U.S. patent application number 09/797493 was filed with the patent office on 2002-09-05 for methods and compositions for suppressing growth of pathogenic microbes.
Invention is credited to Cheung, Ling Y..
Application Number | 20020123126 09/797493 |
Document ID | / |
Family ID | 25170980 |
Filed Date | 2002-09-05 |
United States Patent
Application |
20020123126 |
Kind Code |
A1 |
Cheung, Ling Y. |
September 5, 2002 |
METHODS AND COMPOSITIONS FOR SUPPRESSING GROWTH OF PATHOGENIC
MICROBES
Abstract
Compositions comprising a plurality of yeast cells, wherein said
plurality of yeast cells have been cultured in the presence of an
alternating electric field having a specific frequency and a
specific field strength for a period of time sufficient to
substantially increase the capability of said plurality of yeast
cells to suppress the growth of pathogenic microbes. Also included
are methods of making such compositions.
Inventors: |
Cheung, Ling Y.; (Hong Kong,
HK) |
Correspondence
Address: |
FISH & NEAVE
1251 AVENUE OF THE AMERICAS
50TH FLOOR
NEW YORK
NY
10020-1105
US
|
Family ID: |
25170980 |
Appl. No.: |
09/797493 |
Filed: |
March 1, 2001 |
Current U.S.
Class: |
435/254.2 |
Current CPC
Class: |
B09C 1/10 20130101; C12N
1/16 20130101; C02F 1/50 20130101; C12N 13/00 20130101 |
Class at
Publication: |
435/254.2 |
International
Class: |
C12N 001/18 |
Claims
What is claimed is:
1. A composition comprising a plurality of yeast cells, wherein
said plurality of yeast cells have been cultured in the presence of
an alternating electric field having a frequency in the range of 30
to 50 MHz and a field strength in the range of 0.5 to 200 mV/cm for
a period of time sufficient to substantially increase the
capability of said plurality of yeast cells to suppress the growth
of pathogenic microbes.
2. The composition of claim 1, wherein said field strength is in
the range of 10 to 180 mV/cm.
3. The composition of claim 1, wherein said yeast cells are cells
of the species Saccharomyces cerevisiae, Saccharomyces
carlsbergensis, Saccharomyces uvarum, or Saccharomyces
willianus.
4. The composition of claim 1, wherein said yeast cells are cells
of the strain deposited at The China General Microbiological
Culture Collection Center with an accession number selected from
the group consisting of ACCC2034, ACCC2043, AS2.70, AS2.119,
AS2.152, AS2.200, AS2.369, AS2.408, AS2.451, AS2.562, AS2.607, IFFI
1021, IFFI 1023, IFFI 1032, IFFI 1037, IFFI 1205, IFFI 1211, IFFI
1221, IFFI1251, IFFI 1301, IFFI 1307, IFFI 1308, IFFI1331, and IFFI
1345.
5. The composition of claim 1, wherein said pathogenic microbe is
Staphylococcus aures.
6. The composition of claim 1, wherein said pathogenic microbe is
Diplococcus pneumonia.
7. The composition of claim 1, wherein said pathogenic microbe is
Bacillus anthracis.
8. The composition of claim 1, wherein said pathogenic microbe is
Mycobacterium tuberculosis.
9. The composition of claim 1, wherein said pathogenic microbe is
E. Coli.
10. The composition of claim 1, wherein said pathogenic microbe is
Salmonella.
11. A composition comprising a plurality of yeast cells, wherein
said plurality of yeast cells have been activated such that they
have a substantially increased capability to suppress the growth of
pathogenic microbes as compared to unactivated yeast cells.
12. A method of preparing a yeast composition, comprising culturing
a plurality of yeast cells in the presence of an alternating
electric field having a frequency in the range of 30 to 50 MHz and
a field strength in the range of 0.5 to 200 mV/cm for a period of
time sufficient to substantially increase the capability of said
plurality of yeast cells to suppress the growth of pathogenic
microbes.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the use of yeast cells to suppress
the growth of pathogenic microbes. These yeasts are useful in waste
treatment, and are obtained by growth in electromagnetic fields
with specific frequencies and field strengths.
BACKGROUND OF THE INVENTION
[0002] Environmental pollution by urban sewage and industrial waste
water has posed a serious health threat to living organisms in the
world. Currently, the most common methods for large-scale waste
treatment, such as water treatment, include the activated sludge
technology and the biomembrane technology. These technologies rely
on the innate abilities of myriad natural microorganisms, such as
fungi, bacteria and protozoa, to degrade pollutants. However, the
compositions of these natural microbial components are difficult to
control, affecting the reproducibility and quality of water
treatment. Moreover, pathogenic microbes existing in these
activated sludge or biomembranes cannot be selectively inhibited,
and such microbes usually enter the environment with the treated
water, causing "secondary pollution."
[0003] Further, most of the current technologies cannot degrade
harmful chemicals such as pesticides, insecticides, and chemical
fertilizers. These technologies also cannot alleviate
eutrophication, another serious environmental problem around the
world. Eutrophication is usually caused by sewage, industrial waste
water, fertilizers and the like. It refers to waters (e.g., a lake
or pond) rich in minerals and organic nutrients that promote a
proliferation of plant life, especially algae, which reduces the
dissolved oxygen content or otherwise deteriorates water quality.
Eutrophication often results in the extinction of other
organisms.
SUMMARY OF THE INVENTION
[0004] This invention is based on the discovery that certain yeast
cells can be activated by electromagnetic fields of specific
frequencies and field strengths to suppress the proliferation of
certain pathogenic microorganisms. Compositions comprising these
activated yeast cells can therefore be used for waste treatment,
for example, treatment of sewage, industrial waste water, surface
water, drinking water, sediment, soil, garbage, and manure, to
reduce the growth of pathogenic microbes in the waste. Waste
treatment methods using these compositions are more effective,
efficient, and economical than the conventional methods.
[0005] This invention embraces a composition comprising a plurality
of yeast cells that have been cultured in an alternating electric
field having a frequency in the range of about 30 to 50 MHz and a
field strength in the range of about 0.5 to 200 mV/cm (e.g., about
10 to 180 mV/cm). The yeast cells are cultured in the presence of
the alternating electric field for a period of time sufficient to
substantially increase the capability of said plurality of yeast
cells to suppress the proliferation of pathogenic microorganisms.
In one embodiment, the frequency and/or the field strength of the
alternating electric field can be altered within the aforementioned
ranges during said period of time. In other words, the yeast cells
can be exposed to a series of electromagnetic fields. An exemplary
period of time is about 12-300 hours (e.g., 144-272 hours).
[0006] Yeast cells that can be included in this composition are
available from the China General Microbiological Culture Collection
Center ("CGMCC"), a depository recognized under the Budapest Treaty
(China Committee for Culture Collection of Microorganisms,
Institute of Microbiology, Chinese Academy of Sciences, Haidian,
P.O. Box 2714, Beijing, 100080, China). Useful yeast species
include, but are not limited to, Saccharomyces cerevisiae,
Saccharomyces carlsbergensis, Saccharomyces uvarum, and
Saccharomyces willianus. For instance, the yeast cells can be of
the strain Saccharomyces cerevisiae Hansen ACCC2034, ACCC2043,
AS2.70, AS2.369, AS2.408, AS2.451, AS2.562, AS2.607, IFFI1021,
EFFI1037, EFFI1211, EFFI1221, IFFI1251, IFFI1301, IFFI1307,
IFFI1308, IFFI1331, or IFFI1345; Saccharomyces carlsbergensis
Hansen AS2.200; Saccharomyces uvarum Beijer IFFI1023, IFFI1032, or
IFFI1205; or Saccharomyces willianus Saccardo AS2.119 or
AS2.152.
[0007] This invention further embraces a composition comprising a
plurality of yeast cells, wherein said plurality of yeast cells
have been activated such that they have a substantially increased
capability to suppress the growth of pathogenic microbes as
compared to unactivated yeast cells. Included in this invention are
also methods of making these compositions.
[0008] As used herein, "suppressing the growth of pathogenic
microbes" means preventing the increase in, or even decreasing, the
number of pathogenic microorganisms. It is to be understood that in
the absence of yeast cells of this invention, the number of
pathogenic microbes will increase naturally over a period of time.
Pathogenic microorganisms include, but are not limited to, bacteria
such as those belonging to the Escherichia, Salmonella, Shigella,
Mycobacterium, Staphylococcus, Bacillus, Streptococcus and
Diplococcus genera.
[0009] A "substantially increase" means an increase of more than 10
(e.g., 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, or 10.sup.6)
fold.
[0010] A "culture medium" refers to a medium used in a laboratory
for selecting and growing a given yeast strain, or to liquid or
solid waste in need of treatment.
[0011] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Exemplary methods and materials are described below, although
methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention. All publications and other references mentioned herein
are incorporated by reference in their entirety. In case of
conflict, the present specification, including definitions, will
control. The materials, methods, and examples are illustrative only
and not intended to be limiting.
[0012] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic diagram showing an exemplary apparatus
for activating yeast cells using electromagnetic fields. 1: yeast
culture; 2: container; 3: power supply.
DETAILED DESCRIPTION OF THE INVENTION
[0014] This invention is based on the discovery that certain yeast
strains can be activated by electromagnetic fields ("EMF") having
specific frequencies and field strengths to become highly efficient
in suppressing the growth of certain pathogenic microbes. Yeast
cells having this function are defined herein as belonging to the
same "functional group." Compositions containing the activated
yeast cells are useful in waste treatment.
[0015] Without being bound by any theory or mechanism, the inventor
believes that EMFs activate or enhance the expression of a gene or
a set of genes in yeast cells such that the yeast cells become
active or more efficient in performing certain metabolic activities
which lead to the desired pathogen-suppressing result. These yeast
cells are believed to create an environment that is unfavorable for
the proliferation of certain pathogenic microorganisms.
[0016] I. Yeast Strains Useful in the Invention
[0017] The types of yeasts useful in this invention include, but
are not limited to, yeasts of the genera of Saccharomyces,
Schizosaccharomyces, Sporobolomyces, Torulopsis, Trichosporon,
Wickerhamia, Ashbya, Blastomyces, Candida, Citeromyces,
Crebrothecium, Cryptococcus, Debaryomyces, Endomycopsis,
Eremothecium, Geotrichum, Hansenula, Kloeckera, Lipomyces, Pichia,
Rhodosporidium, and Rhodotorula.
[0018] Exemplary species within the above-listed genera include,
but are not limited to, Saccharomyces cerevisiae, Saccharomyces
bailii, Saccharomyces carlsbergensis, Saccharomyces chevalieri,
Saccharomyces delbrueckii, Saccharomyces exiguus,
Saccharomycesfermentati, Saccharomyces logos, Saccharomyces mellis,
Saccharomyces microellipsoides, Saccharomyces oviformis,
Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces sake,
Saccharomyces uvarum, Saccharomyces willianus, Saccharomyces sp.,
Saccharomyces ludwigii, Saccharomyces sinenses, Saccharomyces
bailii, Saccharomyces carlsbergensis, Schizosaccharomyces
octosporus, Schizosaccharmryces pombe, Sporobolomyces roseus,
Sporobolomyces salmonicolor, Torulopsis candida, Torulopsisfamta,
Torulopsis globosa, Torulopsis inconspicua, Trichosporon behrendoo,
Trichosporon capitatum, Trichosporon cutaneum,
Wickerhamiafluoresens, Ashbya gossypii, Blastomyces dermatitidis,
Candida albicans, Candida arborea, Candida guilliermondii, Candida
krusei, Candida lambica, Candida lipolytica, Candida parakrusei,
Candida parapsilosis, Candida pseudotropicalis, Candida
pulcherrima, Candida robusta, Candida rugousa, Candida tropicalis,
Candida utilis, Citeromyces matritensis, Crebrothecium ashbyii,
Cryptococcus laurentii, Cryptococcus neoformans, Debaryomyces
hansenii, Debaryomyces kloeckeri, Debaryomyces sp.,
Endomycopsisfibuligera, Eremothecium ashbyii, Geotrichum candidum,
Geotrichum ludwigii, Geotrichum robustum, Geotrichum suaveolens,
Hansenula anomala, Hansenula arabitolgens, Hansenulajadinii,
Hansenula saturnus, Hansenula schneggii, Hansenula subpelliculosa,
Kloeckera apiculata, Lipomyces starkeyi, Pichiafarinosa, Pichia
membranaefaciens, Rhodosporidium toruloides, Rhodotorula
aurantiaca, Rhodotorula glutinis, Rhodotorula minuta, Rhodotorula
rubar, and Rhodotorula sinesis.
[0019] Yeast strains useful for this invention can be obtained from
laboratory cultures, or from publically accessible culture
depositories, such as CGMCC and the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209.
Non-limiting examples of useful strains (with accession numbers of
CGMCC) are Saccharomyces cerevisiae Hansen ACCC2034, ACCC2043,
AS2.70, AS2.369, AS2.408, AS2.451, AS2.562, AS2.607, IFFI1021,
IFFI1037, IFFI1211, IFFI1221, IFFI1251, IFFI1301, IFFI1307,
IFFI1308, IFFI1331, and IFFI1345; Saccharomyces carlsbergensis
Hansen AS2.200; Saccharomyces uvarum Beijer IFFI1023, IFFI1032, and
IFFI1205; and Saccharomyces willianus Saccardo AS2.119 and
AS2.152.
[0020] Although it is preferred, the preparation of the yeast
compositions of this invention is not limited to starting with a
pure strain of yeast. A yeast composition of the invention may be
produced by culturing a mixture of yeast cells of different species
or strains that have the same pathogen-suppressing function. The
ability of any species or strain of yeasts to perform this function
can be readily tested by methods known in the art.
[0021] Certain yeast species that can be activated according to the
present invention are known to be pathogenic to human and/or other
living organisms. These yeast species include, for example, Ashbya
gossypii, Blastomyces dermatitidis, Candida albicans, Candida
parakrusei, Candida tropicalis, Citeromyces matritensis,
Crebrothecium ashbyii, Cryptococcus laurentii, Cryptococcus
neoformans, Debaryomyces hansenii, Debaryomyces kloeckeri,
Debaryomyces sp., and Endomycopsis fibuligera. Under certain
circumstances, it may be less preferable to use such pathogenic
yeasts in this invention. If use of these species is necessary,
caution should be exercised to minimize the leak of the yeast cells
into the final treatment product that enters the environment.
[0022] II. Application of Electromagnetic Fields
[0023] An electromagnetic field useful in this invention can be
generated and applied by various means well known in the art. For
instance, the EMF can be generated by applying an alternating
electric field or an oscillating magnetic field.
[0024] Alternating electric fields can be applied to cell cultures
through electrodes in direct contact with the culture medium, or
through electromagnetic induction. See, e.g., FIG. 1. Relatively
high electric fields in the medium can be generated using a method
in which the electrodes are in contact with the medium. Care must
be taken to prevent electrolysis at the electrodes from introducing
undesired ions into the culture and to prevent contact resistance,
bubbles, or other features of electrolysis from dropping the field
level below that intended. Electrodes should be matched to their
environment, for example, using Ag-AgCl electrodes in solutions
rich in chloride ions, and run at as low a voltage as possible. For
general review, see Goodman et al., Effects of EMF on Molecules and
Cells, International Review of Cytology, A Survey of Cell Biology,
Vol. 158, Academic Press, 1995.
[0025] The EMFs useful in this invention can also be generated by
applying an oscillating magnetic field. An oscillating magnetic
field can be generated by oscillating electric currents going
through Helmholtz coils. Such a magnetic field in turn induces an
electric field.
[0026] The frequencies of EMFs useful in this invention range from
30 MHz to 50 MHz. Exemplary frequencies are 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 MHz.
The field strength of the electric field useful in this invention
ranges from about 0.5 to 200 mV/cm, e.g., 10 to 180 mV/cm.
Exemplary field strengths are 26 and 150 mV/cm.
[0027] When a series of EMFs are applied to a yeast culture, the
yeast culture can remain in the same container while the same set
of EMF generator and emitters is used to change the frequency
and/or field strength. The EMFs in the series can each have a
different frequency or a different field strength; or a different
frequency and a different field strength. Such frequencies and
field strengths are preferably within the above-described ranges.
In one embodiment, an EMF at the beginning of the series has a
field strength identical to or lower than that of a subsequent EMF,
such that the yeast cell culture is exposed to EMFs of
progressively increasing field strength. Although any practical
number of EMFs can be used in a series, it may be preferred that
the yeast culture be exposed to a total of 2, 3, 4, 5, 6, 7, 8, 9
or 10 EMFs in a series.
[0028] By way of example, the yeast cells can be cultured in a
first series of alternating electric fields each having a frequency
in the range of 30 to 50 MHz and a field strength in the range of
10 to 180 mV/cm. The yeast cells are exposed to each EMF for about
12 hours. After culturing in the first series of EMFs, the
resultant yeast cells are further incubated under substantially the
same conditions in a second series of alternating electric fields
for a total of 24 to 96 hours. It may be preferred that the
frequencies in the second series of alternating electric fields are
identical to those of the first series in sequence and the field
strengths in the second series are increased to a higher level
within the range of 10 to 180 mV/cm.
[0029] Although the yeast cells can be activated after even a few
hours of culturing in the presence of an EMF, it may be preferred
that the activated yeast cells be allowed to multiply and grow in
the presence of the EMF(s) for a total of 144-272 hours.
[0030] FIG. 1 illustrates an exemplary apparatus for generating
alternating electric fields. An electric field of a desired
frequency and intensity is generated by an AC source (3) capable of
generating an alternating electric field, preferably in a
sinusoidal wave form, in the frequency range of 5 to 5000 MHz.
Signal generators capable of generating signals with a narrower
frequency range can also be used. If desirable, a signal amplifier
can also be used to increase the output. The alternating electric
field can be applied to the culture by a variety of means including
placing the yeast culture in close proximity to the signal
emitters. In one embodiment, the electric field is applied by
electrodes submerged in the culture (1). In this embodiment, one of
the electrodes can be a metal plate placed on the bottom of the
container (2), and the other electrode can comprise a plurality of
electrode wires evenly distributed in the culture (1) so as to
achieve even distribution of the electric field energy. The number
of electrode wires used depends on the volume of the culture as
well as the diameter of the wires. In a preferred embodiment, for a
culture having a volume up to 5000 ml, one electrode wire having a
diameter of 0.1 to 1.2 mm can be used for each 100 ml of culture.
For a culture having a volume greater than 1000 L, one electrode
wire having a diameter of 3 to 30 mm can be used for each 1000 L of
culture.
[0031] III. Culture Media
[0032] Culture media useful in this invention contain sources of
nutrients assimilable by yeast cells. In this invention, a culture
medium refers to a laboratory culture medium, or liquid or solid
waste in need of treatment. Complex carbon-containing substances in
a suitable form, such as carbohydrates (e.g., sucrose, glucose,
fructose, dextrose, maltose, xylose, cellulose, starches, etc.) and
coal, can be the carbon sources for yeast cells. The exact quantity
of the carbon sources utilized in the medium can be adjusted in
accordance with the other ingredients of the medium. In general,
the amount of carbohydrates varies between about 0.1% and 5% by
weight of the medium and preferably between about 0.1% and 2%, and
most preferably about 1%. These carbon sources can be used
individually or in combination. Among the inorganic salts which can
be added to the culture medium are the customary salts capable of
yielding sodium, potassium, calcium, phosphate, sulfate, carbonate,
and like ions. Non-limiting examples of nutrient inorganic salts
are (NH.sub.4).sub.2HPO.sub.4, KH.sub.2PO.sub.4, K.sub.2HP0.sub.4,
CaCO.sub.3, MgSO.sub.4, NaCl, and CaSO.sub.4.
[0033] IV. Electromagnetic Activation of Yeast Cells
[0034] Yeasts of this invention can suppress the natural
proliferation of pathogenic microbes. Normally, in the presence of
ample nutrients, the number of pathogenic microbes would increase
naturally over a period of time. These pathogenic microbes include,
but are not limited to, bacteria such as those belonging to the
Escherichia, Salmonella, Shigella, Mycobacterium, Staphylococcus,
Bacillus, Streptococcus and Diplococcus genera.
[0035] To activate the innate ability of yeast cells to suppress
the growth of pathogenic microbes, these yeast cells can be
cultured in an appropriate medium under sterile conditions at
25.degree. C.-30.degree. C., e.g., 28.degree. C., for a sufficient
amount of time, e.g., 12-300 hours (for example, 144-272 hours), in
an alternating electric field or a series of alternating electric
fields as described above. The culturing process may preferably be
conducted under conditions in which the concentration of dissolved
oxygen is between 0.025 to 0.8 mol/m.sup.3, preferably 0.4
mol/m.sup.3. The oxygen level can be controlled by, for example,
stirring and/or bubbling.
[0036] An exemplary culture medium is made by mixing 400 ml of
sterile water, 8 g of soluble starch, 5 g of sucrose, 0.2 g of
NaCl, 0.2 g of MgSO.sub.4.7H.sub.2O, 0.5 g of CaCO.sub.3.5H.sub.2O,
0.2 g of CaSO.sub.4.2H.sub.2O, 0.5 g of K.sub.2HPO.sub.4, 1.5 g of
peptone, and 600 ml of sludge extract containing pathogenic
microbes.
[0037] Subsequently, the yeast cells can be measured for their
ability to suppress the growth of pathogenic microbes using
standard methods known in the art for counting microorganisms, such
as optical density, plating out dilutions on solid media for
counting, or counting individual cells under a microscope. Stains
may be applied to distinguish or identify different strains or
species of microorganisms present in a sample, or to determine
their viability.
[0038] In one exemplary method, sewage containing more than
10.sup.8 cells/ml Gram-positive Escherichia coli, 10.sup.9 cells/ml
Salmonella, and 10.sup.9 cells/ml Shigella dysenteriae is used as a
substrate. The sewage is inoculated with a dry yeast cell
preparation at a concentration of 0.3-0.6 g/L, and cultured for 24
hours at 10-40.degree. C. The difference between the numbers of the
above-mentioned live bacteria before and after 24 hours indicates
the pathogen-suppressing capacity of the yeast cells.
[0039] Essentially the same protocol as described above can be used
to grow activated yeast cells. To initiate the process, each 100 ml
of culture medium is inoculated with yeast cells of the same
functional group at a density of 10.sup.2-10.sup.5 cells/ml,
preferably 3.times.10.sup.2-10.sup.4 cell/ml. The culturing process
is carried out at about 20-40.degree. C., preferably at about
25-28.degree. C., for 48-96 hours. The process can be scaled up or
down according to needs. For an industrial scale of production,
seventy-five liters of a sterile culture medium are inoculated with
the yeast cells. This culture medium consists of 10 L of the
culture medium described above for this particular yeast functional
group, 30 kg of starch, and 65 L of distilled water. At the end of
the culturing process, the yeast cells may preferably reach a
concentration of 2.times.10.sup.10 cells/ml. The cells are
recovered from the culture by various methods known in the art, and
stored at about 15-20.degree. C. The yeast should be dried within
24 hours and stored in powder form.
[0040] V. Acclimatization of Yeast Cells to Waste Environment
[0041] In yet another embodiment of the invention, the yeast cells
may also be cultured under certain conditions so as to acclimatize
the cells to a particular type of waste. This acclimatization
process results in better growth and survival of the yeasts in a
particular waste environment.
[0042] To achieve this, the yeast cells of a given functional group
can be mixed with waste material from a particular source at
10.sup.6 to 10.sup.8 cells (e.g., 10.sup.7 cells) per 1000 ml. The
yeast cells are then exposed to an alternating electric field as
described above. The strength of the electric field can be 100 to
400 mV/cm (e.g., 120 to 250 mV/cm). The culture is incubated at
temperatures that cycle between about 5.degree. C. to about
45.degree. C. at a 5.degree. C. increment. For example, in a
typical cycle, the temperature of the culture may start at
5.degree. C. and be kept at this temperature for about 1-2 hours,
then adjusted up to 10.degree. C. and kept at this temperature for
1-2 hours, then adjusted to 15.degree. C. and kept at this
temperature for about 1-2 hours, and so on and so forth, until the
temperature reaches 45.degree. C. Then the temperature is brought
down to 40.degree. C. and kept at this temperature for about 1-2
hours, and then to 35.degree. C. and kept at this temperature for
about 1-2 hours, and so on and so forth, until the temperature
returns to 5.degree. C. The cycles are repeated for about 48-96
hours. The resulting yeast cells are then dried and stored at
0-4.degree. C.
[0043] VI. Manufacture of the Waste Treatment Compositions
[0044] Yeast cells of this invention can be mixed with an
appropriate filler, such as rock powder and coal ash at the
following ratio: 600 L of yeast cell culture at 2.times.10.sup.10
cells/ml and 760 kg of filler materials. The mixture is quickly
dried at a temperature below 65.degree. C. for 10 minutes in a
dryer, and then further dried at a temperature below 70.degree. C.
for no more than 30 minutes so that the water content is less than
7%. The dried composition is then cooled to room temperature for
packaging.
[0045] These dried yeast compositions may be used to treat polluted
surface water, sewage, or any other type of waste water. To treat
polluted surface water, a yeast solution may be prepared by adding
1 kg of the dried yeast composition to 30 L of clean water. The
yeast solution is then sprayed onto the polluted surface water at
about 1-3 L of the solution per square meter of the polluted
surface water. To treat sewage or any other type of waste water, a
yeast solution may be prepared by adding about 1 kg of the dried
yeast composition to 10-30 L of clean water. The yeast solution is
incubated at 10-35.degree. C. for 24-48 hours. The resultant yeast
solution is then added to the waste water at about 3-20 L of the
solution per liter of waste water.
VII. EXAMPLES
[0046] In order that this invention be more fully understood, the
following examples are set forth. These examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in any way.
Example 1
[0047] Suppression of the Growth of Staphylococcus aures
[0048] Saccharomyces cerevisiae Hansen IFFI1037 cells were cultured
in the presence of a series of alternating electric fields in the
following sequence: the yeast cells were exposed to (1) an
alternating electric field having a frequency of 30 MHz and a field
strength of 26 mV/cm for 12 hours; (2) then to an alternating
electric field having a frequency of 36 MHz and a field strength of
26 mV/cm for 12 hours; (3) then to an alternating electric field
having a frequency of 43 MHz and a field strength of 26 mV/cm for
12 hours; (4) then to an alternating electric field having a
frequency of 47 MHz and a field strength of 26 mV/cm for 12 hours;
(5) then to an alternating electric field having a frequency of 30
MHz and a field strength of 150 mV/cm for 24 hours; (6) then to an
alternating electric field having a frequency of 36 MHz and a field
strength of 150 mV/cm for 24 hours; (7) then to an alternating
electric field having a frequency of 43 MHz and a field strength of
150 mV/cm for 24 hours; and (8) finally to an alternating electric
field having a frequency of 47 MHz and a field strength of 150
mV/cm for 24 hours.
[0049] To test the ability of the EMF-treated IFFI1037 cells to
suppress the growth of Staphylococcus aures, waste water or
filtrate from animal manure or garbage containing Staphylococcus
aures was incubated under routine conditions to reconstitute a
solution containing Staphylococcus aures at more than 10.sup.10
cells/ml. One milliliter of the EMF-treated IFFI1037 cells at a
concentration of 2.times.10.sup.8-5.times.10.sup.8 cells/ml was
added to 1 L of the Staphylococcus aures solution and cultured at
30.degree. C. for 24 hours (solution A). One liter of the
Staphylococcus aures solution containing the same number of
non-treated yeast cells (solution B) or containing no yeast cells
(solution C) was used as controls. After 24 hours of incubation,
the solutions were examined using a flow cytometer. The results
showed that after 24 hours of incubation, the number of live
Staphylococcus aures in solution A decreased more than 2.7%
relative to solution C. In contrast, the number of live
Staphylococcus aures in solution B showed no significant change
relative to solution C.
Example 2
[0050] Suppression of the Growth of Diplococcus pneumonia
[0051] Saccharomyces cerevisiae Hansen IFFI1021 cells were cultured
in the presence of a series of alternating electric fields in the
following sequence: the yeast cells were exposed to (1) an
alternating electric field having a frequency of 30 MHz and a field
strength of 26 mV/cm for 12 hours; (2) then to an alternating
electric field having a frequency of 36 MHz and a field strength of
26 mV/cm for 12 hours; (3) then to an alternating electric field
having a frequency of 42 MHz and a field strength of 26 mV/cm for
12 hours; (4) then to an alternating electric field having a
frequency of 49 MHz and a field strength of 26 mV/cm for 12 hours;
(5) then to an alternating electric field having a frequency of 30
MHz and a field strength of 150 mV/cm for 24 hours; (6) then to an
alternating electric field having a frequency of 36 M and a field
strength of 150 mV/cm for 24 hours; (7) then to an alternating
electric field having a frequency of 42 MHz and a field strength of
150 mV/cm for 24 hours; and (8) finally to an alternating electric
field having a frequency of 49 MHz and a field strength of 150
mV/cm for 24 hours.
[0052] To test the ability of the EMF-treated IFFI1021 cells to
suppress the growth of Diplococcus pneumonia, waste water or
filtrate from animal manure or garbage containing Diplococcus
pneumonia was incubated under routine conditions to reconstitute a
solution containing Diplococcus pneumonia at more than 10.sup.10
cells/ml. One milliliter of the EMF-treated IFFI1021 cells at a
concentration of 2.times.10.sup.8-5.times- .10.sup.8 cells/ml was
added to 1 L of the Diplococcus pneumonia solution and cultured at
30.degree. C. for 24 hours (solution A). One liter of the
Diplococcus pneumonia solution containing the same number of
non-treated yeast cells (solution B) or containing no yeast cells
(solution C) was used as controls. After 24 hours of incubation,
the solutions were examined using a flow cytometer. The results
showed that after 24 hours of incubation, the number of live
Diplococcus pneumonia in solution A decreased more than 2.8%
relative to solution C. In contrast, the number of live Diplococcus
pneumonia in solution B showed no significant change relative to
solution C.
Example 3
[0053] Suppression of the Growth of Bacillus anthracis
[0054] Saccharomyces cerevisiae Hansen IFFI1251 cells were cultured
in the presence of a series of alternating electric fields in the
following sequence: the yeast cells were exposed to (1) an
alternating electric field having a frequency of 35 MHz and a field
strength of 26 mV/cm for 12 hours; (2) then to an alternating
electric field having a frequency of 39 MHz and a field strength of
26 mV/cm for 12 hours; (3) then to an alternating electric field
having a frequency of 43 MHz and a field strength of 26 mV/cm for
12 hours; (4) then to an alternating electric field having a
frequency of 47 MHz and a field strength of 26 mV/cm for 12 hours;
(5) then to an alternating electric field having a frequency of 35
MHz and a field strength of 150 mV/cm for 24 hours; (6) then to an
alternating electric field having a frequency of 39 MHz and a field
strength of 150 mV/cm for 24 hours; (7) then to an alternating
electric field having a frequency of 43 MHz and a field strength of
150 mV/cm for 24 hours; and (8) finally to an alternating electric
field having a frequency of 47 MHz and a field strength of 150
mV/cm for 24 hours.
[0055] To test the ability of the EMF-treated IFFI1251 cells to
suppress the growth of Bacillus anthracis, waste water or filtrate
from animal manure or garbage containing Bacillus anthracis was
incubated under routine conditions to reconstitute a solution
containing Bacillus anthracis at more than 10.sup.10 cells/ml. One
milliliter of the EMF-treated IFFI1251 cells at a concentration of
2.times.10.sup.8-5.times- .10.sup.8 cells/ml was added to 1 L of
the Bacillus anthracis solution and cultured at 30.degree. C. for
24 hours (solution A). One liter of the Bacillus anthracis solution
containing the same number of non-treated yeast cells (solution B)
or containing no yeast cells (solution C) was used as controls.
After 24 hours of incubation, the solutions were examined using a
flow cytometer. The results showed that after 24 hours of
incubation, the number of live Bacillus anthracis in solution A
decreased more than 3.1% relative to solution C. In contrast, the
number of live Bacillus anthracis in solution B showed no
significant change relative to solution C.
Example 4
[0056] Suppression of the Growth of Mycobacterium tuberculosis
[0057] Saccharomyces cerevisiae Hansen IFFI1331 cells were cultured
in the presence of a series of alternating electric fields in the
following sequence: the yeast cells were exposed to (1) an
alternating electric field having a frequency of 33 MHz and a field
strength of 26 mV/cm for 12 hours; (2) then to an alternating
electric field having a frequency of 36 MHz and a field strength of
26 mV/cm for 12 hours; (3) then to an alternating electric field
having a frequency of 45 MHz and a field strength of 26 mV/cm for
12 hours; (4) then to an alternating electric field having a
frequency of 47 MHz and a field strength of 26 mV/cm for 12 hours;
(5) then to an alternating electric field having a frequency of 33
MHz and a field strength of 150 mV/cm for 24 hours; (6) then to an
alternating electric field having a frequency of 36 MHz and a field
strength of 150 mV/cm for 24 hours; (7) then to an alternating
electric field having a frequency of 45 MHz and a field strength of
150 mV/cm for 24 hours; and (8) finally to an alternating electric
field having a frequency of 47 MHz and a field strength of 150
mV/cm for 24 hours.
[0058] To test the ability of the EMF-treated IFFI1331 cells to
suppress the growth of Mycobacterium tuberculosis, waste water or
filtrate from animal manure or garbage containing Mycobacterium
tuberculosis was incubated under routine conditions to reconstitute
a solution containing Mycobacterium tuberculosis at more than
10.sup.10 cells/ml. One milliliter of the EMF-treated IFFI1331
cells at a concentration of 2.times.10.sup.8-5.times.10.sup.8
cells/ml was added to 1 L of the Mycobacterium tuberculosis
solution and cultured at 30.degree. C. for 24 hours (solution A).
One liter of the Mycobacterium tuberculosis solution containing the
same number of non-treated yeast cells (solution B) or containing
no yeast cells (solution C) was used as controls. After 24 hours of
incubation, the solutions were examined using a flow cytometer. The
results showed that after 24 hours of incubation, the number of
live Mycobacterium tuberculosis in solution A decreased more than
2.9% relative to solution C. In contrast, the number of live
Mycobacterium tuberculosis in solution B showed no significant
change relative to solution C.
Example 5
[0059] Suppression of the Growth of E. Coli
[0060] Saccharomyces cerevisiae Hansen IFFI1345 cells were cultured
in the presence of a series of alternating electric fields in the
following sequence: the yeast cells were exposed to (1) an
alternating electric field having a frequency of 30 MHz and a field
strength of 26 mV/cm for 12 hours; (2) then to an alternating
electric field having a frequency of 34 MHz and a field strength of
26 mV/cm for 12 hours; (3) then to an alternating electric field
having a frequency of 38 MHz and a field strength of 26 mV/cm for
12 hours; (4) then to an alternating electric field having a
frequency of 49 MHz and a field strength of 26 mV/cm for 12 hours;
(5) then to an alternating electric field having a frequency of 30
MHz and a field strength of 150 mV/cm for 24 hours; (6) then to an
alternating electric field having a frequency of 34 MHz and a field
strength of 150 mV/cm for 24 hours; (7) then to an alternating
electric field having a frequency of 38 MHz and a field strength of
150 mV/cm for 24 hours; and (8) finally to an alternating electric
field having a frequency of 49 MHz and a field strength of 150
mV/cm for 24 hours.
[0061] To test the ability of the EMF-treated IFFI1345 cells to
suppress the growth of E. Coli, waste water or filtrate from animal
manure or garbage containing E. Coli was incubated under routine
conditions to reconstitute a solution containing E. Coli at more
than 10.sup.10 cells/ml. One milliliter of the EMF-treated IFFI1345
cells at a concentration of 2.times.10.sup.8-5.times.10.sup.8
cells/ml was added to 1 L of the E. Coli solution and cultured at
30.degree. C. for 24 hours (solution A). One liter of the E. Coli
solution containing the same number of non-treated yeast cells
(solution B) or containing no yeast cells (solution C) was used as
controls. After 24 hours of incubation, the solutions were examined
using a flow cytometer. The results showed that after 24 hours of
incubation, the number of live E. Coli in solution A decreased more
than 48% relative to solution C. In contrast, the number of live E.
Coli in solution B showed no significant change relative to
solution C.
Example 6
[0062] Suppression of the Growth of Salmonella
[0063] Saccharomyces cerevisiae Hansen IFFI1211 cells were cultured
in the presence of a series of alternating electric fields in the
following sequence: the yeast cells were exposed to (1) an
alternating electric field having a frequency of 30 MHz and a field
strength of 26 mV/cm for 12 hours; (2) then to an alternating
electric field having a frequency of 33 MHz and a field strength of
26 mV/cm for 12 hours; (3) then to an alternating electric field
having a frequency of 36 MHz and a field strength of 26 mV/cm for
12 hours; (4) then to an alternating electric field having a
frequency of 38 MHz and a field strength of 26 mV/cm for 12 hours;
(5) then to an alternating electric field having a frequency of 30
MHz and a field strength of 150 mV/cm for 24 hours; (6) then to an
alternating electric field having a frequency of 33 MHz and a field
strength of 150 mV/cm for 24 hours; (7) then to an alternating
electric field having a frequency of 36 MHz and a field strength of
150 mV/cm for 24 hours; and (8) finally to an alternating electric
field having a frequency of 38 MHz and a field strength of 150
mV/cm for 24 hours.
[0064] To test the ability of the EMF-treated IFFI1211 cells to
suppress the growth of Salmonella, waste water or filtrate from
animal manure or garbage containing Salmonella was incubated under
routine conditions to reconstitute a solution containing Salmonella
at more than 10.sup.10 cells/ml. One milliliter of the EMF-treated
IFFI1211 cells at a concentration of
2.times.10.sup.8-5.times.10.sup.8 cells/ml was added to 1 L of the
Salmonella solution and cultured at 30.degree. C. for 24 hours
(solution A). One liter of the Salmonella solution containing the
same number of non-treated yeast cells (solution B) or containing
no yeast cells (solution C) was used as controls. After 24 hours of
incubation, the solutions were examined using a flow cytometer. The
results showed that after 24 hours of incubation, the number of
live Salmonella in solution A decreased more than 66% relative to
solution C. In contrast, the number of live Salmonella in solution
B showed no significant change relative to solution C.
[0065] While a number of embodiments of this invention have been
set forth, it is apparent that the basic constructions may be
altered to provide other embodiments which utilize the compositions
and methods of this invention.
* * * * *