an
alumina wafer having a few extraneous dust particles.
[0052] FIG. 7 graphically depicts the permeate levels of
Cryptosporidium oocysts adsorbed after repeated washings through a
silica column.
[0053] FIG. 8 graphically depicts the permeate levels of
Cryptosporidium oocysts adsorbed after repeated washings through an
alumina column.
EXAMPLES
Materials and Methods
[0054] The .gamma. irradiated bovine Cryptosporidium oocyst samples
were supplied by Australian Water Technologies (AWT) at a
concentration of 5.times.10.sup.6 and 3.times.10.sup.8 oocysts/ml
in distilled water which was stored at 4.degree. C. prior to
use.
[0055] The water used in this study was produced from tap water,
which was fed through a Memtec Krystal Kleen.TM. unit using a
three-stage purification process (prefilter, reverse osmosis and
activated charcoal) before being distilled, collected and stored in
a positive pressure, dust free laminar flow cabinet to prevent air
borne contamination.
[0056] Pure alumina (.alpha.-Al.sub.2O.sub.3) in the form of flat,
optically smooth 4 inch discs, was obtained from Silica Source
Technology, Temple, Ariz., USA.
[0057] Alumina powder (63-200 .mu.m diameter) was obtained from
Merck (art. 1077). The alumina sample was fractionated in water to
separate out only the largest of the particles for column
separation investigations. The purpose of selecting the largest
particles was to prepare a packed column of alumina in which the
pores between the individual spheres would be large compared with
the size of Cryptosporidium oocyst (see FIG. 4). Therefore any
Cryptosporidium retention would be solely due to adsorption rather
than a physical retention based on a size exclusion alone. The
alumina was dispersed in aqueous solution, shaken and quickly
allowed to settle, the fine dispersed phase was decanted off and
the remaining material redispersed. This process was repeated
20.times., each time discarding the dispersed material until only
the largest particles were retained.
[0058] In order to mimic conventional sand filter, parallel column
filtration experiments were carried out using ballotini (glass)
spheres of diameters greater than around 150 .mu.m which were
prepared in a similar fashion to the alumina particles. This
enabled the preparation of columns with sufficiently large pores
that would prevent oocysts capture by size exclusion (see FIG.
4).
Adsorption Studies
[0059] Alumina substrates, immediately after cleaning using UV
irradiation, were directly exposed to 100 .mu.l of a
5.times.10.sup.6 oocysts/ml Cryptosporidium solution. The treated
substrates were placed into a covered petrie dish housed inside
another larger moist petrie dish in a refrigerator for several
hours allowing oocysts to adsorb to the surface. After this time
the liquid was decanted off and the substrate was rinsed with
distilled water. Any residual liquid removed by way of capillary
action using filter paper. The substrate were then examined under a
Kombistereo Wild M32 light microscope having a magnification range
162-1000.times. using a Intralux 5000 optical fibre light source.
The adsorption density was photographed with a Kodak 400 flexi
clear film at a film speed of 200 ASA using a Nikon camera.
Column Separations
[0060] (1) A slurry of the larges alumina particles (prepared as
described above) was transferred to a glass chromatography column
(28 mm O.D) containing a #3 glass sinter the excess liquid was run
from the column leaving a packed column having a depth of 5 mm (see
FIG. 5). A 100 .mu.l of the Cryptosporidium sample at
3.times.10.sup.8 oocysts/ml was diluted to 25 mls in a volumetric
flask, 10 ml of this solution was then transferred to the alumina
packed column, and the solution allowed to stand 15 minutes before
being allowed to percolate through the alumina at a rate (10 ml/hr)
into glass vials. After the entire contents had come through, the
column was washed twice times with 10 ml of distilled water
allowing it to percolate through at a similar rate to that of the
original Cryptosporidium sample. Each 10 ml fraction was collected,
transferred to a microelectrophoresis cell and examined using dark
field illumination. This determination of the density and charge of
any particles present.
[0061] (2) A slurry of the largest Ballotini spheres was
transferred to a glass chromatography column (28 mm O.D) containing
a #3 glass sinter the excess liquid was run from the column leaving
a packed column having a depth of 5 mm (see FIG. 5). A 100 .mu.l of
the Cryptosporidium sample at 3.times.10.sup.8 oocysts/ml was
diluted to 25 mls in a volumetric flask, 10 ml of this solution was
then transferred to the packed silica column, and the solution
allowed to stand 15 minutes before being allowed to percolate
through the silica at a rate (10 ml/hr) into glass vials. After the
entire contents had come through, the column was washed three times
with 10 ml of distilled water allowing it to percolate through at a
similar rate to that of the original Cryptosporidium sample. Each
10 ml fraction was collected, transferred to a microelectrophoresis
cell and examined using dark field illumination. This allowed
determination of the density and charge of any particles
present.
Example 1
Direct Adsorption on Alumina
[0062] The Cryptosporidium oocyst sample received from AWT was
5.times.10.sup.6 oocysts/ml. Since the oocysts are approximately 5
.mu.m in diameter, the area per oocyst of 1.9.times.10.sup.-7
cm.sup.2 means the number of oocysts required to cover a 1 cm.sup.2
substrate is 5.times.10.sup.6. In these studies we have used 0.1 ml
samples of the original 5.times.10.sup.6 oocysts/ml sample.
[0063] Pure alumina (.alpha.-Al.sub.2O.sub.3) wafers were cut to an
appropriate size and UV irradiated at (.lamda.185 and 254 mm) for 1
hr in the presence of water vapour to remove any organic
contamination. The UV irradiation in the presence of water vapour
and oxygen produces ozone and hydroxyl radicals which clean the
alumina surfaces rendering them hydrophilic. After UV irradiation a
small droplet of distilled water was placed on the substrates to
ensure the surfaces were hydrophilic, then blown dry under a gentle
stream of nitrogen.
[0064] The blank substrates were examined under a Kombistereo Wild
M32 light microscope having a magnification range 162-1000.times..
The alumina wafer was highly polished and so only dust
contamination and the roughness of the reverse side of the wafer
enabled us to focus on the smooth surface. FIG. 6 is a photograph
of the microscope focussed on the optically smooth surface having a
few extraneous dust particles attached. This indicated that the
wafer would provide an excellent, optically smooth surface for
adsorption investigations using light microscopy.
[0065] The hydrated alumina wafer was directly exposed to 0.1 ml of
a 5.times.10.sup.6 oocysts/ml solution. The treated substrate was
placed into a covered petrie dish housed inside another larger
moist petrie dish in a refrigerator for several hours allowing
oocysts to adsorb to the positively charged surface. After this
time the liquid was decanted off the substrate and any residual
liquid removed by way of capillary action using a filter paper. The
substrate was examined in a similar manner to that of the blank.
When focussed on the alumina surface, a uniform layer of about
1/10th monolayer coverage of oocysts was observed (see FIG. 3)
which is consistent with the original oocyst density (i.e.
5.times.10.sup.5 oocysts), suggesting that all or most of the
oocysts were recovered from solution by direct adsorption to the
alumina substrate.
[0066] To test whether adsorbed oocysts could be easily desorbed,
the coated alumina substrate was placed into a covered beaker
containing distilled water for approximately 20 hours. After this
time, the substrate was taken out with the aid of tweezers and any
residual liquid removed via capillary action using a filter paper,
it was then examined under the microscope. The adsorption density
of oocysts was unchanged, indicating that the adsorbed oocysts were
stable in water at pH 5.7. This result is consistent with the
observation that alumina is positively charged at this pH.
Furthermore, it also indicates that Cryptosporidium oocysts have a
high affinity for the alumina surface.
[0067] To test whether the oocysts would remain adsorbed to alumina
substrates at higher pH values, the coated alumina was placed into
buffer pH 9.2. At this pH the alumina becomes negatively charged
which might cause oocyst desorption if the adsorption was purely
electrostatic in origin.
[0068] However, after leaving the coated substrate for 16 hours at
this pH the substrate was examined under the microscope, again no
oocyst desorption was found to occur. This could be either because
at this pH the (negative) surface potential is still too weak to
overcome the strong van der Waals attraction or because once the
oocysts have adsorbed, strong short range ligand bonds are formed
between the surface aluminium and the carboxylate or phosphate
groups present on the oocyst surface. To test whether the low
surface potential could be the reason for the failure to desorb, we
placed the coated substrate in a pH 10 buffer (where the alumina
should acquire a high negative surface potential) for several
hours, again no oocyst desorption was observed. This provides
further evidence that the resultant oocyst adsorption was due to a
relatively strong specific chemisorption.
Example 2
Column Separators
[0069] A glass chromatography column (28 mm O.D) containing a #3
glass sinter was chosen for column separations (see FIG. 5). In
order to ensure the pores of the glass sinter were large enough to
allow the Cryptosporidium oocysts through, a 10 ml sample of
1.5.times.10.sup.6 oocysts/ml was passed through the glass sinter
of the unpacked column.
[0070] The column permeate was collected and transferred to a
microelectrophoresis cell and examined using dark field
illumination. At the first stationary level about 125 negatively
charged particles (oocysts) were observed. This demonstrated that
oocysts could easily pass through the glass sinter.
(2.2) Silica Blank
[0071] The silica Ballotini) spheres were fractionated to a size
approx 200 .mu.m in diameter as described earlier. The glass column
was packed to a depth of 5 mm with the fractionated large Ballotini
spheres. 10 ml of distilled water was allowed to percolate through
the packed column and the permeate collected. The permeate was
transferred to a microelectrophoresis cell. This not only allowed
us to test whether the sinter was of sufficient porosity to retain
the silica support but it also allowed determination of the
background levels. The sinter was found to adequately retain the
packing support since the permeate typically had about 8 negatively
charged particles in the field of view.
(2.3) Silica Packed Column
[0072] The silica (Ballotini) spheres were fractionated to a size
approximately 200 .mu.m in diameter. A slurry of these spheres was
transferred to a glass chromatography column (25 mm O.D) containing
a #3 glass sinter the excess liquid was run from the column leaving
a packed column having a depth of 5 mm. A 100 .mu.l of the
Cryptosporidium at 3.times.10.sup.8 oocysts/ml was diluted to 25
mls in a volumetric flask, 10 ml of this solution was then
transferred to the packed silica column, and the solution allowed
to stand 15 mins before being allowed to percolate through the
silica at a rate (10 ml/hr) into glass vials. After the entire
contents had come through, the column was washed three times with
10 ml of distilled water allowing it to percolate through at a
similar rate to that of the original Cryptosporidium sample. Each
10 ml fraction was collected, transferred to a microelectrophoresis
cell and examined using dark field illumination.
[0073] The Cryptosporidium sample before passing through the packed
support contained about 75-78 negatively charged particles at a
given plane of view within the cell. The permeate which was passed
through the packed column was seen to have about 70 negatively
charged particles. Three further 10 ml washings were put through
the column to see whether the Cryptosporidium would desorb or
whether there was a lag time involved in total "Cryptosporidium"
recovery. The first, second and third washings were found to
contain 34, 13 and 8 negatively charged particles, respectively.
The results obtained are shown graphically in FIG. 7. This
demonstrates that Cryptosporidium was not retained in the column
support (i.e. no adsorption evident) thus allowing the oocysts to
travel through large intra-pore spacing between adjacent silica
particles.
(2.4) Alumina Blank
[0074] The alumina powder was fractionated to a size approximately
200 .mu.m in diameter as described in materials and methods. The
glass column was packed to a depth of 5 mm with the fractionated
alumina powder. 10 ml of distilled water was allowed to percolate
through the packed column and the permeate collected. The permeate
was transferred to microelectrophoresis cell and set at the first
stationary level. This allowed determination of whether the sinter
was of sufficient porosity to retain the alumina support as well as
background levels. The sinter was found to adequately retain the
packing support, since the permeate typically had about 14
negatively charged particles at any plane of view.
(2.5) Alumina Packed Column
[0075] The alumina powder was fractionated to a size approximately
200 .mu.m in diameter as previously described. A slurry of this
hydrated alumina powder was transferred to a glass chromatography
column (28 mm O.D) containing a #3 glass sinter. The excess liquid
was run from the column leaving a packed column having a depth of 5
mm. A 100 .mu.l of the Cryptosporidium at 3.times.10.sup.8
oocysts/ml was diluted to 25 mls in a volumetric flask, 10 ml of
this solution was then transferred to the alumina packed column,
and the solution allowed to stand 15 mins before being allowed to
percolate through the alumina at a rate (10 ml/hr) into glass
vials. After the entire contents had come through, the column was
washed twice with 10 ml of distilled water allowing it to percolate
through at a similar rate to that of the original Cryptosporidium
sample. Each 10 ml fraction was collected, transferred to a
microelectrophoresis cell and examined using dark field
illumination. This permitted determination of the density and
charge of any particles present.
[0076] The Cryptosporidium sample before passing through the packed
support contained about 70 negatively charged particles at a given
plane within the cell. By comparison, the permeate which was passed
through the packed column was seen to have only 4 negatively
charged particles (i.e below background level) at the given plane.
Two further 10 ml washings were put through the column to see
whether the Cryptosporidium could be easily desorbed. The second
and third washings were found to contain only 1 negatively charged
particle (see FIG. 8).
[0077] This demonstrated that Cryptosporidium was retained in the
column due to adsorption, as the intra-pore spacing between
adjacent alumina particles would have presented no barrier based on
size exclusion alone. Also, repeated washings provided good
evidence that the oocysts could not be easily desorbed.
Example 3
[0078] To investigate the various rapid rehydroxylation methods the
alumina was heated to 610.degree. C. and the powder treated either
with H.sub.2O.sub.2 or NaOH. The powder was then dried in the oven
at 110.degree. C. to remove surface water and tested to see whether
the powder fines floated or sank. The method developed involved
taking a sample of the powder on the end of a spatula and then as
approach the surfaces at an angle of 45.degree. the powder was
slowly moved through the air/water interface, the spatula was then
slowly withdrawn and if the powder was not fully hydroxylated the
powdered fines floated, whereas if the powder was fully
hydroxylated in appeared to be wetted and ran off the end of the
spatula.
NaOH Treated Alumina.
[0079] We tested the powders treated with 1.times.10.sup.-5,
5.times.10.sup.-4 and 1.times.10.sup.-2M NaOH for an hour. The
powders treated with 1.times.10.sup.-5, 5.times.10.sup.-4 floated
whereas the 1.times.10.sup.-2 M NaOH treated sample sank indicating
that the particles of alumina had become hydrophilic.
H.sub.2O.sub.2 Treated Alumina.
[0080] We tested the powders treated with 10% w/v H.sub.2O.sub.2
for 1/2 Hr and 1 hr and with 30% w/v H.sub.2O.sub.2 for 5, 10, 15,
30, 60, 120 and 225 mins. We found that the powders<1 hr floated
whereas times greater than 1 hr sank indicating that these
experiments demonstrate that hydroxylation of alumina may be
accomplished in a rapid and efficient manner using treatment with
either sodium hydroxide of hydrogen peroxide solutions.
[0081] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0082] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
* * * * *