U.S. patent application number 13/327073 was filed with the patent office on 2012-04-12 for devices for water treatment.
This patent application is currently assigned to EMBRO CORPORATION. Invention is credited to Vance D. Fiegel, David R. Knighton.
Application Number | 20120085700 13/327073 |
Document ID | / |
Family ID | 35798614 |
Filed Date | 2012-04-12 |
United States Patent
Application |
20120085700 |
Kind Code |
A1 |
Knighton; David R. ; et
al. |
April 12, 2012 |
DEVICES FOR WATER TREATMENT
Abstract
A device for use in water comprising a carrier having an
interior cavity and one or more openings allowing ingress to and
egress from the interior cavity; and a moss contained within the
interior cavity in which the carrier completely encloses the
moss.
Inventors: |
Knighton; David R.;
(Minneapolis, MN) ; Fiegel; Vance D.; (Shakopee,
MN) |
Assignee: |
EMBRO CORPORATION
St. Louis Park
MN
|
Family ID: |
35798614 |
Appl. No.: |
13/327073 |
Filed: |
December 15, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13048239 |
Mar 15, 2011 |
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13327073 |
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12854397 |
Aug 11, 2010 |
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13048239 |
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12625876 |
Nov 25, 2009 |
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12854397 |
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12392241 |
Feb 25, 2009 |
7625486 |
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12625876 |
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11106049 |
Apr 14, 2005 |
7497947 |
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12392241 |
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60562196 |
Apr 14, 2004 |
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Current U.S.
Class: |
210/602 |
Current CPC
Class: |
C02F 3/327 20130101;
Y02W 10/15 20150501; Y02W 10/18 20150501; C02F 1/50 20130101; C02F
2101/10 20130101; E04H 4/1281 20130101; C02F 2101/20 20130101; C02F
2303/04 20130101; Y02W 10/10 20150501; A01N 65/00 20130101; C02F
3/10 20130101; C02F 2103/42 20130101; C02F 3/32 20130101; A01N
65/00 20130101; A01N 25/34 20130101; A01N 65/00 20130101 |
Class at
Publication: |
210/602 |
International
Class: |
C02F 3/32 20060101
C02F003/32 |
Claims
1. A method of treating water comprising contacting water with an
amount of a non-decomposed moss effective to remove cations other
than hydrogen ions from the water.
Description
[0001] This application is a continuation of U.S. Ser. No.
13/048,239, filed Mar. 15, 2011, which is a continuation of U.S.
Ser. No. 12/854,397, filed Aug. 11, 2010, now abandoned, which is a
continuation of U.S. Ser. No. 12/625,876, filed Nov. 25, 2009, now
abandoned, which is a continuation of U.S. Ser. No. 12/392,241,
filed Feb. 25, 2009, now U.S. Pat. No. 7,625,486 B2, issued Dec. 1,
2009, which is a continuation of U.S. Ser. No. 11/106,049, filed
Apr. 14, 2005, now U.S. Pat. No. 7,497,947 B2, issued Mar. 3, 2009,
which claims the benefit of provisional application Ser. No.
60/562,196, filed Apr. 14, 2004, the contents of each of which are
hereby incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to devices for water treatment, and
in particular relates to devices comprising sphagnum moss.
BACKGROUND OF THE INVENTION
[0003] There are many types of water treatment systems, such as
filtration and cleaning systems for swimming pools and aquariums.
Many of these systems filter the water to remove suspended matter
and reduce the cloudy appearance of the water. Preventing bacterial
growth in water and removing contaminants from water are
significant industrial, as well as household, problems. For
example, industrial effluent should be cleaned to remove toxic
compounds as well as to remove bacteria before it is dumped into
lakes and rivers. Containers of water such as swimming pools, hot
tubs, aquariums and the like must be kept clean to prevent the
water from becoming cloudy and/or the container walls from becoming
slimy. The water may be treated by active means such as a filter to
remove particles and bacteria, and it may also be treated by
passive means whereby a biocide is placed in a container and
floated in the water.
[0004] It is common to use chemical means to keep the water clean
and reduce growth of bacteria and other microorganisms. Ultraviolet
light, chlorination, bromination, treatment with ions of copper and
silver as well as treatment with ozone can be used to treat and/or
disinfect water. These are typical biocides, that is, substances or
energies that destroy living organisms. Of course care must be
taken with all these methods because of the possible toxicity or
damage to the user. Chemicals require careful handling to avoid
environmental contamination as well as contact with the user.
[0005] "Sphagnum moss" is a generic expression that designates a
range of botanical species that co-exist in a sphagnous bog. It
should be noted that "peat moss" refers generally to a decomposed
or composted sphagnum moss. Sphagnum moss is commonly harvested for
use in various products. The petals, and not the stems, of the moss
preferably may be harvested. Typically large pieces of plant
material (roots, twigs, etc.) are removed and the moss may be
processed further after harvesting by forming an aqueous slurry to
extract very fine particles. Water is removed from the slurry and
the moss is dried. The moss may be compressed prior to packaging or
shipment. Various additives may be used to alter the absorption
characteristics or mechanical properties of the moss. Because
sphagnum moss is readily available and relatively inexpensive, it
has been used in a variety of products, primarily for the
absorption of fluids.
[0006] There is substantial need in the art for products that
inhibit the growth of microorganisms such as bacteria, yeast, and
algae. It would be desirable to have a means to maintain the
clarity of water in a swimming pool, whirlpool bath, aquarium, and
the like, for long periods of time, without shutting a system down
for cleaning. The most desirable system would require very little
maintenance and would be relatively inexpensive.
SUMMARY OF THE INVENTION
[0007] The invention provides a device for use in water comprising:
(i) a carrier having an interior cavity and one or more openings
allowing ingress to and egress from the interior cavity; and (ii) a
moss contained within the interior cavity, wherein the carrier
completely encloses the moss.
[0008] The invention provides a method of inhibiting microorganism
growth comprising placing in water susceptible to bacterial growth
a device for inhibiting microorganism growth in water, the device
comprising: (i) a carrier having an interior cavity and one or more
openings allowing ingress to and egress from the interior cavity;
and (ii) a moss contained within the interior cavity, wherein the
carrier completely encloses the moss, and wherein the device
comprises an amount of the moss effective to inhibit microorganism
growth in the water.
[0009] The invention provides a kit comprising sterilized,
non-decomposed moss and a device for use in water comprising a
carrier having an interior cavity and one or more openings allowing
ingress to and egress from the interior cavity, wherein the
interior cavity can completely enclose the moss.
[0010] The invention provides a method of inhibiting microorganism
growth comprising placing in water susceptible to microorganism
growth a device for inhibiting microorganism growth in water, the
device comprising: (i) a carrier having an interior cavity and one
or more openings allowing ingress to and egress from the interior
cavity; and (ii) a moss contained within the interior cavity,
wherein the carrier completely encloses the moss, wherein the
device comprises an amount of the moss effective to inhibit
microorganism growth in the water, and periodically shocking the
water with an appropriate chemical agent.
[0011] The invention provides a method of treating water comprising
placing in water a device comprising: (i) a carrier having an
interior cavity and one or more openings allowing ingress to and
egress from the interior cavity; and (ii) a moss contained within
the interior cavity, wherein the carrier completely encloses the
moss, and wherein the device comprises an amount of the moss
effective to remove cations other than hydrogen ions from the
water.
[0012] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory and are intended to provide further explanation of
the invention as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A illustrates a perspective view of one embodiment of
the device of this invention. FIG. 1B illustrates a side view, and
FIG. 1C illustrates a cross-sectional view along line C-C of FIG.
1B.
[0014] FIG. 2A illustrates a perspective view of another embodiment
of the device of this invention and FIG. 2B shows a side view of
the moss used within the device shown in FIG. 2A.
[0015] FIG. 3 illustrates a perspective view of another embodiment
of the device of this invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0016] We have discovered species of sphagnum moss that can be used
to treat water such as in a swimming pool, spa, aquarium,
whirlpool, and the like. It is believed that particular species of
moss are particularly effective at inhibiting and/or preventing the
growth of bacteria and other microorganisms.
[0017] In this invention, "bacteriostatic" refers to a material
that inhibits the growth of bacteria. In common lexicography, the
term "antibacterial" generally refers to a bacterial growth
inhibitor. Both terms should be distinguished from "bactericidal"
which refers to materials that kill bacteria upon contact.
[0018] In this invention, "water treatment" refers to a process by
which water is kept clean, clear, and pleasant smelling in swimming
pools, aquariums, spas, and the like. Spas are also known as
whirlpools or hot tubs. When the water is agitated, less foaming is
observed. The moss is believed to inhibit growth of bacteria and
other microorganisms and it also may absorb compounds and
substances that decrease water clarity.
[0019] In this invention, sphagnum papillosum (S. papillosum)
and/or sphagnum cristatum (S. cristatum) can be used in water
treatment devices. In preferred embodiments, the moss is enclosed
or encapsulated in a mesh material that prevents the moss from
disintegrating in an aqueous environment. Thus the moss can be held
in a desired place in a pool, hot tub, whirlpool bath, and the
like. Preferred mesh materials include those comprising polymers
such as nylon or polypropylene, with mesh sizes ranging from about
0.1 to 1 mm. Polymers are generally preferred because they are
inexpensive and may be resistant to degradation.
[0020] Suitable for use in this invention are S. papillosum, which
can be harvested from bogs in northern Minnesota, U.S.A., and S.
cristatum, which is commercially available as a compressed bale
from Sutton's Moss of Dobson, Westland, New Zealand. These species
of moss can be used by themselves or together in the devices and
systems of this invention. Typically and preferably the moss is
cleaned to remove small particles, such as dirt, and larger debris,
such as roots and leaves. Commercially available moss may be
fumigated before it is packaged by a manufacturer in order to
destroy seeds.
[0021] In a preferred embodiment, the moss is cut by mechanical
means into a desired size and shape. The moss preferably is then
sterilized by autoclaving, exposure to ethylene oxide, or by other
means known to one of skill in the art. Sterilization destroys
living organisms in the moss and thus avoids any problems of
undesirable or foreign bacteria being introduced into the
environment where a device of this invention is used. The moss is
then ready for use in a water treatment system or other
applications.
[0022] We have found that a convenient, easy, effective, and
inexpensive way of treating water is to place a portion of S.
papillosum or S. cristatum in a floatation device that permits
water to flow around and through the moss. Another way to use it is
to encapsulate it in mesh and weight the mesh so that the moss will
remain in the water. Any suitable means that will maintain contact
of the moss with water is suitable for use. This device is then
placed in the swimming pool, whirlpool, hot tub, etc., where it can
come into contact with the water. We have found that treatment is
remarkably effective in preventing growth of microorganisms and in
keeping the water clean, clear and free of odor and foam. This is
all the more remarkable because this is a passive system when
compared to a filtration system which forces water through the
moss. Of course it is to be understood that active filtration could
be done with the device of this invention to treat the water.
[0023] When used in swimming pools, hot tubs, and the like, the
water treatment devices described herein are preferably used in
conjunction with materials that kill microorganisms. This is
because these environments may have large loads of microorganisms,
particularly bacteria, introduced at various times. Accordingly,
standard practice is to filter the water, flush water lines, and
test the water as necessary. The pH can be adjusted by using
commercially available solutions. The water treatment devices of
this invention are most desirably used in conjunction with an
oxidizer, such as potassium monopersulfate, referred to as
"chlorine free shock". Potassium monopersulfate is known to
increase the efficiency of chlorine purification products, but we
have found that it is also particularly effective when used with
the sphagnum moss devices described above.
[0024] The sphagnum moss of this invention can be used in any
composition, material, device, or method where the inhibition of
bacteria is desirable. Uses include the inhibition of microorganism
growth, the reduction and/or prevention of odors, water treatment,
and control of mold and fungal growth; and control of fermentation.
Such devices and materials include absorbent products, such as
diaper liners, feminine hygiene products, bandages, and wound
dressings. In such products, the moss can be enclosed between
membranes of differing liquid transmission characteristics. That
is, for example, one membrane may be permeable to fluid and another
membrane may be permeable to vapor. The moss can be incorporated
into polymers and used as face masks. The moss can be encapsulated
in membranes and used in food preservation products such as
packaging wraps and liners to absorb liquid and odors. The moss can
be used in water treatment products to keep water clean in storage
tanks, aquariums, swimming pools, whirlpool baths, spas, and the
like, as well as in water filtration devices. The moss can be used
for waste water and sewage treatment. The moss can be shaped into,
for example, discs or pellets, and used to absorb water from grain
and other food products. The moss also can be used for fermentation
control (such as in liquids or grains). The moss can be used for
the control of fungal or bacterial diseases in lawns and gardens.
The moss can be used for mold control products such as in storage
containers or ductwork linings.
[0025] The invention provides a device for use in water comprising:
(i) a carrier having an interior cavity and one or more openings
allowing ingress to and egress from the interior cavity; and (ii) a
moss contained within the interior cavity, wherein the carrier
completely encloses the moss. The carrier can comprise a float. The
carrier can comprise a float and a cylindrical portion beneath the
float, the cylindrical portion having an interior cavity and one or
more openings allowing ingress to and egress from the interior
cavity. The moss can be enclosed within a mesh bag. The carrier can
comprise one or more weights.
[0026] The moss can be non-decomposed moss. The moss can be
sphagnum moss. The moss can be selected from the group consisting
of sphagnum papillosum, sphagnum cristatum, and mixtures thereof.
The moss can be compressed and can be in the form of strips. The
moss can be sterilized by autoclaving, sterilized by chemical
treatment, or sterilized by treatment with ethylene oxide. The moss
can be washed with an acidic solution, especially a solution of
acetic acid. The moss can be washed with an acidic solution and
then washed with a salt solution.
[0027] The invention provides a method of inhibiting microorganism
growth comprising placing in water susceptible to microorganism
growth a device for inhibiting microorganism growth in water, the
device comprising: (i) a carrier having an interior cavity and one
or more openings allowing ingress to and egress from the interior
cavity; and (ii) a moss contained within the interior cavity,
wherein the carrier completely encloses the moss, and wherein the
device comprises an amount of the moss effective to inhibit
microorganism growth in the water. The water can be in a spa, pool,
or aquarium. The water can be pumped through the device.
[0028] The invention provides a kit comprising sterilized,
non-decomposed moss and a device for use in water comprising a
carrier having an interior cavity and one or more openings allowing
ingress to and egress from the interior cavity, wherein the
interior cavity can completely enclose the moss. The kit can
comprise one or more pH test strips and/or potassium
monopersulfate.
[0029] The invention provides a method of inhibiting microorganism
growth comprising placing in water susceptible to microorganism
growth a device for inhibiting microorganism growth in water, the
device comprising: (i) a carrier having an interior cavity and one
or more openings allowing ingress to and egress from the interior
cavity; and (ii) a moss contained within the interior cavity,
wherein the carrier completely encloses the moss, wherein the
device comprises an amount of the moss effective to inhibit
microorganism growth in the water, and periodically shocking the
water with an appropriate chemical agent. The chemical agent can be
potassium monopersulfate.
[0030] The invention provides a method of treating water comprising
placing in water a device comprising: (i) a carrier having an
interior cavity and one or more openings allowing ingress to and
egress from the interior cavity; and (ii) a moss contained within
the interior cavity, wherein the carrier completely encloses the
moss, and wherein the device comprises an amount of the moss
effective to remove cations other than hydrogen ions from the
water. The cations can be calcium or iron ions, and substantially
all of the calcium or iron ions can be removed from the water. The
moss can be compressed and can be in the form of strips. The moss
can be sterilized by autoclaving, sterilized by chemical treatment,
or sterilized by treatment with ethylene oxide. The moss can be
washed with an acidic solution, especially a solution of acetic
acid. The moss can be washed with an acidic solution and then
washed with a salt solution. The water can be in a spa, pool, or
aquarium.
[0031] FIGS. 1A to 1C illustrate a suitable device of this
invention. FIG. 1A shows device 10 floating in water and FIGS. 1B
and 1C shows side and cross sectional views, respectively. Device
10 is adapted to receive a segment of compressed sphagnum moss 15
that has been cut into a desired dimension. The moss is shown in
phantom in FIGS. 1A and 1B. A convenient dimension for the moss
used in device 10 is about 6.times.1/4.times.1/4 inches
(15.2.times.0.63.times.0.63 cm). A piece of moss this size weighs
about 5 grams. Moss 15 is enclosed in nylon mesh 16, sized to
permit the compressed moss to expand. The mesh size is such that it
will retain even small particles of moss and prevent it from
breaking apart and floating away.
[0032] Device 10 comprises a plastic material that is impact
resistant, does not dissolve in water, and can be shaped into a
desired shape. Device 10 is commercially available as a "floater"
from MP Industries of Huntington Beach, Calif. It should be noted
that floaters of this type are commonly used with pellets or discs
of pool cleaning agents, such as those containing chlorine. Device
10 has been adapted for use with sphagnum moss by adding holes to
facilitate passage of water into the device.
[0033] Device 10 comprises float portion 20 and flow through
portion 30. Float portion 20 is cylindrical, and may be any desired
dimension, though typically it is larger in diameter than
flow-through portion 30. A useful dimension for the float portion
is about 5 inches (12.7 cm) in diameter.
[0034] Flow-through portion 30 is a two-part elongated cylinder
having core or hollow center 32. First part 33 is attached to
floatation portion 20 and is provided with screw threads onto which
second part 35 affixes. In this way the length of the flow-through
portion can be changed. Second part 35 is fixed in position by
means of adjustable collar 34. Second part 35 also has removable
cap 37, which is weighted so that device 10 floats in the water as
illustrated in FIG. 1.
[0035] Slots 38 and holes 39 permit water to flow through the
cylinder. The slots and holes may be any desired dimension and can
be positioned as desired. A useful length of the flow through
portion is about 7 inches (17.8 cm). Cap 37 is removable so that
the desired size of the sphagnum moss can be inserted into portion
30. Once exposed to water, the compressed moss expands. The density
of expanded moss is such that water can permeate it. Device 10 is
sufficient to treat up to about 500 gallons of water (for example,
in a whirlpool or spa) for up to 30 days.
[0036] FIG. 2A illustrates device 50 floating in water. Device 50
comprises cylindrical portion 60 having core or hollow center 62.
Slots 64 and holes 66 permit water to enter the hollow center. Moss
55, shown in phantom in FIG. 2A, is encapsulated by mesh 52, as
most clearly shown in FIG. 2B. The moss expands when in contact
with the water, filling hollow center 62. Cylindrical portion 60 is
shown sealed at one end, with removable cap 57 at the other end.
Cap 57 may be weighted so that the maximum length of device 60
stays in contact with the water.
[0037] FIG. 3 shows device 70 attached to wall W of a swimming
pool, aquarium, hot tub, or the like. Moss 75 is encapsulated by
mesh 72 and the mesh is affixed to bracket 77. The mesh is of a
sufficient size that particles or fragments of moss will stay
within the mesh. The bracket hangs from the wall and the device can
remain fixed at this location. Alternatively, device 70 could lie
on the bottom of the pool or tub. It could be affixed there or
could be held down by a weight. It could also be placed in-line
with a filter.
EXAMPLES
Example 1
[0038] S. papillosum moss, harvested from northern Minnesota, and
was prepared for bacterial inhibition testing. The moss species was
validated by the University of Minnesota and again upon
receipt.
[0039] All samples were placed in plastic bags. All raw moss was
stored at 4.degree. C. until processed by lab personnel. All
pre-dried outside moss samples were stored at room temperature
until processed.
[0040] The following equipment was used: [0041] a) Blender, 1.25 L
capacity (commercially available as Osterizer.RTM. from Oster)
[0042] b) Distilled Water (available from Premium Water, Inc.)
[0043] c) Tissue Sieve, 1 cup capacity (commercially available as
Cellector.RTM. E-C Apparatus Corp.) [0044] d) 1 L Glass Beaker
(commercially available as Pyrex.RTM.) [0045] e) Sterile
Polystyrene Petri Dishes 100.times.15 mm (commercially available
from Falcon) [0046] f) Sterile Polystyrene Petri Dishes
150.times.15 mm (commercially available from VWR) [0047] g)
Autoclave (commercially available from Market Forge) [0048] h)
Metal Lab Scoop (16.5 cm)/commercially available as Adison Tissue
Forceps from VWR) [0049] i) Laminar Flow Hood (commercially
available from Baker)
Procedure:
[0049] [0050] 1) Raw moss was taken out of the bag by hand and
picked clean of any visible, roots, leafs and debris and placed in
the blender. The blender was then filled with moss until it reached
approximately the 1 L mark [0051] 2) The blender was then filled
with 1 L of distilled water and shaken manually with the lid on for
30 seconds and drained to remove any remaining dirt and debris. The
process was repeated 2 more times to thoroughly wash the moss.
[0052] 3) The blender was then filled with distilled water again
and the moss was blended using the pulse mode on settings 4 and 5
for 30 seconds each until the moss was homogeneous throughout the
sample. [0053] 4) The blender was then drained of water. Any
remaining water was then squeezed out by hand from the moss using
the tissue sieve. The squeezed moss was then placed in a clean 1L
beaker. Steps 1 to 4 were repeated until the 1L beaker was filled
with processed moss. The beaker was then autoclaved for 20 minutes
at 121.degree. C. at 15 psi (103.4 MPa) using the liquid setting
and allowed to cool at room temperature. [0054] 5) Once cooled, the
moss was brought to the laminar flow hood and carefully placed in
labeled, pre-weighed Petri dishes using a sterile lab scoop and
forceps. Special care was taken not contaminate the moss and to
pack each dish in a uniform manner. Once packed, the dishes remain
uncovered for at least 72 hours until the moss was dry. The dried
dishes were covered and kept in the flow hood until used.
Example 2
[0055] S. cristatum moss, obtained from Sutton's Moss, Canada,
(harvested in New Zealand) was prepared for bacterial inhibition
testing. The moss species was validated upon receipt. Handling of
the moss samples was identical to that described above in Example
1.
[0056] The following equipment was used: [0057] a) Sterile
syringes, individually wrapped 30 cc or 60 cc (commercially
available from Becton Dickenson) [0058] b) Sterile syringe filters,
0.45 .mu.m (micrometer) and 0.20 .mu.m (commercially available as
Acrodisc from Pall Gellman) [0059] c) Adison tissue forceps
(commercially available from VWR) [0060] d) 50 cc polypropylene
graduated test tubes, sterile pack (commercially available from
Falcon) [0061] e) Wax film (Parafilm.RTM., commercially available
from American National Can) [0062] f) Laminar flow hood
(commercially available from Baker Company) [0063] g) Pipetman with
sterile graduated polypropylene tips, 25 mL (commercially available
from Becton Dickenson) [0064] h) 10 M HCl and 10 M NaOH [0065] i)
pH Meter (commercially available from Beckman Omega 40)
Reagents and Solutions Preparations: (Depending on Liquid Used for
Treatment)
[0065] [0066] a) Bacton.TM. Tryptic Soy Broth, 30 g/L (Becton
Dickenson)/MEM-Alpha (Gibco) [0067] b) Phosphate Buffered Saline
(1.times.), pH 7.1 (commercially available from Gibco.TM.) [0068]
c) HPLC Grade Water (commercially available from J. T. Baker)
Procedure
[0068] [0069] a) All liquids used for treatment were either
autoclaved or filter sterilized and stored at a proper temperature
prior to use in this protocol. All treatment steps were done with
in the laminar flow hood using aseptic technique. Tissue forceps
used were autoclaved prior to use. [0070] b) After weighing the
dried moss, the treatment liquid was pipetted into the Petri dish
at a concentration of one milliliter of treatment liquid to every
50 mg dried moss. If a concentration other that 50 mg/mL was used,
the process changed accordingly. [0071] c) The Petri dish of moss
was wrapped in wax film (Parafilm.RTM.) and refrigerated at
4.degree. C. for one hour. [0072] d) The Petri dish was removed
from the refrigerator and the moss was scooped and packed into a 30
cc or 60 cc syringe using sterile tissue forceps. The plunger of
the syringe was re-inserted and the liquid was squeezed into a
sterile 50 mL polypropylene test tube until all possible liquid was
extracted. [0073] e) The treatment liquid was filtered through a
0.45 .mu.m syringe filter, then a 0.2 .mu.m syringe filter and
stored at 4.degree. C. until used. [0074] f) Filtered samples were
pH adjusted using 1.0 M HCl/NaOH and sterile filtered with a 0.2
.mu.m syringe filter and stored again at 4.degree. C. until used in
the bacteria inhibition assay.
Example 3
[0075] This experiment determined the amount of bacterial growth in
Tryptic Soy Broth (TSB) by an inhibition assay.
[0076] All S. papillosum and S. cristatum moss treatment samples
used in this assay were prepared as described above. TSB was also
prepared, autoclaved and stored at 4.degree. C. prior to use.
[0077] The following equipment was used: [0078] a) Beckman.RTM.
DU-64 Spectrophotometer [0079] b) Incubator Oven (commercially
available from Boekel Instruments Model #133000) [0080] c) 5 mL and
50 mL Polystyrene Tubes (commercially available from Falcon) [0081]
d) 10 .mu.L and 1 mL polypropylene tips (commercially available
from Pipetman)
Reagents and Solutions:
[0081] [0082] a) Bacto.RTM. Tryptic Soy Broth (TSB), 30 g/L
(commercially available from Becton Dickenson) [0083] b)
Escherichia Coli frozen culture stock grown in TSB for a minimum of
3 log growth phases (Clinical isolate) [0084] c) Staphylococcus
Aureus frozen culture stock grown in TSB for a minimum of 3 log
growth phases. (ATCC Strain #29213 (American Type Culture
Collection of Manassas, Va.)) [0085] d) Distilled Water
(commercially available from Premium Water Inc.) [0086] e) TSB
treatment sample of S. papillosum moss and New Zealand moss
Procedure:
[0086] [0087] 1) TSB nutrient broth was prepared by adding 30 g/L
Bacto.RTM. Tryptic Soy Broth to distilled water. The solution was
stirred with a stir-bar until all the powder was dissolved and
autoclaved at 121.degree. C. for 20 minutes. The S. papillosum
sample was prepared as described in B, above. One mL of the
solutions, TSB or moss-treated TSB sample, was pipetted into 3 to 5
mL polystyrene test tubes. [0088] 2) Frozen aliquots of E. coli and
S. aureus stored at -20.degree. C. were allowed to thaw to room
temperature. Once thawed, 100 .mu.L of each bacterial stock was
added to a 10 mL aliquot of TSB. Each tube was then capped,
thoroughly mixed, labeled and placed in the incubator at 37.degree.
C. [0089] 3) Ten .mu.L of bacteria stock was pipetted into the one
mL solutions and the tubes were incubated at 37.degree. C. for the
desired time. One tube in each sample and time point did not
receive bacteria in order to serve as the blank.
[0090] 4) The solutions were removed from the 37.degree. C. oven at
the assigned time points and placed in an ice bath. Samples were
then immediately read on the spectrophotometer at 550 nm. The
absolute OD value was sample minus the blank. Inhibition was
measured as percent decrease in OD value vs. the appropriate TSB
control sample.
Example 4
[0091] The following data illustrate the effect of treatment of
bacterial growth media (i.e., Tryptic Soy Broth) with various moss
species according to the procedure in Example 3. Non-sphagnum
species are very poor at preventing E. coli growth. Of the moss
tested below, the most effective sphagnum mosses to prevent E. coli
growth were S. papillosum and S. cristatum. "N" refers to the
number of tests, "Range" presents the highest and lowest numbers
obtained for these tests; and "Mean" refers to the mean value of
the tests.
TABLE-US-00001 % Inhibition of Bacterial Growth Mean N Range
Sphagnum Species S. papillosum (MN) 48 10 26-71 S. cristatum
(NZ--Sutton Moss) 45 8 31-62 S. magellanicum (WI--Mosser Lee) 34 7
21-43 S. fuscum (MN) 20 1 -- S. falcatulum (NZ) 7 2 2-11 Non-
Sphagnum Species Sheet Moss (WI--Mosser Lee) 4 1 -- Spanish Moss
(WI--Mosser Lee) -2 1 --
Example 5
[0092] TSB was treated with S. papillosum and the ability of this
solution to support bacterial growth was measured according to
Example 3. The percent inhibition of bacterial growth for E. coli
(clinical isolate) and S. aureus (ATCC Strain #29213) is reported.
A TSB Control sample and moss-treated TSB solutions (MT-TSB) are
reported below. The OD of a blank (B) is subtracted from the
measured OD (Meas.) of the sample to obtain the reported Value.
TABLE-US-00002 E. coli S. aureus Test Material B Meas. Value B
Meas. Value TSB 0.063 0.744 0.681 0.063 0.250 0.187 (Control) 0.726
0.663 0.257 0.194 Mean 0.672 0.191 SD 0.013 0.005 MT-TSB 0.109
0.662 0.553 0.109 0.279 0.170 (5 mg/mL) 0.714 0.605 0.285 0.176
Mean 0.579 0.173 SD 0.037 0.004 % Inhibition 13.84 9.19 MT-TSB
0.131 0.575 0.444 0.131 0.278 0.147 (10 mg/mL) 0.748 0.617 0.274
0.143 Mean 0.531 0.145 SD 0.122 0.003 % Inhibition 21.06 23.88
MT-TSB 0.173 0.662 0.489 0.173 0.276 0.101 (25 mg/mL) 0.652 0.479
0.284 0.111 Mean 0.484 0.107 SD 0.007 0.006 % Inhibition 27.98
43.83 MT-TSB 0.243 0.599 0.356 0.243 0.355 0.112 (50 mg/mL) 0.564
0.321 0.352 0.109 Mean 0.339 0.111 SD 0.025 0.002 % Inhibition
49.63 41.99 MT-TSB 0.284 0.388 0.104 0.284 0.361 0.077 (75 mg/Ml)
0.430 0.146 0.355 0.071 Mean 0.125 0.074 SD 0.030 0.004 %
Inhibition 81.40 61.15 MT-TSB 0.274 0.322 0.048 0.274 0.322 0.048
(100 mg/mL) 0.339 0.065 0.310 0.036 Mean 0.057 0.042 SD 0.012 0.008
% Inhibition 91.59 77.95
Example 6
[0093] This example demonstrates that treatment with moss does not
kill the bacteria but it does inhibit their growth. A fluorescence
assay, commercially available from Molecular Probes, Eugene,
Oregon, Kit No. L-7012, was used to determine the viability of
bacteria. This system uses mixtures of green and red fluorescent
nucleic acid stains that have differing ability to penetrate viable
and non-viable bacterial cells. The green fluorescent strain, which
emits at 500 nm, binds to both viable and non-viable bacteria. The
red fluorescent strain, which emits at 650 nm, binds only to
non-viable bacteria. Therefore, a bacterial sample containing a
higher proportion of non-viable bacteria will have an altered
staining ratio. The data show that for both E. coli and S. aureus,
the ratio of viable to non-viable bacteria remains the same as in
the control sample.
Procedure:
[0094] 1. 100 .mu.L was incubated in 10 mL of media (pH controlled
TSB and S. papillosum sample, as prepared in Examples 1 and 2,
respectively) for 3 hours at 37.degree. C. [0095] 2. This mixture
was centrifuged to concentrate the bacteria, which was then
resuspended in 10 mL phosphate buffered saline (PBS). [0096] 3.
Three mL of resuspended bacteria were added to each of three
cuvettes. [0097] 4. 40 .mu.L Styo-9 dye was mixed with 40 .mu.L
Propidium Iodide dye. Caution should be used as these compounds are
believed to be carcinogens. [0098] 5. 9 .mu.L of the mixed dye
solution was added to each cuvette and stored in the dark for 15
minutes. [0099] 6. Two PBS cuvettes were prepared with no dye and
two PBS cuvettes were prepared with dye to be used as blanks.
[0100] 7. The solutions were mixed thoroughly. The fluorescence
intensity is measured at 500 nm (with absorption at 480 nm) and at
650 nm (with absorption at 490 nm). The ratio of these two values
relates to the degree of viability of the bacterial culture. The
following tables report the fluorescence intensity at two
wavelengths for bacterial samples in two different media. The
intensity of fluorescence at 500 nm over the intensity of the
fluorescence at 650 nm creates a ratio which relates to the degree
of viability of the bacterial culture. The mean of three TSB and
three moss-treated TSB samples (MT-TSB) is reported below. In each
case, the percent of inhibition is compared to a control
sample.
TABLE-US-00003 [0100] Viability Assay for E. coli (clinical
isolate) Media 500 nm 650 nm Ratio 500/650 TSB 505.117 5.783
87.3228 MT-TSB of 130.1833 1.5167 85.8352 S. papillosum
TABLE-US-00004 Viability Assay for S. aureus (ATCC Strain #29213)
Media 500 nm 650 nm Ratio 500/650 TSB 80.117 3.533 22.6745 MT-TSB
of 45.055 2.0667 21.7984 S. papillosum
The data show that there is no significant change in the ratios
between TSB and the moss-treated TSB, indicating that the effect of
the moss is bacteriostatic rather than bactericidal.
Example 7
[0101] Various bacteria were treated with S. papillosum TSB
(concentration of 50 mg/mL) according to Example 3. The percent
inhibition of bacterial growth is reported. Both a TSB Control
sample and moss-treated TSB solutions (MT-TSB) are reported below.
The "Value" is obtained by subtracting the optical density (OD) of
a blank (B) from the measured OD (Meas.) of a sample.
[0102] Two studies were done and are denoted (1) and (2) below.
TABLE-US-00005 E. coli S. aureus (clinical isolate) ATCC # 29213
Test Material (1) B Meas. Value B Meas. Value TSB 0.034 0.694 0.660
0.034 0.273 0.239 (Control) 0.718 0.684 0.257 0.253 Mean 0.672
0.246 SD 0.017 0.010 MT-TSB 0.219 0.542 0.323 0.219 0.362 0.143
0.572 0.353 0376 0.157 Mean 0.338 0.150 SD 0.021 0.010 % Inhibition
49.7 39.02 E. coli S. aureus Test Material (2) B Meas. Value B
Meas. Value TSB 0.043 0.693 0.650 0.043 0.332 0.289 (Control) 0.667
0.624 0.327 0.284 Mean 0.637 0.287 SD 0.018 0.004 MT-TSB 0.247
0.492 0.245 0.247 0.394 0.147 0.498 0.251 0.428 0.181 Mean 0.248
0.164 SD 0.004 0.024 % Inhibition 61.07 42.76
TABLE-US-00006 S. epidermidis P. aeruginosa ATTC # 12228 ATTC
#10145 Test Material(1) B Meas. Value B Meas. Value TSB 0.034 0.388
0.354 0.034 0.175 0.141 (Control) 0.412 0.375 0.167 0.133 Mean
0.366 0.137 SD 0.017 0.006 MT-TSB 0.219 0.542 0.323 0.219 0.321
0.102 0.572 0.353 0.331 0.112 Mean 0.338 0.107 SD 0.021 0.007 %
Inhibition 53.26 21.90 S. epidermidis P. aeruginosa Test
Material(2) B Meas. Value B Meas. Value TSB 0.043 0.349 0.306 0.043
0.204 0.161 (Control) 0.327 0.284 0.186 0.143 Mean 0.295 0.152 SD
0.016 0.013 MT-TSB 0.247 0.428 0.181 0.247 0.371 0.124 0.444 0.197
0.340 0.093 Mean 0.189 0.109 SD 0.011 0.022 % Inhibition 35.93
28.62
TABLE-US-00007 C. albicans A. amsterodami ATTC # 10231 ATTC # 1001
Test Material(1) B Meas. Value B Meas. Value TSB 0.034 0.068 0.034
0.034 0.053 0.019 (Control) 0.069 0.035 0.047 0.013 Mean 0.035
0.016 SD 0.001 0.004 MT-TSB 0.219 0.249 0.030 0.219 0.231 0.012
0.245 0.026 0.229 0.010 Mean 0.028 0.011 SD 0.003 0.001 %
Inhibition 18.84 31.25 C. albicans A. amsterodami Test Material(2)
B Meas. Value B Meas. Value TSB 0.043 0.1019 0.058 0.043 0.067
0.024 (Control) 0.057 0.044 0.071 0.028 Mean 0.051 0.026 SD 0.010
0.003 MT-TSB 0.247 0.275 0.028 0.247 0.268 0.021 0.292 0.045 0.254
0.007 Mean 0.037 0.014 SD 0.012 0.010 % Inhibition 28.43 46.15
Example 8
Effect of Acid Treatment of the Moss
[0103] Compressed sticks of S. cristatum moss (obtained from
Sutton's Moss, Canada (harvested in New Zealand)) were soaked in
four increasing concentrations of Fe (Fe standard in concentrated
HCl, 0, 0.5,5,50 mg/L, available from Ricca Chemicals, Arlington,
Tex.) at 50 mg moss/ml in distilled water. The soaked moss was
stored overnight at 4 C. The Fe solutions were extracted by
syringe, filtered and measured for Fe by inductively coupled plasma
atomic emission spectrometry analysis. The results showed that the
moss bound significant amounts of Fe, up to 15 mg/L in the 50 mg/L
sample. This experiment was run with distilled water washed moss
resulting in similar results. However, it was noted that the Fe
spiked samples had a low pH. Since optimal binding is at pH 4 to 6,
we adjusted the pH up to between 4 and 7 on the next experiment.
When the pH of the Fe samples was brought up, the Fe started to
precipitate. The moss removed all of the Fe from the sample (up to
25 mg/L). It was then decided that ions bound to the moss before
use could affect the cation binding tests and methods to remove the
cations from the moss should be investigated.
[0104] When developing the method for acid washing Sphagnum moss
(not peat moss, which is decomposed moss), two different acids were
first used. One batch of moss (approx. 5 g) was constantly stirred
in 3.5 L of distilled water pH adjusted to 1 with concentrated
HNO.sub.3. For the other batch of moss, a 2% solution of glacial
acetic acid was used. The washes were stirred for 1 hour, then the
supernatant was filtered off and new wash solution was put on the
moss. This was repeated for a total of four acid washes. The acid
washes were followed by 4 distilled water washes carried out the
same way. At the end of the distilled water washes, the
conductivity was similar to that of distilled water. The two acids
removed similar amounts of ions; therefore acetic acid was
routinely used for washing the moss thereafter.
[0105] It was decided that a metal that was soluble at neutral pH
would be better suited to test binding capacity and test for
improvement of binding with acid washed Sphagnum moss vs.
non-washed moss. Calcium was chosen for this purpose. The moss used
was the S. cristatum moss described earlier in this example, the
acid used was a 2% solution of glacial acetic acid, and the wash
was performed as described in the preceding paragraph. The first
test showed the opposite of what was expected, the non-washed moss
bound the most and the acid washed moss bound the least. When the
pH of the samples was checked, it was discovered that the binding
ability correlated with pH. The acid washed moss reduced the pH of
the solutions, which affected the ion binding. To eliminate this
problem, acid washed moss was washed with a high salt solution to
displace the H.sup.+ ions with Na. The high salt solution was 1M
sodium acetate and approximately 5 g of moss was constantly stirred
in 3.5 L of this sodium acetate solution for one hour, and then the
wash was repeated. These two sodium acetate washes were followed by
four washes with distilled water, each wash for one hour, and each
was carried out the same way. The acid/salt-washed moss was tested;
the pH of the extract did not drop and showed a significant
improvement of binding over distilled water and non-washed moss as
can be seen in the following table (concentrations of Ca are in
ppm).
TABLE-US-00008 Starting Resulting Moss treatment [Ca] Average [Ca]
Stdev pH Control 0 -1.08 0.61 6.02 Acetic Acid, Acetate 0 -0.97
0.45 7.62 Washed No wash 0 -0.14 0.51 5.26 Distilled water washed 0
0.36 1.32 5.13 Acetic Acid, Acetate 100 -1.40 0.25 6.62 washed No
wash 100 11.24 0.00 4.81 Distilled water washed 100 16.89 1.17 4.54
Control 100 88.76 0.05 6.65 Acetic Acid, Acetate 200 -0.33 0.87
6.17 washed No wash 200 46.47 5.88 4.82 Distilled water washed 200
53.55 4.42 4.61 Control 200 198.10 1.83 6.76
[0106] The pH of the extracts varied by up to 2 pH units, so it was
still difficult to distinguish between the effects of acid washing
and pH. Therefore a new method to test for binding of the moss was
developed that would allow adjustment of pH while the moss was
still in the sample solutions. This was accomplished by using
multi-well plates, small stir bars and 10 mg/ml moss
concentrations. After the solutions were on the moss for
approximately one-half hour, the pH's of the solutions were all
adjusted to within 0.25 pH units of 6.5. This allowed for the
testing of acid/salt washed moss and acid only washed moss. The
results are shown below. The averages shown are the averages of two
samples.
TABLE-US-00009 Starting Resulting Moss treatment [Ca] Average [Ca]
Stdev Acetic Acid/Na acetate 0.00 6.93 0.72 washed Acetic Acid
washed 0.00 6.16 0.16 Distilled water washed 0.00 6.85 0.31 No wash
0.00 8.54 1.56 Control 0.00 8.10 0.31 Acetic Acid/Na acetate 100.00
6.78 0.31 washed Acetic Acid washed 100.00 6.82 0.26 Distilled
water washed 100.00 20.73 1.09 No wash 100.00 26.25 0.72 Control
100.00 92.27 1.56 Acetic Acid/Na acetate 500.00 307.28 7.25 washed
Acetic Acid washed 500.00 319.25 8.13 Distilled water washed 500.00
375.18 3.36 No wash 500.00 382.85 4.38 Control 500.00 454.30
8.91
With the pH's adjusted to be within 0.5 pH units of each other; the
pH effect is eliminated and the significantly improved binding
capacity of the acid washed mosses can be seen. The acid/salt
washed moss bound slightly better than the acid only washed moss
and the distilled washed slightly better than the non-washed moss,
but barely enough to be significant.
[0107] Thus, the moss was shown to bind both Fe and Ca ions, and
therefore is effective in water treatment because the removal of
one or both of these ions is a goal of water treatment.
[0108] The above description and the drawings are provided for the
purpose of describing embodiments of the invention and are not
intended to limit the scope of the invention in any way. It will be
apparent to those skilled in the art that various modifications and
variations can be made without departing from the spirit or scope
of the invention. Thus, it is intended that the present invention
cover the modifications and variations of this invention provided
they come within the scope of the appended claims and their
equivalents.
* * * * *