Note: Descriptions are shown in the official language in which they were submitted.
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A BIOLOGICAL FERTILIZER BASED ON YEASTS
1. FIELD OF THE INVENTION
The invention relates to a biological fertilizer that comprises yeasts for
fixing
atmospheric nitrogen, and decomposing insoluble compounds containing
phosphorus,
potassium and/or carbon. The invention also relates to methods for
manufacturing the
biological fertilizer, and methods for using the biological fertilizer to
increase crop yields.
2. BACKGROUND OF THE INVENTION
Use of fertilizer is essential in supporting the growth of high yield crops.
Of
the basic nutrients that plants need for healthy growth, large amounts of
nitrogen (taken up
as NO; or NH,~+), phosphorus (taken up as HZP04 ), and potassium (taken up as
K~)
nutrients are required by most crops on most soils (Wichmann, W., et al., IFA
World
Fertilizer Use Manual). Such large amounts of nitrogen, phosphorus, and
potassium
nutrients are supplied mainly in the form of mineral fertilizers, either
processed natural
minerals or manufactured chemicals (K.F. Isherwood, 1998, Mineral Fertilizer
Use and the
Environment, United Nations Environmental Programme Technical Report No. 26.).
The
development and use of mineral fertilizers since the 1940s has permitted
significant
increases in crop yields on the same to slightly less amount of cropland to
support today's
enormous population. Without such advances in agriculture, a great amount of
pastures and
forests would have been converted into cropland. (K.F. Isherwood, 1998,
Mineral Fertilizer
Use and the Environment, United Nations Environmental Programme Technical
Report No.
26.)
Despite the importance of mineral fertilizers in providing mankind with
abundant agricultural products, the harm done to the environment has been
recognized in
the recent years. Mineral fertilizers may incurred damages to soils. For
example, most
nitrogen fertilizers may acidify soils, thereby adversely affecting the growth
of plants and
other soil organisms. Extensive use of chemical nitrogen fertilizers may also
inhibit the
activity of natural nitrogen fixing microorganisms, thereby decreasing the
natural fertility of
soils. Mineral fertilizers may also introduce toxic substances into soil and
produce. For
example, phosphate fertilizers processed from rock phosphate often contain
small amounts
of toxic elements, such as cadmium, which may build up in soil and be taken up
by plants.
The long term use of mineral fertilizers may also cause severe environmental
pollution. For
example, the loss of nitrogen and phosphate fertilizers due to leaching and
soil erosion has
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led to contamination of soil and ground water, and eutrophication of surface
water.
Cleaning up polluted soil and water has been a complicated and difficult task.
The cost for
such a task is also astronomical.
In search for a solution to the problem, some are going back to organic
fertilizers. As is well known, organic fertilizers come from many different
sources. Types
of organic fertilizer include farm wastes, such as crop residues and animal
manures;
residues from plant and animal products, such as wood materials; and town
wastes, such as
sewage (Wichmann, W., et al., IFA World Fertilizer Use Manual). Organic
fertilizers are
usually low in nutrients and less effective in supporting plant growth. For
example, the total
nutrients in cattle manure is less than 2%, and the nitrogen nutrients therein
are more
difficult to be effectively utilized due to their losses into the environment
(K.F. Isherwood,
1998. Mineral Fertilizer Use and the Environment, United Nations Environmental
Programme Technical Report No. 26.). Normally, very large amount of organic
fertilizers
have to be applied to soil. To reach high crop yield, organic fertilizers have
only been used
to supplement mineral fertilizers. Therefore, the problems with mineral
fertilizers cannot be
satisfactorily solved by substituting mineral fertilizer with organic
fertilizer. Furthermore,
organic fertilizers also have created environmental problems. For example,
some organic
fertilizers, if unprocessed, contains pathogenic microorganisms, such as E.
coli,
Salmonella, and Coccidae. Organic fertilizers may also contain toxic chemicals
and may
produce undesirable odor. The use of organic fertilizer also contribute to the
contamination
and eutrophication of the natural water system. Therefore, in many parts of
the world,
including the United States, laws and regulations have been established
imposing
considerable restriction on both the composition and the usage of organic
fertilizers.
Biological fertilizers utilizing microorganisms have been proposed as
alternatives to mineral fertilizers. Naturally occurnng nitrogen fixing
microorganisms
including bacteria, such as Rhizobium, Azotobacter, and Azospirillum, (See for
example, U.
S. Patent No. 5,071,462) and fungi, such as Aspergillus flavus-ory~ae, (See,
for example, U.
S. Patent No. 4,670,037) have been utilized in biological fertilizers.
Naturally occurring
microorganisms capable of solubilizing rock phosphate ore or other insoluble
phosphates
into soluble phosphates have also been utilized in biological fertilizers
either separately
(e.g., U. S. Patent No. 5,912,398) or in combination with nitrogen fixing
microorganisms
(e.g., U..S. Patent No. 5,484,464). Genetically modified bacterial strains
have also been
developed and utilized in biological fertilizers. An approach based on
recombinant DNA
techniques has been developed to create more effective nitrogen fixing,
phosphorus
decomposing, and potassium decomposing bacterial strains for use in a
biological fertilizer,
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see, fox example, U.S. Patent No. 5,578,486; PCT publication WO 95/09814;
Chinese
patent publication: CN 1081662A; CN 1082016A; CN 1082017A; CN 1103060A; and CN
1I09595A.
However, the biological fertilizers that are based on naturally occurring
microorganisms are generally not efficient enough to effectively replace
mineral fertilizers.
It is therefore important to develop biological fertilizers that can replace
mineral fertilizers
in supplying nitrogen, phosphorus, and potassium to crops for producing high
quality
agricultural products while avoiding the problems associated with mineral
fertilizers. The
present invention provides a biological fertilizer based on yeasts, which can
replace mineral
fertilizers.
Citation of documents herein is not intended as an admission that any of the
documents cited herein is pertinent prior art, or an admission that the cited
documents are
considered material to the patentability of the claims of the present
application. All
statements as to the date or representations as to the contents of these
documents are based
on the information available to the applicant and does not constitute any
admission as to the
correctness of the dates or contents of these documents.
3. SUMMARY OF THE INVENTION
The present invention relates to biological fertilizers. The biological
fertilizer compositions of the invention may comprise up to six different
yeast cell
components, an organic substrate component andlor an inorganic substrate
component. In
particular, the yeast cell components of the composition are capable of fixing
atmospheric
nitrogen, decomposing insoluble minerals or compounds, decomposing complex
carbon
materials or compounds, overproducing growth factors, or overproducing ATP,
respectively.
The present invention uses yeasts that are commercially available and/or
accessible to the public, such as but not limited to Saccharomyces cerevisiae.
The yeast cell
components of the invention are produced by culturing yeast cells under
activation
conditions such that the abilities of the cells to fix atmospheric nitrogen,
to decompose
insoluble phosphorus minerals ox compounds, to decompose insoluble potassium
minerals
or compounds, and to decompose complex carbon materials or compounds are
activated or
enhanced. The yeast cells can also be cultured under conditions such that
their abilities to
produce excess growth factors or ATP are activated or enhanced. Yeast cells
exhibiting
such activities are useful in converting nitrogen from the atmosphere to
nitrogenous
compounds that can be used by plants as nutrients, releasing the otherwise
insoluble
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phosphorus, potassium and carbon from minerals and complex molecules, such
that these
elements become available in a form that the plant can utilize for growth.
Some yeast cells
in the fertilizer are used for supporting other plant nutrient-providing yeast
cells by
supplying them with growth factors and ATP.
The present invention also involves the use of a wide variety of organic and
inorganic materials in the fertilizer to support the growth of the yeast
strains of the present
invention. In one embodiment, the fertilizer is produced by mixing coal-mine
waste and
rock phosphate with the yeast strains. In another embodiment, the fertilizer
is produced by
mixing animal manures, and optionally, a biological disinfectant, with the
yeast strains. In
yet another embodiment, the fertilizer is produced by mixing sludge from
sewage water
treatment plant and a biological disinfectant with the yeast strains.
The invention also relates to methods for manufacturing the fertilizer
comprising mixing, drying, and packing the yeast strains of the present
invention and the
organic and/or inorganic materials.
The invention further relates to methods for using the fertilizer of the
present
invention. The biological fertilizers of the present invention are used to
support and
enhance the growth and maturation of a wide variety of plants.
4. BRIEF DESCRIPTION OF FIGURES
Fig. 1. Activation of yeast cells. 1 yeast culture; 2 container; 3
electromagnetic field source.
Fig. 2. Formation of symbiosis-like relationships among yeast strains. 4
electromagnetic field source for nitxogen-fixing yeast; 5 electromagnetic
field source for P-
2$ decomposing yeast; 6 electromagnetic field source for K-decomposing yeast;
7
electromagnetic field source for C-decomposing yeast; 8 yeast culture; 9
container.
Fig. 3. Adaptation of yeast cells to a soil type. 10 electrode; 11 container;
12
electrode; 13 yeast culture; 14 electromagnetic field source; 15 temperature
controller.
Fig. 4. Organic material grinding process. 16 organic raw material; 17
crusher; 18 grinder; 19 organic material in powder form.
Fig. 5. Inorganic material grinding process. 20 inorganic raw material; 21
c~sher; 22 grinder; 23 inorganic material in powder form.
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Fig. 6. Yeast fermentation process. 24 activated yeast cells; 25 tank for
culturing yeast cells, starch : water (35 °C ) = 1 : 2.5, semi-aerobic
fermentation at 28 to
30°C for 48 to 72 hours; 26 harvested culture.
Fig. 7. Mixing organic and inorganic raw materials. 27 inorganic materials;
28 starch; 29 organic materials; 30 mixer; 31 mixture; 32 mixture to be
transported to
fertilizer production stage.
Fig. 8. Mixing yeast cells. 33 inlets for nitrogen-fixing, P-decomposing, K-
decomposing, and C-decomposing yeasts; 34 mixing tank; 35 ATP-producing yeast;
36 GP-
producing yeast; 37 mixture of yeasts; 38 mixture to be transported to
fertilizer production
stage.
Fig. 9. Fertilizer production process. 3 9 mixture of yeast; 40 mixture of
organic and inorganic materials; 41 granulizer; 42 fertilizer granules.
Fig. 10. Drying process. 43 fertilizer granules; 44 first dryer; 45 second
dryer; 46 dried fertilizer.
Fig. 11. Cooling and packaging process. 47 dried fertilizer; 48 cooler; 49
separator; 50 bulk bag filler; 51 final product.
5. DETAILED DESCRIPTION OF THE INVENTION
In one embodiment, the present invention provides biological fertilizer
compositions that comprise yeast cells. The present invention also provides in
various
embodiments, methods for manufacturing the biological fertilizer compositions
as well as
methods for using the biological fertilizer compositions.
The biological fertilizer compositions of the invention can replace
chemical/mineral fertilizers in supplying nitrogen (N), phosphorus (P), and
potassium (K) to
plus, especially crop plants. The biological fertilizer compositions of the
present
invention can increase crop yields by 10-60%. Because the biological
fertilizers of the
present invention utilize metabolic activities of living yeasts to convert raw
materials, such
as atmospheric nitrogen and phosphorus and potassium minerals, into plant
nutrients, the
conversion and release of such nutrients by the yeast cells is regulated in
part by the nutrient
content of the soil. The nutrient content of the soil in turn depends in part
on both the
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environment and the changing needs of plants. Therefore, the release of plant
nutrients by
the biological fertilizer compositions is adaptable to the soil condition and
can be sustained
over a period of time.
In one embodiment, the biological fertilizer compositions of the invention
comprise one or more yeast cell components. A yeast cell component of the
biological
fertilizer compositions comprises a plurality of yeast cells which are capable
of performing
one of the following functions, each of which results in the provision of one
type of
nutrients to plants : (1) fixation of atmospheric nitrogen; (2) decomposition
of phosphorus
minerals or compounds; (3) decomposition ofpotassium minerals or compounds;
(4)
decomposition of complex or high molecular weight carbon materials or
compounds.
Additional yeast cell components can be included to produce growth factors and
ATP to
support the other yeasts in the fertilizer compositions.
The biological fertilizer compositions of the invention can further comprises
an organic substrate component, and/or an inorganic substrate component. The
organic
substrate component of the fertilizer compositions is a primary carbon source
for the yeast
cells in the fertilizer. The inorganic substrate component provides the yeast
cells in the
fertilizer compositions minerals, materials, and compounds containing
phosphorus and/or
potassium. The organic and inorganic substrate component may also provide the
plants
with other minerals such as but not limited to calcium, magnesium, and sulfur;
and
0 micronutrients, such as but not limited to boron, copper, iron, manganese,
molybdenum, and
zinc.
As used herein, the term "nitrogen fixation" or "fixation of atmospheric
nitrogen" encompasses biological processes in which molecular nitrogen or
nitrogen in the
atmosphere is converted into one or more nitrogenous (N) compounds, including
but not
5 limited to, ammonia, ammonium salts, urea, and nitrates.
As used herein, the phrase "decomposition of phosphorus minerals or
compounds" xefers to biological processes which convert phosphorus (P)
compounds, such
as but not limited to those water-insoluble phosphorus compounds present in
rock
phosphate, into one or more different phosphorus compounds) which can be more
readily
30 used for survival and/or growth by plants and other yeasts. For example,
the resulting
phosphorus compounds may be more soluble in water, and can thus be taken up by
the roots
of plants.
As used herein, the phrase "decomposition of potassium minerals or
compounds" refers biological processes which convert potassium (K) compounds,
such as
35 but not limited to those water-insoluble potassium compounds present in
potassium mica,
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into one or more different potassium compounds) which can be more readily used
for
survival and/or growth by plants and other yeasts. For example, the resulting
potassium
compounds may be more soluble in water, and can thus be taken up by the roots
of plants.
As used herein, the phrase "decomposition of complex or high molecular
weight carbon minerals, materials or compounds" refers to the biological
conversion of a
complex organic or inorganic carbon molecule into one or more carbon
molecules) which
usually are of a lower molecular weight, and can be more readily used for
survival and/or
growth by plants and other organisms, including other yeasts. For example, it
encompasses
the conversion of high molecular weight carbon compounds in weathered coal to
simple
carbohydrates, such as pentose and hexose. This process includes those
reactions where
long chains of carbon atoms in a polymeric carbon compound are cleaved.
As used herein, the term "growth factors" refers to molecules commonly
required for growth of yeasts, including but not limited to vitamins, in
particular, vitamin B
complexes, e.g., vitamin B-1, riboflavin (vitamin B-2), vitamin B-12, niacin
(B-3),
pyridoxine (B-6), pantothenic acid (B-5); folic acid; biotin; para-
aminobenzoic acid;
choline; and inositol.
A wide variety of organic and inorganic materials may be used to supply the
phosphorus, potassium, and complex high molecular weight carbon minerals,
materials and
compounds to be converted by the yeast cells into nutrients for use by the
yeasts and the
plants. The organic and inorganic materials that may be used in conjunction
with the
present invention include, but not limited to, minerals, such as but not
limited to phosphate
rock or rock phosphate, apatite, phosphorite, sylvinite, halite, carnalitite,
potassium mica,
lignite; industrial materials or wastes, such as but not limited to coal-mine
waste, weathered
coal, coal-powder, and hydrocarbon waste; environmental materials and wastes,
such as but
not limited to sludge from sewage water treatment plant and land fills, muds,
such as turf
mud, mud from river and lake bed; organic wastes, such as but not limited to
waste and
manure from urban areas and animal manure, such as poultry manure, cattle
manure, hog
manure, sheep manure, and guano, waste materials from plants, waste material
from animals
including fish meal, bone meal, human waste, dried blood, etc., and products
or by-products
from fermentation of plant materials containing cellulose, starch and/or other
carbohydrates.
In addition, depending on needs, a disinfectant may be included in the
biological fertilizer compositions. An environmentally safe disinfectant is
preferred. For
example, a biological disinfectant, super-CM6~ can be used with environmental
and organic
wastes, such as waste and manure from urban areas and animal manure.
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In various embodiments, the biological fertilizer compositions of the present
invention comprises at least one yeast cell component, and preferably six
yeast cell
components. The inventor discovered that, under certain culture conditions,
various yeast
strains can be induced to exhibit the following six activities: (1) fixation
of atmospheric
nitrogen; (2) decomposition of phosphorus minerals or compounds; (3)
decomposition of
potassium minerals or compounds; (4) decomposition of complex or high
molecular weight
carbon materials or compounds; (5) production of excess growth factors in an
amount that is
sufficient to support the needs of other yeast strains in the fertilizer
composition; and (6)
production of excess ATP in an amount that is sufficient to support the needs
of other yeast
strains in the fertilizer composition. The culture condition determines the
activity which is
activated or enhanced in the cultured yeasts. The specific culture conditions
for each of the
six activities are described in details in sections 5.1-5.6 respectively.
According to the invention, a yeast cell component of the biological
fertilizer
is produced by culturing a plurality of yeast cells in an appropriate culture
medium in the
presence of an electromagnetic field. The electromagnetic field can be
generated by various
means well known in the art. A schematic illustration of an exemplary setup is
depicted in
Fig. 1. The electromagnetic field of a desired frequency and amplitude is
generated by an
electromagnetic source (3) which comprises one or more signal generators that
are capable
of generating electromagnetic waves, preferably sinusoidal waves, iri the
frequency range of
100 MHz - 2000 MHz. If desirable, a signal amplifier can also be used to
increase the
output. The electromagnetic field can be applied to the culture by a variety
of means
including placing the culture in close proximity to the signal emitters. In
one embodiment,
the electromagnetic field is applied by electrodes that are submerged in the
culture (1). In a
preferred embodiment, one of the electrodes is a metal plate, and the other
electrode
comprises a plurality of wires configured inside the container (2) so that the
energy of the
electromagnetic field can be evenly distributed in the culture. The number of
electrode
wires used depends on both the volume of the culture and the diameter of the
wire. In
preferred embodiments, for a culture having a volume up to 5000 ml, one
electrode wire
having a diameter of between 0.1-1.2 mm can be used for each 100 ml of
culture; for a
culture having a volume greater than 10001, one electrode wire having a
diameter of
between 3-30 mm can be used for each 10001 of culture.
The types of yeasts contemplated for use in the invention include without
limitation, yeasts of the genera of Saccharomyces, Schizosaccharomyces,
Sporobolomyces,
Torulopsis, Trichosporon, Wickerhamia, Ashbya, Blastomyces, Candida,
Citeromyces,
Crebrothecium, Cryptococcus, Debaryomyces, E~domycopsis; Geotrichum,
Hansenula,
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Kloeckera, Lipomyces, Pichia, Rhodosporidium, and Rhodotorula. Non-limiting
examples
of yeast strains include Saccharomyces cerevisiae Hansen, ACCC2034, ACCC2035,
ACCC2036, ACCC2037, ACCC2038, ACCC2039, ACCC2040, ACCC2041, ACCC2042,
AS2.1, AS2.4, AS2.11, AS2.14, AS2.16, AS2.56, AS2.69, AS2.70, AS2.93, AS2.98,
AS2.101, AS2.109, AS2.110, AS2.112, AS2.139, AS2.173, AS2.174, AS2.182,
AS2.196,
AS2.242, AS2.336, AS2.346, AS2.369, AS2.374, AS2.375, AS2.379, AS2.380,
AS2.382,
AS2.390, AS2.393, AS2.395, AS2.396, AS2.397, AS2.398, AS2.399, AS2.400,
AS2.406,
AS2.408, AS2.409, AS2.413, AS2.414, AS2.415, AS2.416, AS2.422, AS2.423,
AS2.430,
AS2.431, AS2.432, AS2.451, AS2.452, AS2.453, AS2.458, AS2.460, AS2.463,
AS2.467,
AS2.486, AS2.501, AS2.502, AS2.503, AS2.504, AS2.516, AS2.535, AS2.536,
AS2.558,
AS2.560, AS2.561, AS2.562, AS2.576, AS2.593, AS2.594, AS2.614, AS2.620,
AS2.628,
AS2.631, AS2.666, AS2.982, AS2.1190, AS2.1364, AS2.1396, IFFI 1001, IFFI 1002,
IFFI
1005, IFFI 1006, IFFI 1008, IFFI 1009, IFFI 1010, IFFI 1012, IFFI 1021, IFFI
1027, IFFI
1037, IFFI 1042, IFFI 1043, IFFI 10451, IFFI 1048, IFFI 1049, IFFI 1050, IFFI
1052, IFFI
1059, IFFI 1060, IFFI 1063, IFFI 1202, IFFI 1203, IFFI 1206, IFFI 1209, IFFI
1210, IFFI
1211, IFFI 1212, IFFI 1213, IFFI 1215, IFFI 1220, IFFI 1221, IFFI 1224, IFFI
1247, IFFI
1251, IFFI 1270, IFFI 1277, IFFI 1287, IFFI 1289, IFFI 1290, IFFI 1291, IFFI
1291, IFFI
1292, IFFI 1293, IFFI 1297, IFFI 1300, IFFI 1301, IFFI 1302, IFFI 1307, IFFI
1308, IFFI
1309, IFFI 1310, IFFI 131 l, IFFI 1331, IFFI 1335, IFFI 1336, IFFI 1337, IFFI
1338, IFFI
1339, IFFI 1340, IFFI 1345, IFFI 1348, IFFI 1396, IFFI 1397, IFFI 1399, IFFI
1411, IFFI
1413, ACCC2043, AS2.2, AS2.3, AS2.8, AS2.53, AS2.163, AS2.168, AS2.483,
AS2.541,
AS2.559, AS2.606, AS2.607, AS2.611, AS2.612; Saccharomyces chevaliers
Guillermond,
AS2.131, AS2.213; Saccharomyces delbrueckii Lindner, AS2.285; Saccharomyces
delbrueckii Lindner ver. mongolicus Lodder, AS2.209, AS2.1157; Saccharomyces
exiguus
Hansen, AS2.349, AS2.1158; Saccharomyces fermentati (Saito) Lodder et van Rij,
AS2.286, AS2.343; Saccharomyces logos van laer et Denamur ex Jorgensen,
AS2.156,
AS2.327, AS2.335; Saccharomyces mellis Lodder et Kreger Van Rij, AS2.195;
Saccharomyces microellipsoides Osterwalder, AS2.699; Saccharomyces oviforrriis
Osterwalder, AS2.100; Saccharomyces roses Lodder et kreger van Rij, AS2.287;
Saccharomyces rouxii Boutroux, AS2.178, AS2.180, AS2.370, AS2.371;
Saccharomyces
sake Yabe, ACCC2045; Saccharomyces uvarum Beijer, IFFI 1023, IFFI 1032, IFFI
1036,
IFFI 1044, IFFI 1072, IFFI 1205, IFFI 1207; Saccharomyces willianus Saccardo,
AS2.5,
AS2.7, AS2.119, AS2.152, AS2.293, AS2.381, AS2.392, AS2.434, AS2.614,
AS2.1189;
Saccharo»Zyces sp., AS2.311; Saccharomyces ludwigii Hansen, ACCC2044, AS2.243,
AS2.508; Saccharomyces sinenses Yue, AS2.1395; Schizosaccharomyces octosporus
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WO 02/20431 PCT/GB00/03399
Beijerinck, ACCC 2046, AS2.1148; Schizosaccharomycespombe Linder, ACCC2047,
ACCC2048, AS2.248, AS2.249, AS2.255, AS2.257, AS2.259, AS2.260, AS2.274,
AS2.994, AS2.1043, AS2.1149, AS2.1178, IFFL1056; Sporobolomyces roseus Klyver
et
van Niel, ACCC 2049, ACCC 2050, AS2.619, AS2.962, AS2.1036; Sporobolomyces
salmonicolor (Fischer et Brebeck) Kluyver et van Niel, ACCC2051, AS2.261,
AS2.262;
Torulopsis candida(SaitO)Lodder, ACCC2052, AS2.270; Torulopsis famta
(Harrison)Lodder et van Rij, ACCC2053, AS2.685; Torulopsis globosa (Olson et
Hammer)Lodder et van Rij, ACCC2054, AS2.202; Torulopsis inconspicua Lodder et
van
Rij, AS2.75; Trichosporon behrendoo Lodder et Kreger van Rij, ACCC2055,
AS2.1193;
Trichosporon capitatum Diddens et Lodder, ACCC2056, AS2.1385; Trichosporon
cutaneum(de Beurm et al.)Ota, ACCC2057, AS2.25, AS2.570, AS2.571, AS2.1374;
Wickerhamia fluoresens (Soneda) Soneda, ACCC2058, AS2.1388; Ashbya gossypii
(Ashby
et Nowell) Guillermond, ACCC2001, AS2.475, AS2.1176; Blastomyces dermatitidis
Gilehrist et Stikes, ID(D 10)23; Candida albicans (Robin) Berkhout, ACCC2002,
AS2.538,
ID 16u(C1)u, ID 61v(C1)v; Candida arborea, AS2.566; Candida
guillermondii(Castellani)
Langeron et guerra, AS2.63, ID 21 a(C5)a, ID 21 b(C5)b; Candida Kratsei
(Castellani)
Berkhout, AS2.1045; Candida lambica(Lindner et GenOUd) van.Uden et Buckley,
AS2.1182; Candida lipolytica (Harrison) Diddens et Lodder, AS2.1207, AS2.1216,
AS2.1220, AS2.1379, AS2.1398, AS2.1399, AS2.1400; Candida parakrusei
(Castellani et
Chalmer) Langeron et Guerra, iD 19 a(C4)a, ID 19 b(C4)b, ID 19 c(C4)c, ID 19
d(C4)d;
Candida parapsilosis (Ashford) Langeron et Talice, AS2.590; Candida
parapsilosis
(Ashford) et Talice Var.imtermedia Van Rij et Verona, AS2.491;
Candidapseudotropicalis
(Castellani) Basgal, AS2.68, ID64(C3); Candida pulcherrima (Lindner) Windisch,
AS2.492; Candida robusta Diddens et Lodder, AS2.1195; Candida rugousa
(Anderson)
Diddens et LOddeer, AS2.51 l, AS2.1367, AS2.1369, AS2.1372, AS2.1373,
AS2.1377,
AS2.1378, AS2.1384; Candida tropicalis (Castellani) Berkout, ACCC2004,
ACCC2005,
ACCC2006, AS2.164, AS2.402, AS2.564, AS2.565, AS2.567, AS2.568, AS2.617,
AS2.637, AS2.1387, AS2.1397, ID 17 a(CZ)a, ID 17 b(CZ)b, ID 17 d(CZ)d; Candida
utilis
Henneberg Lodder et Kreger Van Rij, AS2.120, AS2.281, AS2.1180; Citeromyces
matritensis (Santa Maria) Santa Maria, AS2.1401; Crebrothecium ashbyii
(Guillermond)
Routein, ACCC2013, ACCC2014, AS2.481, AS2.482, AS2.1197; Cryptococcus
laurentii
(Kufferath) Skinner, ACCC2007, AS2.114, ID 95 (y2); Cryptococcus neoformans
(Sanfelice) Vuillemin, ID 25 u(DZ)u, ID 25 v(DZ)v, ID 25 w(DZ)w; Debaryomyces
hansenii
(Zopf) Lodder et Kreger-van Rij, ACCC2010, AS2.45; Debaryomyces kloeckeri
Guilliermond et Peju, ACCC2008, ACCC2009, AS2.33, AS2.34,.AS2.494;
Debaryomyces
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sp., ACCC201 l, ACCC2012; Endomycopsis fibuligera (Lindner) Dekker, ACCC2015,
AS2.1145;,Eremothecium ashbyii Guilliermond; Geotrichum candidum Link,
ACCC2016,
AS2.361, AS2.498, AS2.616, AS2.1035, AS2.1062, AS2.1080, AS2.1132, AS2.1175,
AS2.1183; Geotrichum ludwigii (Hansen) Fanf et al., AS2.363; Geotrichum
robustum Fang
et al., ACCC2017, AS2.621; Geotrichum suaveolens (Krzemecki) Fang et al.,
AS2.364;
Hansenula anomala (Hansen) H et P sydow, ACCC2018, AS2.294, AS2.295, AS2.296,
AS2.297, AS2.298, AS2.299, AS2.300, AS2.302, AS2.338, AS2.339, AS2.340,
AS2.341,
AS2.470, AS2.592, AS2.641, AS2.642, AS2.735, AS2.782, AS2.794; Hansenula
arabitolgens Fang, AS2.887; Hansenula jadinii Wickerham, ACCC2019; Hansercula
saturnus (Klocker) H et P sydow, ACCC2020, AS2.303; Hansenula schneggii
(Weber)
Dekker, AS2.304; Hansenula subpelliculosa Bedford, AS2.740, AS2.760, AS2.761,
AS2.770, AS2.783, AS2.790, AS2.798, AS2.866; Kloeckera apiculata (Reess emend.
Klocker) Janke, ACCC2021, ACCC2022, ACCC2023, AS2.197, AS2.496, AS2.711,
AS2.714; Lipomyces starkeyi Lodder et van Rij, ACCC2024, AS2.1390; Pichia
farinosa
(Lindner) Hansen, ACCC2025, ACCC2026, AS2.86, AS2.87, AS2.705, AS2.803; Pichia
membranaefaciens Hansen, ACCC2027, AS2.89, AS2.661, AS2.1039; Rhodosporidium
toruloides Banno, ACCC2028, AS2.1389; Rhodotorula aurantiaca (Saito) Lodder,
ACCC2029, AS2.280; Rhodotorula glutinis (Fresenius) Harrison, ACCC2030,
AS2.102,
AS2.107, AS2.278, AS2.499, AS2.694, AS2.703, AS2.704, AS2.1146; Rhodotorula
minuta
(Saito) Harrison, AS2.277; Rhodotorula rubar (Demure) Lodder, ACCC2031,
AS2.21,
AS2.22, AS2.103, AS2.I05, AS2.108, AS2.140, AS2.I66, AS2.272, AS2.279,
AS2.282;
Rhodotorula sinesis Lee, AS2.1391; Saccharomyces bailiff Lindner, AS2.312; and
Saccharomyces carlsbergensis Hansen, ACCC2032, ACCC2033, AS2.113, AS2.116,
AS2.118, AS2.121, AS2.132, AS2.162, AS2.189, AS2.200, AS2.216, AS2.265,
AS2.377,
AS2.417, AS2.420, AS2.440, AS2:441, AS2.443, AS2.444, AS2.459, AS2.595,
AS2.605,
AS2.638, AS2.742, AS2.745, AS2.748, AS2.1042.
Certain yeast species that can be activated according to the present invention
and are included in the present invention are known to be pathogenic to human
and/or other
living organisms, for example, Ashbya gossypii (Ashby et Nowell) Guillermond,
ACCC2001, AS2.475, AS2.1176; Blastomyces dermatitidis Gilehrist et Stikes,
ID(D 10)23;
Candida albicar~s (Robin) Berkhout, ACCC2002, AS2.538, ID 16u(C1)u, ID
61v(C1)v;
Candida parakrusei (Castellani et Chalmer) Langeron et Guerra, ID 19 a(C4)a,
ID 19
b(C4)b, ID 19 c(C4)c, ID 19 d(C4)d; Candida tropicalis (Castellani) Berkout,
ID 17 a(CZ)a,
ID 17 b(CZ)b, ID 17 d(Cz)d; Citeromyces matritensis (Santa Maria) Santa Maria,
AS2.1401;
Crebrothecium ashbyii (Guillermond) Routein, ACCC2013, ACCC2014; Cryptococcus
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laurentii (Kufferath) Skinner, ACCC2007, AS2.114, ID 95 (y2); Cryptococcua
neoformans
(Sanfelice) Vuillemin, ID 25 u(DZ)u, ID 25 v(Dz)v, ID 25 w(Dz)w; Debaryomyces
hansercii
(Zopf) Lodder et Kreger-van Rij, ACCC2010; Debaryomyces Kloeckeri Guilliermond
et
Peju, ACCC2008, ACCC2009; Debaryomyces sp., ACCC2011, ACCC2012; Endomycopsis
fibuligera (Lindner) Dekker, ACCC2015, AS2.1145. Under certain circumstances,
it may
be less preferable to use such pathogenic yeasts in the biological fertilizer
of the invention,
for example, if such use in an open field may endanger the health of human
and/or other
living organisms.
Yeasts of the Saccharomyces genus are generally preferred. Among strains
of Saccharomyces cerevisiae, Saccharomyces cerevisiae Hansen is a preferred
strain. The
most preferred strains of yeast are Saccharomyces cerevisiae Hansen strains
having
accession numbers AS2.501, AS2.535, AS2.441, AS2.406, AS2.382, and AS2.16 as
deposited at the China General Microbiological Culture Collection Center
(CGMCC).
Generally, the yeast strains can be obtained from private or public laboratory
cultures, or
publically accessible culture deposits, such as the American Type Culture
Collection, 10801
University Boulevard, Manassas, VA 20110-2209 and the China General
Microbiological
Culture Collection Center (CGMCC), China Committee for Culture Collection of
Microorganisms, Institute of Microbiology, Chinese Academy of Sciences,
Haidian, P.O.
Box 2714, Beijing, 100080, China.
Although it is preferred, the preparation of the yeast cell components of the
invention is not limited to starting with a pure strain of yeast. Each yeast
cell component
may be produced by culturing a mixture of yeast cells of different species or
strains. The
constituents of a yeast cell component can be determined by standard yeast
identification
techniques well known in the art.
Some yeasts may perform one of the desired functions more efficiently than
others. The ability of any species or strain of yeast to perform one of the
six desired
functions before or after culturing under the conditions of the invention can
readily be tested
by methods known in the art. For example, the amount of nitrogen fixed can be
determined
by a modified acetylene reduction method as described in U.S. Patent No.
5,578,486 which
is incorporated herein by reference in its entirety. The modified acetylene
reduction method
determines the amount of nitrogen fixed by measuring the decrease in molecular
nitrogen in
a volume of air. The amount of nitrogen fixed can also be determined by
measurement of
the ammonia and nitrates produced by the yeast cells (see, for example,
Grewling et al.,
1965, Cornell Agr Exp Sta Bull 960:22-25). For the other functions, the amount
of
phosphorus available to plants as a result of conversion from insoluble or
biologically-
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unavailable phosphorus compounds can be determined by the molybdenum blue
method
(see, for example, Murphy et al., 1962, Analytica Chimica Acta 27:31-36) or
the UV
absorption method; whereas the amount of available potassium converted from
insoluble or
biologically-unavailable potassium compounds can be determined, for example,
by flame
atomic absorption spectroscopy (see, for example, Puchyr, et al., 1986, J.
Assoc. Off. Anal.
Chem. 69:868-870). The ability of the yeasts to supply plant available N, P,
and K after the
biological fertilizer composition has been added to soil can be tested by many
techniques
known in the art. For example, plant-available ammonia, nitrates, P, and K
produced by the
yeast cells in soil can be extracted and quantitatively analyzed by the Morgan
soil test
system (see, for example, Lunt et al., 1950, Conn Agr Exp Sta Bull 541).
Without being bound by any theory or mechanism, the inventor believes that
the culture conditions activate and/or enhance the expression of a gene or a
set of genes in
yeast such that the yeast cells become active or more efficient in performing
the respective
functions.
According to the invention, the biological fertilizer compositions comprises
at least one yeast cell component capable of performing one of the following
biological
functions: (1) fixation of atmospheric nitrogen; (2) decomposition of
insoluble or
biologically-unavailable phosphorus minerals or compounds present in the
fertilizer
composition or in soil; (3) decomposition of insoluble or biologically-
unavailable potassium
minerals or compounds present in the fertilizer composition or in soil; (4)
decomposition of
complex or high molecular weight carbon materials or compounds present in the
fertilizer
composition or in soil; (5) production of excess growth factors in an amount
that is
sufficient to support the needs of other yeast strains in the fertilizer
composition; and (6)
production of excess ATP in an amount that is sufficient to support the needs
of other yeast
strains in the fertilizer composition. In preferred embodiments, the
biological fertilizer
compositions can comprise from ane yeast strain to up to six different yeast
species or
strains, each cultured under specific conditions to induce or maximize its
ability to perform
the respective functions. It will be understood that alternative formulations
are also
contemplated. Thus, if desired, the biological fertilizer composition may omit
one or more
of the above-described yeast cell components. For example, in soil rich in
biologically-
available phosphorus, a fertilizer composition may be formulated to lack the
component
consisting of phosphorus compounds-decomposing yeast. In the most preferred
embodiments of the present invention, a biological fertilizer composition that
contains all
six yeast cell components as well as the organic and/or inorganic substrates
is contemplated.
In another embodiment of the invention, where the yeast cells of the various
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yeast cell components are present in a mixture, the yeast cells can be
cultured under certain
conditions,such that the yeast cells with different functions can supply each
other with
and/or rely on each other for nutrients and growth factors. As a result, a
symbiosis-like
relationship is established among the various yeast cell components in the
fertilizer
compositions of the invention. This culturing process is optional but can
improve the
stability and efficiency of the biological fertilizer such that the fertilizer
is made more
suitable for long term use in natural soil environments. The culturing
conditions fox this
optional process are described in Section 5.7.
In yet another embodiment of the invention, the yeast cells may also be
cultured under certain conditions so as to adapt the yeast cells to a
particular type of soil.
This culturing process is optional, and can be applied to each yeast cell
component
separately or to a mixture of yeast cell components. The result is better
growth and survival
of the yeasts in a particular soil environment. The culturing conditions for
this optional
process are described in Section 5.8.
As used herein, the biological fertilizer composition supports or enhances
plant growth, if in the presence of the biological fertilizer in the soil, or
applied to the roots,
stems, leaves or other parts of the plant, the plant or a part of the plant
gains viability, size,
weight, rate of germination, rate of growth, or rate of maturation. Thus, the
biological
fertilizer compositions have utility in any kind of agricultural,
horticultural, and forestry
practices. The biological fertilizer compositions can be used for large scale
commercial
farming, in open fields or in greenhouse, or even in interiors for decorative
plants.
Preferably, the biological fertilizer is used to enhance the growth of crop
plants, such as but
not limited to cereal crops, vegetable crops, fruit crops, flower crops, and
grass crops. For
example, the biological fertilizer may be used with wheat, barley, corn,
soybean, rice, oat,
potato, apple, orange, tomato, melon, cherry, lemon, lettuce, carrot, sugar
cane, tobacco,
cotton, etc.
The biological fertilizer compositions may be applied in the same manner as
conventional fertilizers. As known to those skilled in the relevant art, many
methods and
appliances may be used. In one embodiment, culture broths of the yeast strains
of the
present invention are applied directly to soil or plants. In another
embodiment, dried
powders of the yeast strains of the present invention are applied to soil or
plants. In yet
another embodiment, mixtures of the yeast cell components and organic and
inorganic
substrate components of the present invention are applied to soil or plants.
The biological
fertilizer compositions may be applied to soil, by spreaders, sprayers, and
other mechanized
means which may be automated. The biological fertilizer compositions may be
applied
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directly to plants, for example, by soaking seeds and/or roots, or spraying
onto leaves. Such
application, may be made periodically, such as once per year, or per growing
season, or
more frequently as desired. The biological fertilizer compositions of the
invention can also
be used in conjunction or in rotation with other types of fertilizers.
S Described respectively in Sections 5.1 - S.6 are the yeast cell components
used for nitrogen fixation, phosphorus compound decomposition, potassium
compound
decomposition, complex carbon compound decomposition, growth factors
production, and
ATP production. Methods for preparing each yeast cell components are
described. Section
S.7 describes the methods for establishing a symbiosis-like relationship among
yeast strains
in a fertilizer composition of the invention. Section S.8 describes methods
for adapting
yeast cells of the invention to a particular type of soil. Section S.9
describes the
manufacture of the biological fertilizer compositions. Methods for the
preparation of
organic and inorganic raw materials and for the manufacture of the biological
fertilizer,
including mixing, drying, cooling, and packing, are also described. In various
embodiments
1 S of the invention, standard techniques for handling, transferring, and
storing microorganisms
are used. Although it is not necessary, sterile conditions or clean
environments are desirable
when carrying out the processes of the invention.
5.1. NITROGEN-FIXING YEAST CELL COMPONENT
Nitrogen fixation is a process whereby atmospheric nitrogen is converted
into ammonia and nitrates. Close to 800 species of naturally occurring
microorganisms,
mostly bacteria and cyanobacteria, from more than 70 genera have been found to
be able to
fix nitrogen. Some of the nitrogen-fixing microorganisms, such as Rhizoboum,
form
symbiotic association with plants, especially in the root of legumes. Others,
such as
Azotobacter, are free-living and capable of fixing nitrogen in soil.
In the present invention, the ability of yeast to fix nitrogen is activated or
enhanced, and the resulting nitrogen-fixing yeast cells can be used as a
component of the
biological fertilizer composition of the invention.
According to the invention, yeast cells that have an enhanced ability to fix
nitrogen are prepared by culturing the cells in the presence of an
electromagnetic field in an
appropriate culture medium. The frequency of the electromagnetic field for
activating or
enhancing nitrogen fixition in yeasts can generally be found within the range
of 800 MHz -
1000 MHz. After the yeast cells have been cultured for a sufficient period of
time, the cells
3S can be tested for their ability to fix nitrogen by methods well known in
the art.
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The method of the invention for making the nitrogen-fixing yeast cells is
carried out in a liquid medium. The medium contains sources of nutrients
assimilable by
the yeast cells. In general, carbohydrates such as sugars, for example,
sucrose, glucose,
fructose, dextrose, maltose, xylose, and the like and starches, can be used
either alone or in
combination as sources of assimilable carbon in the culture medium. The exact
quantity of
the carbohydrate source or sources utilized in the medium depends in part upon
the other
ingredients of the medium but, in general, the amount of carbohydrate usually
varies
between about 0. I % and 5% by weight of the medium and preferably between
about 0.5%
and 2%, and most preferably about 1 %. These carbon sources can be used
individually, or
several such carbon sources may be combined in the medium.
Among the inorganic salts which can be incorporated in the culture media are
the customary salts capable of yielding sodium, potassium, calcium, phosphate,
sulfate,
carbonate, and like ions. Non-limiting examples of nutrient inorganic salts
are CaCO~,
KHzPO4, MgS04, NaCI, and CaS04.
Table I: Composition for a culture medium for nitrogen-fixing yeast
Medium Composition Quantity
KHzP04 0.2g
~zHPOa 0.2g
MgS047H20 0.25g
CaC03SH20 3.5g
CaS042Hz0 O,Sg
2$ NaCI 0.25g
Yeast extract paste 0.3g
Sucrose 12.0g
Distilled water or autoclaved1000m1
water
It should be noted that the composition of the media provided in Table I is
not intended to be limiting. Various modifications of the culture medium may
be made by
those skilled in the art, in view of practical and economic considerations,
such as the scale
of culture and local supply of media components.
The process is initiated by inoculating each 100m1 of medium with lml of an
inoculum of the selected yeast strains) at a cell density of 10'-105 cell/ml,
preferably 3x1 Oz-
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104 cell/ml. The process can be scaled up or down according to needs. The
yeast culture is
grown for about 12-24 hours, preferably for about 24 hours, in the presence of
an
electromagnetic field. The electromagnetic field, which can be applied by any
means
known in the art, has a frequency in the range of 860 to 8?0 MHz, preferably
at about 865
MHz, more preferably in the range of 865.522 to 865.622 MHz, and most
preferably at
865:572 MHz. The amplitude of the field is in the range of 1000-2000mV,
preferably at
about 1250mV. After this first period of culture, the yeast cells are further
incubated under
substantially the same conditions for approximately another 24 hours, except
that the
amplitude is increased to a higher level in the range of 4000-5000 mV,
preferably to about
4656 mV. An exemplary set-up of the culture process is depicted in Figure 1.
The process
of the invention is carried out at temperatures ranging from about 25°
to 30°C; however, it
is preferable to conduct the process at 28°C. The culturing process may
preferably be
conducted under conditions in which the concentration of dissolved oxygen is
between
0.025 to 0.8 mol/m3, preferably 0.4 mol/m3. The oxygen level can be controlled
by any
conventional means known to one skilled in the art, including but not limited
to stirring
and/or bubbling.
At the end of the culturing process, the nitrogen-fixing yeast cells may be
recovered from the culture by various methods known in the art, and stored at
a temperature
below about 0°C to 4°C. The nitrogen-fixing yeast cells may also
be dried and stored in
powder form.
Any methods known in the art can be used to test the cultured yeast cells for
their ability to fix nitrogen. For example, a modified acetylene reduction
method for
measuring nitrogen fixed by microorganisms is used to evaluate the nitrogen-
fixing
capability of the prepared yeast. The modified acetylene reduction method is
described in
U.S. Patent No. 5,578,486 which is incorporated herein by reference in its
entirety. For
example, 1 ml of the prepared yeast culture is inoculated into 30 ml of a
medium according
to Table I in a sealed 250 ml flask. The culture is incubated at a temperature
in the range of
20-28 ° C for 24-56 hours in the presence of air containing about 20%
by volume oxygen and
80% by volume nitrogen. The amount of nitrogen fixed can then be determined by
measuring the decrease in nitrogen from the air by any means known in the art,
such as but
not limited to gas chromatography. The amount of nitrogen fixed by the yeast
cells of the
invention is at least about 10 mg for each gram of yeast dry weight. For
example, after
activation, the amount of nitrogen fixed by Saccharomyces cerevisiae Hansen
strain
AS2.501, can reach about 11200 mg/g.
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5.2. PHOSPHORUS-DECOMPOSING YEAST CELL COMPONENT
The phosphorus compound-decomposing (P-decomposing) yeast of the
invention converts insoluble or biologically-unavailable phosphorus-containing
substances,
such as rock phosphate, into soluble phosphorous compounds so that they become
available
to plants.
In the present invention, the ability of yeast to decompose insoluble
phosphorus-containing substances is activated or enhanced, and the resulting P-
decomposing yeast cells can be used as a component of the biological
fertilizer composition
of the invention.
According to the invention, yeast cells that are capable of P-decomposing are
prepared by culturing the cells in the presence of an electromagnetic field in
an appropriate
culture medium. The frequency of the electromagnetic field for activating or
enhancing P-
decomposition in yeasts can generally be found in the range of 300 MHz to 500
MHz. After
the cells have been cultured for a sufficient period of time, the cells can be
tested for their
ability to decompose phosphorus-containing substances by methods well known in
the art.
The method of the invention for making the P-decomposing yeast cells is
carried out in a liquid medium. The medium contains sources of nutrients
assimilable by
the yeast cells. In general, carbohydrates such as sugars, for example,
sucrose, glucose,
fructose, dextrose, maltose, xylose, and the like and starches, can be used
either alone or in
combination as sources of assimilable carbon in the culture medium. The exact
quantity of
the carbohydrate source or sources utilized in the medium depends in part upon
the other
ingredients of the medium but, in general, the amount of carbohydrate usually
varies
between about 0.1% and 5% by weight of the medium and preferably between about
0.5%
and 2%, and most preferably about 1.5%. These carbon sources can be used
individually, or
several such carbon sources may be combined in the medium.
Among the inorganic salts which can be incorporated in the culture media are
the customary salts capable of yielding sodium, potassium, calcium, sulfate,
carbonate, and
like ions. Non-limiting examples of nutrient inorganic salts are CaC03, MgS04,
NaCI, and
CaS04. Insoluble phosphorus-containing substances in a suitable form are also
included in
the media. Non-limiting examples include powder of rock phosphate of >_ 200
mesh. Other
insoluble phosphorus-containing substances can also be used either separately
or in
combination.
Table II: Composition for a culture medium for P-decomposing yeast
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Medium Composition Quantity
Sucrose ' 15g
NaCI 1.2g
MgSO47Hz0 0.2g
CaC03SH20 3.0g
CaS042H20 0.3g
KN03 0.3 g
f east extract paste . O. S g
Rock phosphate 1.2g; Powder of > 200 mesh
Autoclaved water 1 OOOmI
It should be noted that the composition of the media provided in Table II is
not intended to be limiting. Various modifications of the culture medium may
be made by
those skilled in the art, in view of practical and economic considerations,
such as the scale
of culture and local supply of media components.
The process is initiated by inoculating each 100m1 of medium with lml of an
inoculum of the selected yeast strains) at a cell density of 10'--105 cell/ml,
preferably 3x102
104 cell/ml. The process can be scaled up or down according to needs. The
yeast culture is
grown for about 12-24 hours, pxeferably for about 24 hours, in the presence of
an
electromagnetic field. The electromagnetic field, which can be applied by any
means
known in the art, has a frequency in the range of 360 to 370MHz, preferably at
about
366MHz, more preferably in the range of 366.199 to 366.287MHz, and most
preferably at
2$ 366.243 MHz. The amplitude of the field is in the range of 1000 to 2000mV,
preferably at
about 1230mV. After this first period of culture, the yeast cells are further
incubated under
substantially the same conditions for approximately another 24 hours, except
that the
amplitude is increased to a higher level in the range of 4000 to 5000 mV,
preferably to
about 4570 mV. An exemplary set-up of the culture process is depicted in
Figure 1. The
process of the invention is carried out at temperatures ranging from about
25° to 30°C;
however, it is preferable to conduct the process at 28 °C. The
culturing process may
preferably be conducted under conditions in which the concentration of
dissolved oxygen is
between 0.025 to 0.8 mol/m3, pxeferably 0.4 mol/m3. The oxygen level can be
controlled by
any conventional means known to one skilled in the art, including but not
limited to stirring
3 5 and/or bubbling.
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At the end of the culturing process, the P-decomposing yeast cells may be
recovered from the culture by various methods known in the art, and stored at
a temperature
below about 0°C to 4°C. The P-decomposing yeast cells may also
be dried and stored in
powder form.
Any methods known in the art can be used to test the cultured yeast cells for
their ability to decompose insoluble phosphorus-containing substances. In one
embodiment,
1 ml of the prepared yeast culture is inoculated into 30 ml of a medium
according to Table
II. The culture is incubated at a temperature in the range of 20-28 °C
for 24-56 hours. The
amount of biologically available phosphorus in the form of P043- in the
culture can then be
determined by any methods known in the art, including but not limited to UV
absorption
spectroscopy. The amount of P043- in the culture is increased by at least lOmg
for each
gram of yeast dry weight. For example, after activation, the amount of P0~,3-
in a culture of
Saccharomyces cerevisiae Hansen strain AS2.535 is increased to about 4460
mg/g.
5.3. POTASSIUM-DECOMPOSING YEAST CELL COMPONENT
The potassium compound-decomposing (K-decomposing) yeast of the
invention converts insoluble potassium-containing substances, such as
potassium mica, into
soluble potassium so that they become available to plants.
In the present invention, the ability of a plurality of yeast cells to
decompose
insoluble potassium-containing substances is activated or enhanced, and the
resulting K-
decomposing yeast cells can be used as a component of the biological
fertilizer composition
of the invention.
According to the present invention, yeast cells that are capable of K-
decomposing are prepared by culturing the cells in the presence of an
electromagnetic field
in an appropriate culture medium. The frequency of the electromagnetic field
for activating
or enhancing K-decomposition in yeasts can generally be found in the range of
100 MHz -
300MHz. After the yeast cells have been cultured for a sufficient period of
time, the cells
can be tested for their ability to decompose potassium-containing substances
by methods
well known in the art.
The method of the invention for making the K-decomposing yeast cells is
carried out in a liquid medium. The medium contains sources of nutrients
assimilable by
the yeast cells. In general, carbohydrates such as sugars, for example,
sucrose, glucose,
fructose, dextrose, maltose, xylose, and the like and starches, can be used
either alone or in
combination as sources of assimilable carbon in the culture medium. The exact
quantity of
the carbohydrate source or sources utilized in the medium depends in part upon
the other
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ingredients of the medium but, in general, the amount of carbohydrate usually
varies
between about 0.1% and 5% by weight of the medium and preferably between about
0.5%
and 2%, and most preferably about 1.5%. These carbon sources can be used
individually, or
several such carbon sources may be combined in the medium.
Among the inorganic salts which can be incorporated in the culture media are
the customary salts capable of yielding sodium, calcium, phosphate, sulfate,
carbonate, and
like ions. Non-limiting examples of nutrient inorganic salts are (NH4)ZHP04,
CaC03,
MgSO~, NaCI, and CaS04. Insoluble potassium-containing substances in a
suitable fornz are
also included in the media. Non-limiting examples include powder of potassium
mica of >_
200 mesh. Other insoluble potassium-containing substances can also be used
either
separately or combined.
Table III: Composition for a culture medium for K-decomposing yeast
Medium Composition Quantity
Sucrose 15g
NaCI 1.2g
MgSO47HZ0 0.2g
CaC03SH20 3.0g
20CaS042Hz0 0.3g
(NH4)ZHP04 0.3g
Yeast extract paste 0.3g
Potassium mica 1.2g, Powder of > 200 mesh
25Autoclaved water 1000m1
It should be noted that the composition of the media provided in Table III is
not intended to be limiting. Various modifications of the culture medium may
be made by
those skilled in the art, in view of practical and economic considerations,
such as the scale
30 of culture and local supply of media components.
The process is initiated by inoculating each 100m1 of medium with lml of an
inoculum of the selected yeast strains) at a cell density of 102-105 celllml,
preferably 3x102-
104 cell/ml. The process can be scaled up or down according to needs. The
yeast culture is
grown for about 12-24 hours, preferably for about 24 hours, in the presence of
an
35 electromagnetic field. The electromagnetic field, which can be applied by
any means
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known in the art, has a frequency in the range of 2~0-260MHz, preferably at
about
255MHz, more preferably in the range of 255.388 to 255.462 MHz, and most
preferably at
255.425 MHz. The amplitude of the field is in the range of 1000-2000mV,
preferably at
about 1340mV. After this first period of culture, the yeast cells are further
incubated under
S substantially the same conditions for approximately another 24 hours, except
that the
amplitude is increased to a higher levellin the range of 4000-5000 mV,
preferably to about
4850 mV. An exemplary set-up of the culture process is depicted in Figure 1.
The process
of the invention is carried out at temperatures ranging from about 25°
to 30°C; however, it
is preferable to conduct the process at 28°C. The culturing process may
preferably be
conducted under conditions in which the concentration of dissolved oxygen is
between
0.025 to 0.8 mol/m3, preferably 0.4 mol/m3. The oxygen level can be controlled
by any
conventional means known to one skilled in the art, including but not limited
to stirring
and/or bubbling.
At the end of the culturing process, the K-decomposing yeast cells may be
recovered from the culture by various methods known in the art, and stored at
a temperature
below about 0-4 °C. The K-decomposing yeast cells may also be dried and
stored in
powder form.
Any methods known in the art can be used to test the cultured yeast cells for
their ability to decompose insoluble potassium-containing substances. In one
embodiment,
1 ml of the prepared yeast culture is inoculated into 30 ml of a medium
according to Table
III. The culture is incubated at a temperature in the range of 20-28 °
C for 24-56 hours. The
amount of biologically available potassium in the form of K+ in the culture
can then be
determined by any methods known in the art, including but not limited to
atomic absorption
spectrometry. The amount of K~ in the culture is increased by at least 10 mg
for each gram
of yeast dry weight. For example, after activation, the amount of K+ in a
culture of
Saccharomyces cerevisiae Hansen strain AS2.441 can reach about 4050 mg/g.
5.4. COMPLEX CARBON-DECOMPOSING YEAST CELL
COMPONENT
The carbon-decomposing (C-decomposing) yeast of the invention converts
complex, usually high molecular weight, carbon compounds and materials, such
as
cellulose, into simple carbohydrates, such as pentoses and hexoses. Such
simple
carbohydrates are utilized by other yeast cells to support their.growth and
activities.
In the present invention, the ability of yeast to decompose complex carbon
compounds very efficiently is activated or enhanced, and the resulting C-
decomposing yeast
cells can be used as a component of the biological fertilizer composition of
the invention.
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According to the present invention, yeast cells that are capable of C-
decomposition are prepared by culturing the cells in the presence of an
electromagnetic field
in an appropriate culture medium. The frequency of the electromagnetic field
for C-
decomposition in yeasts can generally be found in the range of 1000 MHz -1200
MHz.
After the yeast cells have been cultured for a sufficient period of time, the
cells can be tested
for their ability to decompose complex carbon compounds by methods well known
in the .
art.
The method of the invention for making the C-decomposing yeast cells is
carried out in a liquid medium. The medium contains sources of nutrients
assimilable by
the yeast cells. Complex carbon-containing substances such as cellulose, coal,
etc., in a
suitable form can be used as sources of carbon in the culture medium. The
exact quantity of
the carbon source or sources utilized in the medium depends in part upon the
other
ingredients of the medium but, in general, the amount of carbohydrate usually
varies
between about 0.1% and 5% by weight of the medium and preferably between about
0.1%
and 1 %, and most preferably about 0.5%. These carbon sources can be used
individually, or
several such carbon sources may be combined in the medium.
Among the inorganic salts which can be incorporated in the culture media are
the customary salts capable of yielding sodium, calcium, phosphate, sulfate,
carbonate, and
like ions. Non-limiting examples of nutrient inorganic salts are (NH4)ZHP04,
CaC03,
MgS04, NaCI, and CaS04.
Table IV: Composition for a culture medium for C-decomposing yeast
Medium Composition Quantity
25Cellulose S.Og; Powder of > 100 mesh
NaCl 0.6g
MgS047H20 0.3 g
CaC035HZ0 1.5g
CaS042HZ0 0.4g
(NH4)2HP04 0.3g
Yeast extract paste 0.5g
KZHP04 0.5g
Autoclaved water 1000m1
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It should be noted that the composition of the media provided in Table IV is
not intended to be limiting. Various modifications of the culture medium may
be made by
those skilled in the art, in view of practical and economic considerations,
such as the scale
of culture and local supply of media components.
The process is initiated by inoculating each 100m1 of medium with 1 ml of an
inoculum of the selected yeast strains) at a cell density of 10'-105 cell/ml,
preferably 3x102-
104 cell/ml. The process can be scaled up or down according to needs. The
yeast culture is
grown for about 12-24 hours, preferably for about 24 hours, in the presence of
an
electromagnetic field. The electromagnetic field, which can be applied by any
means
known in the art, has a frequency in the range of 1087-1097MHz, preferably
about at 1092,
more preferably in the range of 1092.346 to 1092.428MHz, and most preferably
at 1092.387
MHz. The amplitude used can be in the range of 1000-2000mV, preferably at
about
1530mV. After this first period of culture, the yeast cells are further
incubated under
substantially the same conditions for approximately another 24 hours, except
that the
amplitude is increased to a higher level in the range of 4000-5000 mV,
preferably to about
4720 mV. An exemplary set-up of the culture process is depicted in Figure 1.
The process
of the invention is carried out at temperatures ranging from about 25 °
to 30 ° C; however, it
is preferable to conduct the process at 28 °C. The culturing process
may preferably be
conducted under conditions in which the concentration of dissolved oxygen is
between
0.025 to 0.8 mollm3, preferably 0.4 mol/m3. The oxygen level can be controlled
by any
conventional means known to one skilled in the art, including but not limited
to stirring
and/or bubbling.
At the end of the culturing process, the C-decomposing yeast cells may be
recovered from the culture by various methods known in the art, and stored at
a temperature
below about 0-4 °C. The C-decomposing yeast cells may also be dried and
stored in
powder form.
Any methods known in the art can be used to test the cultured yeast cells for
their ability to decompose complex-carbon containing substances. In one
embodiment, 1 ml
of the prepared yeast culture is inoculated into 30 ml of a medium according
to Table IV.
The culture is incubated at a temperature in the range of 20-28°C for
24-56 hours. The
amount of simple carbohydrates in the culture can then be determined by any
methods
known in the art, including but not limited to chromatography and molecular
fluorescence
spectroscopy. Preferably, the amount of simple carbohydrates in the culture is
increased by
at least 10 mg for each gram of yeast dry weight. For example, after
activation, the amount
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of simple carbohydrates in a culture of Saccharomyces cerevisiae Hansen
AS2.406 can
reach 27200 mglg.
5.5. GROWTH FACTORS PRODUCING YEAST CELL COMPONENT
The growth factors producing (GP-producing) yeast of the present invention
produces vitamins and other nutrients, such as but not limited to, vitamin B-
l, riboflavin
(vitamin B-2), vitamin B-12, niacin (B-3), pyridoxine (B-6), pantothenic acid
(B-5), folic
acid, biotin, para-aminobenzoic acid, choline, inositol, in such amounts that
can support the
growth of other yeast strains. Such growth factors are produced by yeast
during the
fermentation process.
In the present invention, the ability of yeast to overproduce growth factors
is
activated or enhanced, and the resulting GP-producing yeast cells can be used
as a
component of the biological fertilizer composition of the invention.
According to the present invention, yeast cells that are capable of GP-
producing are prepared by culturing the cells in the presence of an
electromagnetic field in
an appropriate culture medium. The frequency of the electromagnetic field for
activating or
enhancing GP-production in yeasts can generally be found in the range of 1300
MHz -1500
MHz. After the yeast cells have been cultured for a sufficient period of time,
the cells can
be tested for their ability to produce growth factors by methods well known in
the art.
The method of the invention for making the GP-producing yeast cells is
carried out in a liquid medium. The medium contains sources of nutrients
assimilable by
the yeast cells. In general, carbohydrates such as sugars, for example,
sucrose, glucose,
fructose, dextrose, maltose, xylose, and the like and starches, can be used
either alone or in
combination as sources of assimilable carbon in the culture medium. The exact
quantity of
the carbohydrate source or sources utilized in the medium depends in part upon
the other
ingredients of the medium but, in general, the amount of carbohydrate usually
varies
between about 0.1% and 5% by weight of the medium and preferably between about
0.5%
and 2%, and most preferably about 0.8%. These carbon sources can be used
individually, or
several such carbon sources may be combined in the medium.
Among the inorganic salts which can be incorporated in the culture media are
the customary salts capable of yielding sodium, calcium, phosphate, sulfate,
carbonate, and
like ions. Non-limiting examples of nutrient inorganic salts are NH4N03,
I~ZHP04, CaC03,
MgS04, NaCI, and CaS04.
Table V: Composition for a culture medium for GP-producing yeast
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Medium Composition Quantity
Starch ~ 8.0g; Powder of > 120 mesh
NaCI 0.3 g
MgS047H20 0.2g
CaC03 SH20 0.5 g
CaS042Hz0 0.2g
NH4N03 0.3 g
KaHPO4 0.8g
Autoclaved water 1000m1
It should be noted that the composition of the media provided in Table V is
not intended to be limiting. Various modifications of the culture medium may
be made by
those skilled in the art, in view of practical and economic considerations,
such as the scale
of culture and local supply of media components.
The process is initiated by inoculating each 100m1 of medium with lml of an
inoculum of the selected yeast strains) at a cell density of 102-105 cell/ml,
preferably 3x10'-
104 cell/ml. The process can be scaled up or down according to needs. The
yeast culture is
grown for about 12-24 hours, preferably for about 24 hours, in the presence of
an
electromagnetic field. The electromagnetic field, which can be applied by any
means
known in the art, has a frequency in the range of 1382-1392MHz, preferably at
about
1387MHz, more preferably in the xange of 1387.517 to 1387.595 MHz, and most
preferably
at 1387.556 MHz. The amplitude used can be in the range of 1000-2000mV,
preferably at
about 1620mV. After this first period of culture, the yeast cells are further
incubated under
substantially the same conditions for approximately another 24 hours, except
that the
amplitude is increased to a higher level in the range of 4000-5000 mV,
'preferably to about
4830 mV. An exemplary set-up of the culture process is depicted in Figure 1.
The process
of the invention is carried out at temperatures ranging from about 25 °
to 30 °C; however, it
is preferable to conduct the process at 28°C. The culturing process may
preferably be
conducted under conditions in which the concentration of dissolved oxygen is
between
0.025 to 0.8 mol/m3, preferably 0.4 mol/m3. The oxygen level can be controlled
by any
conventional means known to one skilled in the art, including but not limited
to stirnng
and/or bubbling.
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At the end of the culturing process, the GP-producing yeast cells may be
recovered from the culture by various methods known in the art, and stored at
a temperature
below about 0-4 °C. The GP-producing yeast cells may also be dried and
stored in powder
form.
S Any methods known in the art can be used to test the cultured yeast cells
for
their ability to overproduce growth factors. In one embodiment, 1 ml of the
prepared yeast
culture is inoculated into 30 ml of a medium according to Table V. The culture
is incubated
at a temperature in the range of 20-28°C for 32-48 hours. The amount of
growth factors as
represented by the total amount of vitamin B 1, B2, B6, and B 12 in the
culture can then be
determined by any methods known in the art, including but not limited to high
performance
liquid chromatography (HPLC). The amount of growth factors in the culture is
increased by
at least 10 mg for each gram of yeast dry weight. For example, after
activation, the amount
of vitamin B 1, B2, B6, and B 12 in a culture of Saccharomyces cerevisiae
Hansen AS2.382
can reach an aggregate of 6120 mg/g.
5.6. ATP-PRODUCING YEAST CELL COMPONENT
The ATP-producing yeast of the present invention is capable of
overproducing ATP in such amounts that can support the growth of other yeast
strains in the
biological fertilizer composition.
In the present invention, the ability of yeast to overproduce ATP is activated
or enhanced, and the resulting ATP-producing yeast cells can be used as a
component of the
biological fertilizer composition of the invention.
According to the present invention, yeast cells that are capable of enhanced
ATP-production are prepared by culturing the cells in the presence of an
electric field in an
appropriate culture medium. The frequency of the electromagnetic field for
activating or
enhancing ATP-production in yeasts can generally be found in the range of 1600
MHz -
1800 MHz. After sufficient time is given for the cells to grow, the cells can
be tested for
their enhanced ability to produce ATP by methods well known in the art.
The method of the invention for making the ATP-producing yeast cells is
carried out in a liquid medium. The medium contains sources of nutrients
assimilable by
the yeast cells. In general, carbohydrates such as sugars, for example,
sucrose, glucose,
fructose, dextrose, maltose, xylose, and the like and starches, can be used
either alone or in
combination as sources of assimilable carbon in the culture medium. The exact
quantity of
the carbohydrate source or sources utilized in the medium depends in part upon
the other
ingredients of the medium but, in general, the amount of carbohydrate usually
varies
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between about 0.1% and 5% by weight of the medium and preferably between about
0.5%
and 2%, and most preferably about 0.8%. These carbon sources can be used
individually, or
several such carbon sources may be combined in the medium.
Among the inorganic salts which can be incorporated in the culture media are
the customary salts capable of yielding sodium, calcium, phosphate, sulfate,
carbonate, and
like ions. Non-limiting examples of nutrient inorganic salts are (NH4)2HPO4,
CaC03,
MgS04, NaCI, and CaS04.
Table VI: Composition for a culture medium for ATP-producing yeast
Medium Composition Quantity
Starch lO.Og
NaCI 0.2g
MgS047H20 0.2g
1 CaC035H20 0.8g
S
CaS042H20 0.2g
NH4N03 0.2g
I~ZHP04 O.Sg
Autoclaved water 1000m1
It should be noted that the composition of the media provided in Table VI is
not intended to be limiting. Various modifications of the culture medium may
be made by
those skilled in the art, in view of practical and economic considerations,
such as the scale
of culture and local supply of media components.
The process is initiated by inoculating each 100m1 of medium with lml of an
inoculum of the selected yeast strains) at a cell density of 102-105 cell/ml,
preferably 3x102-
104 cell/ml. The process can be scaled up or down according to needs. The
yeast culture is
grown for about 12-24 hours, preferably for about 24 hours, in the presence of
an
electromagnetic field. The electromagnetic field, which can be applied by any
means
known in the art, has a frequency in the range of 1690-1700MHz, preferably at
about
1694MHz, more preferably in the range of 1694.328 to 1694.402 MHz, and most
preferably
at 1694.365 MHz. The amplitude of the field is in the range of 1000-2000mV,
preferably
at about 1470mV. After this first period of culture, the yeast cells are
further incubated
under substantially the same conditions for approximately another 24 hours,
except that the
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amplitude is increased to a higher level in the range of 4000-5000 mV,
preferably to about
4780 mV. An exemplary set-up of the culture process is depicted in Figure 1.
The process
of the invention is carried out at temperatures ranging from about 25 °
to 30 ° C; however, it
is preferable to conduct the process at 28 ° C. The culturing process
may preferably be
conducted under conditions in which the concentration of dissolved oxygen is
between
0.025 to 0.8 mol/m3, preferably 0.4 mol/m3. The oxygen level can be controlled
by any
conventional means known to one skilled in the art, including but not limited
fo stirring
and/or bubbling.
At the end of the culturing process, the ATP-producing yeast cells may be
recovered from the culture by various methods known in the art, and stored at
a temperature
below about 0-4 °C. The ATP-producing yeast cells may also be dried and
stored in
powder form.
Any methods known in the art can be used to test the cultured yeast cells for
their ability to overproduce ATP. In one embodiment, 1 ml of the prepared
yeast culture is
inoculated into 30 ml of a medium according to Table VI. The culture is
incubated at a
temperature in the range of 20-28°C for 36-56 hours. The amount of ATP
in the culture can
then be determined by any methods known in the art, including but not limited
to HPLC.
The amount of ATP produced is increased by at least about 10 mg for each gram
of yeast
dry weight. For example, after activation, the amount of ATP produced in a
culture of
Saceharomyces cerevisiae Hansen strain AS2.16 can reach about 3320 mg/g.
5.7. FORMATION OF SYMBIOSIS-LIKE RELATIONSHIPS
In another embodimemt of the present invention, yeast strains with the newly
activated or enhanced ability to fix nitrogen, decompose phosphorus-containing
minerals or
compounds, decompose insoluble potassium-containing minerals or compounds, and
decompose complex carbon materials as described in Sections 5.1-5.4 are
combined and
cultured so that they form a symbiosis-like relationship whereby they can grow
together
without substantially relying on outside supplies of biological available
nitrogen,
phosphorus, potassium, and carbon nutrients. The nutrients needed for growth
are supplied
by the respective nutrient-producing yeast strain within the fertilizer
composition by
converting biologically-unavailable nutrients from various sources into
available nutrients.
The activity of each of the yeast strains in producing the respective types of
nutrient relates
in part to the needs of other yeast cells as well as the plants. As a result,
soluble,
biologically-available nutrients will be converted when needed, thereby
avoiding excess
losses due to, for example, leaching.
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The optional process which can be used to improve the performance of the
biological fertilizer is described as follows. Four strains of yeasts prepared
according to
Sections 5.1-5.4 are mixed and cultured in the presence of an electromagnetic
field in an
appropriate liquid medium. The medium contains nitrogen, phosphorus,
potassium, and
carbon nutrients in biologically unavailable forms. As non-limiting examples,
atomospheric
nitrogen is used as the source of nitrogen nutrient, powder of phosphate rock
is used as the
source of phosphorus nutrient, powder of potassium mica is used as the source
of potassium
nutrient, and powdered cellulose is used as the source of complex carbon
nutrient. Other
forms of insoluble phosphorus- and potassium-containing substances and complex
carbon
compounds may also be used in place of or in combination with any of the above-
identified
minerals as sources of phosphorus, potassium, and carbon nutrients. Among the
inorganic
salts which can be incorporated in the culture media are the customary salts
capable of
yielding sodium, calcium, sulfate, carbonate, and like ions. Non-limiting
examples of
nutrient inorganic salts are CaC03, MgS04, NaCI, and CaSO4.
Table VII: Composition for a culture medium for formation of symbiosis-like
relation
Medium Composition Quantity
NaCI O.Sg
MgS047H20 0.4g
CaC03SHZO 3.0g
CaS042H~0 0.3g
Yeast extract paste 0.3g
Potassium mica 1.2g; Powder of > 200 mesh
Rock phosphate 1.2g; Powder of > 200 mesh
Cellulose S.Og; Powder of > 200 mesh
Autoclaved water 1000m1
It should be noted that the composition of the media provided in Table VII is
not intended to be limiting. Various modifications of the culture medium may
be made by
those skilled in the art, in view of practical and economic considerations,
such as the scale
of culture and local supply of media components.
The culturing process may preferably be conducted under conditions in
3$ which the concentration of dissolved oxygen is between 0.025 to 0.8 mollm3,
preferably 0.4
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mol/m3. The oxygen level can be controlled by any conventional means known to
one
skilled in the art, including but not limited to stirring andlor bubbling. The
process of the
invention is carried out at temperatures ranging from about 25° to
30°C; however, it is
preferable to conduct the process at 28°C. The process is initiated in
sterilized medium by
inoculating typically about 20m1 of each inoculum of the four strains of yeast
cells, each at a
cell density of about 108 celllml. The optional process can be scaled up or
down according
to needs.
The yeast culture is grown for 12-72 hours, preferably for about 48 hours, in
the presence of four independent electromagnetic fields. The electromagnetic
fields, which
can be applied by a variety of means, each has the following respective
frequencies: (I) in
the range of 860 to 870 MHz, preferably at about 865 MHz, more preferably in
the range of
865.522 to 865.622 MHz, and most preferably at 865.572 MHz, for nitrogen-
fixing; (2) in
the range of 360-370MHz, preferably at about 366MHz, more preferably in the
range of
366.199 to 366.287MHz, and most preferably at 366.243 MHz, for phosphorus-
decomposing; (3) in the range of 250-260MHz, preferably at about 255MHz, more
preferably in the range of 255.388 to 255.462 MHz, and most preferably at
255.425 MHz,
for potassium-decomposing; and (4) in the range of 1087-1097MHz, preferably
about at
1092, more preferably in the range of 1092.346 to 1092.428MHz, and most
preferably at
1092.387 MHz, for complex carbon-decomposing. The amplitude of each
electromagnetic
field is repeatedly cycled between 0-3000mV, preferably between 20-1800mV, in
steps of
1mV at a rate of 18-23 minutes per complete cycle. An exemplary set-up of the
culture
process is depicted in Figure 2.
5.8. SOIL ADAPTATION
The yeast strains of the invention must also be able to grow and perform their
respective functions in various types of soils. The ability of the yeast
strains to survive and
grow can be enhanced by adapting the yeast strains of the invention to a
particular soil
condition.
In another embodiment of the invention, yeast cells prepared according to
any one of Sections 5.1-5.6 can be cultured separately or in a mixture in a
solid or semi-
solid medium containing soil from one or more soil sources. This optional
process which
can be used to improve the performance of the biological fertilizer is
described by way of an
example as follows.
A suspension containing l Oml of yeasts at a density of 106 cell/ml is mixed
with a 1000cm3 of the soil medium. The process can be scaled up or down
according to
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needs. The mixture of yeast and soil is cultured for about 48-96 hours,
preferably for about
48 hours, in the presence of an electromagnetic field. The electromagnetic
field, which can
be applied by a variety of means, has a frequency that, depending on the
strain of yeast,
corresponds to one of the frequencies described in Sections 5.1-5.6. A field
amplitude in
the range of 100-3000mV, preferably 2100mV, can be used. The culture is
incubated at
temperatures that cycle between about 3 ° C to about 48 ° C. For
example, in a typical cycle,
the temperature of the culture may start at 35-48°C and be kept at this
temperature for about
1-2 hours, then adjusted up to 42-45 °C and kept at this temperature
foal-2 hours, then
adjusted to 26-30°C and kept at this temperature fox about 2-4 hours,
and then brought
down to 5 -10°C and kept at this temperature for about 1-2 hours, and
then the temperature
may be raised again to 35-45°C for another cycle. The cycles are
repeated until the process
is completed. After the last temperature cycle is completed, the temperature
of the culture is
lowered to 3-4°C and kept at this temperature for about 5-6 hours.
After adaptation, the
yeast cells may be isolated and recovered from the medium by conventional
methods, such
as filtration. The adapted yeast cells can be stored under 4°C. An
exemplary set-up of the
culture process is depicted in Figure 3.
5.9. SEPARATION OR ENRICHMENT OF YEAST CELLS
Yeast cells that have been adapted to form a symbiosis-like relationship
according to Section 5.7. can be separated or enriched in such a way that each
strain of yeast
cells keep their acquired or enhanced functions. Separation of yeast cells is
carried out
according to methods described in U.S. Patent No. 5,578,486 and Chinese patent
publication CN 1110317A which are incorporated herein by reference in its
entirety. The
frequency used for activating the yeast cells may be used during the
separation process. The
2$ separated yeast cells can then be dried, and stored.
5.10. MANUFACTURE OF THE BIOLOGICAL FERTILIZERS
In addition to yeast cell components, various organic and inorganic raw
materials can also be included in the biological fertilizer compositions of
the invention. The
preparation of such materials as well as the steps involved in the manufacture
of the
biological fertilizer are described herein.
5.10.1. Preparation of the Organic and Inorganic Substrate Components
A wide range of organic and inorganic materials can be used in the biological
fertilizer compositions of the present invention. Organic materials, such as
but not limited
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to coal-mine waste and weathered coal, or any materials that contain more than
20% of
organic substances, can be used as sources of carbon to support the growth of
plants and
yeasts. Combinations and mixtures of such organic materials can also be used.
Organic
compounds present in such materials are decomposed by the yeast capable of
breaking
complex or high molecular weight carbon-chain molecules into simple carbon
compounds
so that they can be used by plants and other yeast cells in the fertilizer.
Inorganic materials, such as but not limited to phosphate rock and potassium
mica, are included as sources of phosphorus and potassium respectively. Other
phosphorous- or potassium-containing materials and minerals can also be used.
These
inorganic compounds are decomposed by K-decomposing and P-decomposing yeast
cells
into biologically available potassium and biologically available phosphorus
that can be used
by the growing plants as well as the yeast cells in the fertilizer. Any
organic or inorganic
material may be used alone or in combination or in substitution with any other
materials in
the present invention. Alternatively, one or more organic or inorganic
ingredients may be
omitted, or substituted by another if it is deemed desirable by the particular
application. For
example, potassium mica can be omitted if the soil contains sufFcient
potassium minerals.
The organic and inorganic materials used in the invention should not contain
amounts of toxic substances or microorganisms that can inhibit the growth of
the yeast cells
or plants.
The organic and inorganic components in the present invention are ground
into suitable forms and sizes before incorporated into the fertilizer.
Typically, the organic or
inorganic material is conveyed into a crusher where it is broken up into
pieces of _< 5 cm in
diameter. Any conventional crusher or equivalent machines can be used for this
purpose.
The pieces are then transferred to a grinder by any conveying means and ground
to a powder
of >_ 150 mesh. Any grinder that allows fine grinding can be used for this
purpose. The
powder is then conveyed to an appropriate storage tank for storage until use
with other
components of the fertilizer. A schematic illustration of the grinding process
is shown in
Figs. 4 and S.
5.10.2. Ferraentation Process Using Growth Factor-Producing Yeast
In the present invention, the preparation of GP-producing yeast is earned out
in a fermentation process using as seed the activated yeast strain as
described in Section 5.5.
A schematic of the fermentation process is illustrated in Fig. 6.
The fermentation medium is prepared according to a ratio of 2.5 liters of
3~ water per kilogram of starch. Clean water, preferably water free of any
microorganisms, is
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used to prepare the fermentation medium. The fermentation is carried out at a
temperature
between 20-30 °C, preferably between 25-28 °C, in a clean
environment and in a space
where there are no strong sources of electromagnetic fields, such as power
lines and power
generators. Any equipments that contact the fermentation broth, including
reactors,
pipelines, and stirrers, must be throughly cleaned before each use. The
fermentation process
normally lasts about 60-72 hours, depending on the fermentation temperature.
At least 90%
of the fermentation substrate is fermented. Fermentation is preferably
conducted under
semi-aerobic conditions or conditions in which the oxygen level is about 20-
60% of the
maximal soluble oxygen concentration. The oxygen level can be controlled by
any
conventional means known to one skilled in the art, including but not limited
to stirring
andlor bubbling. After fermentation, the cell counts should reach about
2x10t° cells/ml.
The fermentation broth is kept at a temperature in the range of 15-28 °
C and must be used
within 24 hours. Alternatively, the GP-producing yeasts can be drained, dried
and stored in
powder form.
5.10.3. Fermentation Process Using ATP-Producing Yeast
In the present invention, the preparation of ATP-producing yeast is carried
out by a fermentation process using as seed the adapted yeast strain as
described in Section
5.6. A schematic of the fermentation process is illustrated in Fig. 6.
The fermentation medium is prepared according to a ratio of 2.5 liters of
water per kilogram of starch. Clean water, preferably water free of any
microorganisms,
most preferably autoclaved water, is used to prepare the fermentation media.
The
fermentation is carried out at a temperature between 20-30°C,
preferably between 25-28°C,
in a clean environment and in a space where there are no strong sources of
electromagnetic
fields, such as power lines and power generators. Any equipments that contact
the
fermentation broth, including reactors, pipelines, and stirrers, must be
throughly cleaned
before each use. The fermentation process normally lasts about 60-72 hours,
depending on
the fermentation temperature. At least 90% of the fermentation substrate is
fermented.
Fermentation is preferably conducted under semi-aerobic conditions or
conditions in which
the oxygen level is about 20-60% of the maximal soluble oxygen concentration.
The
oxygen level can be controlled by any conventional means known to one skilled
in the art,
including but not limited to stirring and/or bubbling. After fermentation, the
cell counts
should reach about 2x10'° cells/ml. The fermentation broth is kept at a
temperature in the
range of 15-28°C and must be used within 24 hours. Alternatively, the
ATP-producing
yeasts can be drained, dried and stored in powder form.
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5.10.4. Preparation of Mixture of Raw Materials
Organic and inorganic raw materials are mixed in exemplary proportions as
shown in Table VIII. Appropriate amount of organic and inorganic materials
prepared
according to Section 5.10.1 and starch are conveyed to a mixer. Any
conventional mixer,
such as but not limited a rotary drum mixer, can be used. The mixing tank is
rotated
constantly so that powders of inorganic material, organic material, and starch
are mixed
evenly. The mixture is then conveyed to a storage tank. The procedure for
mixing organic
and inorganic substrate material is illustrated in FIG. 7.
Table VIII Ratio of raw materials
Material Percentage Requirement
Powder of organic 60-71% z 150 mesh, water
materials content
<_ 5%
Powder of inorganic15-20% >_ 150 mesh, water
content
materials <_ 3%
Starch 10-15% regular starch powder,
water
content <_ 8%
5.10.5. Preparation of Yeast Mixture
A yeast mixture is prepared in the exemplary proportions as shown in Table
IX. Appropriate amounts of the six yeast strains in dried powder form prepared
according
to Section 5.1-5.6 are conveyed to a mixing tank. The yeasts are allowed to
mix for about
10-20 minutes. The mixture is then transferred to a storage tank. Any
equipments used for
mixing yeasts, including the mixing tank and the storage tank, must be
throughly cleaned,
preferably sterilized, before each use. The yeast mixture is stored at a
temperature below 20
°C and must be used within 24 hours. The procedure for mixing yeasts is
illustrated in FIG.
8. Alternatively, the mixture of six yeasts can be dried and stored in powder
form.
Table IX Ratio of microorganisms
Yeast Quantity Percentage Note
(dry weight)
Nitrogen-fixing yeast1.0-2.Okg 0.1-0.2% Dry yeast powder
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Phosphorus-decomposing1.0-2.Okg 0.1-0.2% Dry yeast powder
yeast
Potassium-decomposing1.0-2.Okg 0.1-0.2% Dry yeast powder
yeast
Carbon-decomposing 1.0-2.Okg 0.1-0.2% Dry yeast powder
yeast
Growth factor-producing25L 1% Yeast fermentation
yeast broth
ATP-producing yeast 75L 3% Yeast fermentation
broth
5.10.6. Manufacture of Biological Fertilizer
The biological fertilizer of the present invention is produced by mixing the
yeast mixture of Section 5.10.5 and the mixture of the organic and inorganic
materials of
Section 5.10.1 at a ratio according to Table X. For example, the yeasts and
the organic and
inorganic materials are conveyed to a granulizer to form granules. The
granules of the
fertilizer are then dried in a two-stage drying process. During the first
drying stage, the
fertilizer is dried in a first dryer at a temperature not exceeding 65
°C for a period of time
not exceeding 10 minutes so that yeast cells quickly become dormant. The
fertilizer is then
send to a second dryer and dried at a temperature not exceeding 70 ° C
for a period of time
not exceeding 30 minutes to further remove water. After the two stages, the
water content
should be lower than 5%. It is preferred that the temperatures and drying
times be adhered
to in both drying stages so that yeast cells do not lose their vitality and
functions. The
fertilizer is then cooled to room temperature. The fertilizer may also be
screened in a
separator so that fertilizer granules of a preferred size are selected. Any
separator, such as
but not limited to a turbo separator with adjustable speed and screen sizes,
can be used. The
fertilizer of the selected size is then sent to a bulk bag filler for packing.
The production process is illustrated in Figs. 9-11. Fig. 9 is a schematic
illustration of the procedure for producing the fertilizer from its
components. Fig. 10 is a
schematic illustration of the drying process. Fig. 11 is a schematic
illustration of the cooling
and packing process.
Table X Composition of the biological fertilizer (for one metric ton of
fertilizer)
Quantity Percentage Note
(dry
3 weight)
5
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Mixture of raw materials 952-956kg 95.2-95.4% Dry weight
Mixture of yeasts 100L 4.4-4.8% Dry weight
6. EXAMPLE
The following example demonstrates the manufacture of a biological
fertilizer composition of the present invention. This example is a preferred
embodiment of
the pxesent invention.
Saccharomyces cerevisiae Hansen strains having accession numbers
AS2.501, AS2.535, AS2.441, AS2.406, AS2.382, and AS2.16, each of which is
deposited in
China General Microbiological Culture Collection Center (CGMCC), China
Committee for
Culture Collection of Microorganisms, were used to prepare the yeast cell
components of
the biological fertilizer. Yeast strain AS2.501 was cultured according to the
method
described in Section 5.1 for nitrogen-fixation. Yeast strain AS2.535 was
cultured according
to the method described in Section 5.2 for P-decomposition. Yeast strain
AS2.441 was
cultured according to the method described in Section 5.3 for K-decomposition.
Yeast
strain AS2.406 was cultured according to the method described in Section 5.4
for C-
decomposition. Yeast strain AS2.382 was cultured according to the method
described in
Section 5.5 for growth factor-production. Yeast strain AS2.16 was cultured
according to the
method described in Section 5.6 for ATP-production.
Coal mine waste and phosphate rock were used as organic and inorganic
materials respectively. The coal mine waste used in the example contained at
least 30% of
organic substances. The phosphate rock used in the example contained at least
25% of
pZps, Coal mine waste and phosphate rock were prepared according to Sections
5.10.1.
The production of growth factor-producing yeast was carried out in a
fermentation process using as seed the activated yeast strain AS2.382 as
described in
Section 5.5. A schematic of the fermentation process is illustrated in Fig. 6.
The
fermentation medium was prepared according to a ratio of 2.5 liters of clean
water per
kilogram of starch. The fermentation medium was inoculated according to a
ratio of l Oml
of seed solution per liter of medium. The fermentation was carried out at a
temperature of
281 °C and an oxygen concentration of 0.4mollm3 in a clean environment
where there
were no sources of electromagnetic fields for about 48 hours. After
fermentation, the cell
counts reached about 2x101° cells/ml.
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The production of ATP-producing yeast was carried out in a fermentation
process using as seed the activated yeast strain AS2.16 as described in
Section 5.6. A
schematic of the fermentation process is illustrated in Fig. 6. The
fermentation medium was
prepared according to a ratio of 2.5 liters of clean water per kilogram of
starch. The
fermentation medium was inoculated according to a ratio of 10m1 of seed
solution per liter
of medium. The fermentation was carn.ed out at a temperature of 281 °C
and an oxygen
concentration of 0.4mo1/m3 in a clean environment where there were no sources
of
electromagnetic fields for about 56 hours. After fermentation, the cell counts
reached about
2x10'° cells/ml.
The mixture of raw materials was prepared according to Table XI and the
procedure in Section 5.10.4.
Table XI Ratio of raw materials
Material Percentage Requirement
Powder of coal mine 65% >_ 150 mesh, water
waste content
<_ 5%
Powder of phosphate 20% > 150 mesh, water
rock content
<3%
20Starch 15% regular starch powder,
water
content <_ 8%
The yeast mixture was prepared according to Table XII and the procedure
described in Section 5.10.5.
Table XII Ratio of yeasts (for 1 metric ton of fertilizer)
Yeast Quantity PercentageNote
(dry weight)
Nitrogen-fixing yeast 2.Okg 0.2% Dry yeast powder
AS2.502
Phosphorus-decomposing2.Okg 0.2% Dry yeast powder
yeast
AS2.535
Potassium-decomposing 2.Okg 0.2% Dry yeast powder
yeast
AS2.441
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Carbon-decomposing yeast2.Okg 0.2% Dry yeast powder
AS2.406
Growth factor producing25L 1 % Yeast fermentation
yeast
AS2.382 broth
ATP producing yeast 75L 3% Yeast fermentation
AS2.16
broth
The biological fertilizer was produced by mixing the yeast mixture, the
organic and inorganic materials at a ratio according to Table XIII. The mixed
yeasts and
organic and inorganic materials were conveyed to a granulizes to form
granules. The
granules of the fertilizer were then dried in a two stage drying process.
During the first
drying stage, the fertilizer was dried in a first dryer at a temperature not
exceeding 602°C
for a period of 5 minutes so that yeast cells quickly became dormant. The
fertilizer was then
1 S Sent to a second dryer and dried at a temperature not exceeding 652
°C for a period of 8
minutes to further remove water. The fertilizer was then cool to room
temperature. The
fertilizer was then sent to a bulk bag filler for packing.
Table XIII Fertilizer composition (for 1 metric ton of fertilizer)
Quantity Percentage (dryNote
weight)
Raw material mixture952kg 95.2% Dry weight
Yeast mixture 100L 4.8% Dry weight
The present invention is not to be limited in scope by the specific
embodiments
described which are intended as single illustrations of individual.aspects of
the invention,
and functionally equivalent methods and components are within the scope of the
invention.
Indeed various modifications of the invention, in addition to those shown and
described
herein will become apparent to those skilled in the art from the foregoing
description and
accompanying drawings. Such modifications are intended to fall within the
scope of the
appended claims.
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