U.S. patent application number 10/999861 was filed with the patent office on 2005-07-21 for biological fertilizer based on yeasts.
This patent application is currently assigned to Ultra Biotech Limited. Invention is credited to Zhang, Lingyu.
Application Number | 20050155400 10/999861 |
Document ID | / |
Family ID | 24627707 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050155400 |
Kind Code |
A1 |
Zhang, Lingyu |
July 21, 2005 |
Biological fertilizer based on yeasts
Abstract
The present invention provides biological fertilizer
compositions that comprise yeast cells that have an enhanced
ability to fix atmospheric nitrogen, decompose phosphorus minerals
and compounds, decompose potassium minerals and compounds,
decompose complex carbon compounds, over produce growth factors,
and over produce ATP. The biological fertilizer composition of the
invention can replace mineral fertilizers in supplying nitrogen,
phosphorus, and potassium to crop plants. Methods of manufacturing
the biological fertilizer compositions and methods of uses are also
encompassed.
Inventors: |
Zhang, Lingyu; (Bethesda,
MD) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
Ultra Biotech Limited
|
Family ID: |
24627707 |
Appl. No.: |
10/999861 |
Filed: |
November 29, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10999861 |
Nov 29, 2004 |
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10192805 |
Jul 9, 2002 |
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6828131 |
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10192805 |
Jul 9, 2002 |
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09655144 |
Sep 5, 2000 |
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6416982 |
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Current U.S.
Class: |
71/11 |
Current CPC
Class: |
C05F 11/08 20130101;
C12N 1/14 20130101; C12N 13/00 20130101 |
Class at
Publication: |
071/011 |
International
Class: |
C05F 001/00 |
Claims
1. A biological fertilizer composition comprising at least one of
the following yeast cell components: (a) a first yeast cell
component comprising a first plurality of yeast cells that fix
nitrogen; (b) a second yeast cell component comprising a second
plurality of yeast cells that decompose phosphorus compounds; or
(c) a third yeast cell component comprising a third plurality of
yeast cells that decompose potassium compounds.
2. The biological composition of claim 1 further comprising: (d) a
fourth yeast cell component comprising a fourth plurality of yeast
cells that convert complex carbon compounds to simple
carbohydrates; (e) a fifth yeast cell component comprising a fifth
plurality of yeast cells that overproduce growth factors; and (f) a
sixth yeast cell component comprising a sixth plurality of yeast
cells that overproduce adenosine triphosphate.
3. A biological fertilizer composition comprising at least one of
the following yeast cell components: (a) a first yeast cell
component prepared by culturing a first plurality of yeast cells in
a first electromagnetic field having a frequency in the range of
860 to 870 MHz and an amplitude in the range of 1000 to 5000 mV for
a period of time sufficient to cause said first plurality of yeast
cells to fix nitrogen; (b) a second yeast cell component prepared
by culturing a second plurality of yeast cells in a second
electromagnetic field having a frequency in the range of 360 to 370
MHz and an amplitude in the range of 1000 to 5000 mV for a period
of time sufficient to cause said second plurality of yeast cells to
decompose phosphorus compounds; or (c) a third yeast cell component
prepared by culturing a third plurality of yeast cells in a third
electromagnetic field having a frequency in the range of 250 to 260
MHz and an amplitude in the range of 1000 to 5000 mV for a period
of time sufficient to cause said third plurality of yeast cells to
decompose potassium compounds.
4. The biological composition of claim 3 further comprising: (d) a
fourth yeast cell component prepared by culturing a fourth
plurality of yeast cells in a fourth electromagnetic field having a
frequency in the range of 1087 to 1097 MHz and an amplitude in the
range of 1000 to 5000 mV for a period of time sufficient to cause
said fourth plurality of yeast cells to convert complex carbon
molecules to simple carbohydrates; (e) a fifth yeast cell component
prepared by culturing a fifth plurality of yeast cells in a fifth
electromagnetic field having a frequency in the range of 1382 to
1392 MHz and an amplitude in the range of 1000 to 5000 mV for a
period of time sufficient to cause said fifth plurality of yeast
cells to overproduce growth factors; and (f) a sixth yeast cell
component prepared by culturing a sixth plurality of yeast cells in
a sixth electromagnetic field having a frequency in the range of
1690 to 1700 MHz and an amplitude in the range of 1000 to 5000 mV
for a period of time sufficient to cause said plurality of yeast
cells to overproduce adenosine triphosphate.
5. The biological fertilizer composition of claim 2 or 4 further
comprising an organic substrate component, an inorganic substrate
component, or both organic and inorganic substrate components.
6. The biological fertilizer composition of claim 2 or 4 wherein
each yeast cell component separately comprises yeast cells that
belongs to a genus selected from the group consisting of
Saccharomyces, Schizosaccharomyces, Sporobolomyces, Torulopsis,
Trichosporon, Wickerhamia, Ashbya, Blastomyces, Candida,
Citeromyces, Crebrothecium, Cryptococcus, Debaryomyces,
Endomycopsis; Geotrichum, Hansenula, Kloeckera, Lipomyces, Pichia,
Rhodosporidium, and Rhodotorula.
7. The biological fertilizer composition of claim 2 or 4 wherein
each yeast cell component comprises cells of a species of yeast
selected from the group consisting of Saccharomyces cerevisiae,
Saccharomyces chevalieri, Saccharomyces delbrueckii, Saccharomyces
exiguus, Saccharomyces fermentati, Saccharomyces logos,
Saccharomyces mellis, Saccharomyces microellipsoides, Saccharomyces
oviformis, Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces
sake, Saccharomyces uvarum Beijer, Saccharomyces willianus,
Saccharomyces sp., Saccharomyces ludwigii, Saccharomyces sinenses,
Saccharomyces bailii, Saccharomyces carlsbergensis,
Schizosaccharomyces octosporus, Schizosaccharomyces pombe,
Sporobolomyces roseus, Sporobolomyces salmonicolor, Torulopsis
candida, Torulopsis famta, Torulopsis globosa, Torulopsis
inconspicua, Trichosporon behrendoo, Trichosporon capitatum,
Trichosporon cutaneum, Wickerhamia fluoresens, Ashbya gossypii,
Blastomyces dermatitidis, Candida albicans, Candida arborea,
Candida guillermondii, Candida Krusei, Candida lambica, Candida
lipolytica, Candida parakrusei, Candida parapsilosis, Candida
parapsilosis, Candida pseudotropicalis, Candida pulcherrima,
Candida robusta, Candida rugousa, Candida utilis, Citeromyces
matritensis, Crebrothecium ashbyii, Cryptococcus laurentii,
Cryptococcus neoformans, Debaryomyces hansenii, Debaryomyces
kloeckeri, Endomycopsis fibuligera, Eremothecium ashbyii,
Geotrichum candidum, Geotrichum ludwigii, Geotrichum robustum,
Geotrichum suaveolens, Hansenula anomala, Hansenula arabitolgens,
Hansenula jadinii, Hansenula saturnus, Hansenula schneggii,
Hansenula subpelliculosa, Kloeckera apiculata, Lipomyces starkeyi,
Pichia farinosa, Pichia membranaefaciens, Rhodosporidium
toruloides, Rhodotorula aurantiaca, Rhodotorula glutinis,
Rhodotorula minuta, Rhodotorula rubar, and Rhodotorula sinesis.
8. The biological fertilizer composition of claim 2 or 4 wherein
each yeast cell component comprises cells of Saccharomyces
cerevisiae.
9. The biological fertilizer composition of claim 2 or 4 wherein
the yeast cells of each yeast cell component are separately cells
of the yeast strain Saccharomyces cerevisiae Hansen deposited at
China General Microbiological Culture Collection Center having an
accession number selected from the group consisting of AS2.501,
AS2.535, AS2.441, AS2.406, AS2.382, and AS2.16.
10. The biological fertilizer composition of claim 2 which
comprises yeast cell components (a), (b), and (c) of claim 1.
11. The biological fertilizer composition of claim 4 which
comprises yeast cell components (a), (b), and (c) of claim 3.
12. The biological fertilizer composition of claim 10 or 11 further
comprising an organic substrate component, an inorganic substrate
component, or both an organic and an inorganic substrate
component.
13. The biological fertilizer composition of claim 12 which
comprises about 0.1 to 0.2% by weight of yeast cell component (a),
about 0.1 to 0.2% by weight of yeast cell component (b), about 0.1
to 0.2% by weight of yeast cell component (c), about 0.1 to 0.2% by
weight of yeast cell component (d), about 1% by weight of yeast
cell component (e), about 3% by weight of yeast cell component (f);
about 65% by weight of organic substrate component; about 19% by
weight of inorganic substrate component; and about 14% by weight of
starch.
14-20. (canceled)
21. A biological fertilizer composition comprising (i) at least one
of the following yeast cell components: (a) a first yeast cell
component prepared by culturing a first plurality of yeast cells in
a first electromagnetic field having a frequency of about 865.522
MHz and an amplitude of about 1250 mV for a period of 24 hours and
culturing said first plurality of yeast cells in the presence of a
second electromagnetic field having a frequency of about 865.522
MHz and an amplitude of about 4656 mV for a period of 24 hours so
that said first plurality of yeast cells can fix nitrogen; (b) a
second yeast cell component prepared by culturing a second
plurality of yeast cells in a first electromagnetic field having a
frequency of about 366.243 MHz and an amplitude of about 1230 mV
for a period of 24 hours and culturing said second plurality of
yeast cells in the presence of a second electromagnetic field
having a frequency of about 366.243 MHz and an amplitude of about
4570 mV for a period of 24 hours so that said second plurality of
yeast cells can decompose phosphorus compounds; or (c) a third
yeast cell component prepared by culturing a third plurality of
yeast cells in a first electromagnetic field having a frequency of
about 255.425 MHz and an amplitude of about 1340 mV for a period of
24 hours and culturing said third plurality of yeast cells in the
presence of a second electromagnetic field having a frequency of
about 255.425 MHz and an amplitude of about 4850 mV for a period of
24 hours so that said plurality of yeast cells can decompose
potassium compounds; (ii) a fourth yeast cell component prepared by
culturing a fourth plurality of yeast cells in a first
electromagnetic field having a frequency of about 1092.387 MHz and
an amplitude of about 1530 mV for a period of 24 hours and
culturing said fourth plurality of yeast cells in the presence of a
second electromagnetic field having a frequency of about 1092.387
MHz and an amplitude of about 4720 mV for a period of 24 hours so
that said fourth plurality of yeast cells can convert complex
carbon molecules to simple carbohydrates; (iii) a fifth yeast cell
component prepared by culturing a fifth plurality of yeast cells in
a first electromagnetic field having a frequency of about 1387.556
MHz and an amplitude of about 1620 mV for a period of 24 hours and
culturing said fifth plurality of yeast cells in the presence of a
second electromagnetic field having a frequency of about 1387.556
MHz and an amplitude of about 4830 mV for a period of 24 hours so
that said fifth plurality of yeast cells can overproduce growth
factors; and (iv) a sixth yeast cell component prepared by
culturing a sixth plurality of yeast cells in a first
electromagnetic field having a frequency of about 1694.365 MHz and
an amplitude of about 1470 mV for a period of 24 hours and
culturing said sixth plurality of yeast cells in the presence of a
second electromagnetic field having a frequency of about 1694.365
MHz and an amplitude of about 4780 mV for a period of 24 hours so
that said plurality of yeast cells can overproduce adenosine
triphosphate; wherein said yeast cell components comprise cells of
Saccharomyces cerevisiae.
22. The biological fertilizer composition of claim 21 wherein the
first yeast cell component comprises cells of the yeast strain
Saccharomyces cerevisiae Hansen AS2.501, the second yeast cell
component comprises cells of the yeast strain Saccharomyces
cerevisiae Hansen AS2.535, the third yeast cell component comprises
cells of the yeast strain Saccharomyces cerevisiae Hansen AS2.441,
the fourth yeast cell component comprises cells of the yeast strain
Saccharomyces cerevisiae Hansen AS2.406, the fifth yeast cell
component comprises cells of the yeast strain Saccharomyces
cerevisiae Hansen AS2.382, and the sixth yeast cell component
comprises cells of the yeast strain Saccharomyces cerevisiae Hansen
AS2.16.
23. The biological fertilizer composition of claim 21, wherein the
pluralities of yeast cells are dried.
24. A method of activating or enhancing the ability of a plurality
of yeast cells to fix atmospheric nitrogen, comprising culturing
said plurality of yeast cells in the presence of an electromagnetic
field having a frequency in the range of 850 to 860 MHz and an
amplitude in the range of 1000 to 5000 mV for a period of time
sufficient to cause said plurality of yeast cells to fix
nitrogen.
25. (canceled)
26. A method of activating or enhancing the ability of a plurality
of yeast cells to decompose phosphorus-containing minerals or
compounds, comprising culturing said plurality of yeast cells in
the presence of an electromagnetic field having a frequency in the
range of 360 to 370 MHz and an amplitude in the range of 1000 to
5000 mV for a period of time sufficient to cause said plurality of
yeast cells to decompose phosphorus compounds.
27. (canceled)
28. A method of activating or enhancing the ability of a plurality
of yeast cells to decompose potassium-containing minerals or
compounds, comprising culturing said plurality of yeast cells in
the presence of an electromagnetic field having a frequency in the
range of 250 to 260 MHz and an amplitude in the range of 1000 to
5000 mV for a period of time sufficient to cause said plurality of
yeast cells to decompose potassium compounds.
29. (canceled)
30. A method of activating or enhancing the ability of a plurality
of yeast cells to decompose high molecular weight carbon
substances, comprising culturing said plurality of yeast cells in
the presence of an electromagnetic field having a frequency in the
range of 1087 to 1097 MHz and an amplitude in the range of 1000 to
5000 mV for a period of time sufficient to cause said plurality of
yeast cells to convert complex carbon molecules to simple
carbohydrates.
31. (canceled)
32. A method of activating or enhancing the ability of a plurality
of yeast cells to overproduce growth factors, comprising culturing
said plurality of yeast cells in the presence of an electromagnetic
field having a frequency in the range of 1382 to 1392 MHz and an
amplitude in the range of 1000 to 2000 mV for a period of between
1000 to 5000 mV for a period of time sufficient to cause said
plurality of yeast cells to overproduce growth factors.
33. The method of claim 32, comprising culturing said plurality of
yeast cells in the presence of a first electromagnetic field having
a frequency of about 1387.556 and an amplitude of about 1620 mV for
a period of 24 hours and culturing said fifth plurality of yeast
cells in the presence of a second electromagnetic field having a
frequency of about 1387.556 MHz and an amplitude of about 4830 mV
for a period of 24 hours so that said fifth plurality of yeast
cells can overproduce growth factors.
34. The method of claim 32 or 33, further comprising inoculating a
fermentation medium comprising a starch solution at a concentration
of about 400 gram/liter with said yeast cells and allowing
fermentation to proceed at a temperature between 20 to 30.degree.
C. until at least 90% of the fermentation substrate has been
fermented.
35. A method of activating the ability of a plurality of yeast
cells to overproduce ATP, comprising culturing said plurality of
yeast cells in the presence of an electromagnetic field having a
frequency in the range of 1690 to 1700 MHz and an amplitude in the
range of 1000 to 5000 mV for a period of time sufficient to cause
said plurality of yeast cells to overproduce adenosine
triphosphate.
36. The method of claim 35, comprising culturing said plurality of
yeast cells in the presence of a first electromagnetic field having
a frequency of about 1694.365 MHz and an amplitude of about 1470 mV
for a period of 24 hours and culturing said sixth plurality of
yeast cells in the presence of a second electromagnetic field
having a frequency of about 1694.365 MHz and an amplitude of about
4780 mV for a period of 24 hours so that said plurality of yeast
cells can overproduce adenosine triphosphate.
37. The method of claim 35 or 36, further comprising inoculating a
fermentation medium comprising a starch solution at a concentration
of about 400 gram/liter with said yeast cells and allowing
fermentation to proceed at a temperature between 20 to 30.degree.
C. until at least 90% of the fermentation substrate has been
fermented.
38. A method of forming a symbiosis-like relationship among yeast
cells components of a biological fertilzer, said method comprises
the steps of: preparing a mixture comprising (a) a first yeast cell
component prepared by culturing a first plurality of yeast cells in
a first electromagnetic field having a frequency in the range of
860 to 870 MHz and an amplitude in the range of 1000 to 5000 mV for
a period of time sufficient to cause said first plurality of yeast
cells to fix nitrogen; (b) a second yeast cell component prepared
by culturing a second plurality of yeast cells in a second
electromagnetic field having a frequency in the range of 360 to 370
MHz and an amplitude in the range of 1000 to 5000 mV for a period
of time sufficient to cause said second plurality of yeast cells to
decompose phosphorus compounds; (c) a third yeast cell component
prepared by culturing a third plurality of yeast cells in a third
electromagnetic field having a frequency in the range of 250 to 260
MHz and an amplitude in the range of 1000 to 5000 mV for a period
of time sufficient to cause said third plurality of yeast cells to
decompose potassium compounds; and (d) a fourth yeast cell
component prepared by culturing a fourth plurality of yeast cells
in a fourth electromagnetic field having a frequency in the range
of 1087 to 1097 MHz and an amplitude in the range of 1000 to 5000
mV for a period of time sufficient to cause said fourth plurality
of yeast cells to convert complex carbon molecules to simple
carbohydrates; and culturing said mixture in the presence of an
electromagnetic field having a plurality of frequencies of 860 to
870 MHz, 360 to370 MHz, 250 to 260 MHz, and 1087 to 1097 MHz and
each frequency having an amplitude of 0 to 3000 mV for a period of
time sufficient to cause said yeast cell components to form
symbiosis-like relationship, wherein the amplitude for each
frequency in said electromagnetic field is cycled between 0 to 3000
mV.
39. The method of claim 24, further comprising: preparing a mixture
comprising said plurality of yeast cells and soil; culturing said
mixture in an electromagnetic field having a frequency in the range
of 850 to 860 MHz and an amplitude in the range of 1000 to 5000 mV
for a period of time sufficient to cause said plurality of yeast
cells to adapt to soil.
40. The method of claim 26, further comprising: preparing a mixture
comprising said plurality of yeast cells and soil; culturing said
mixture in an electromagnetic field having a frequency in the range
of 360 to 370 MHz and an amplitude in the range of 1000 to 5000 mV
for a period of time sufficient to cause said plurality of yeast
cells to adapt to soil.
41. The method of claim 28, further comprising: preparing a mixture
comprising said plurality of yeast cells and soil; culturing said
mixture in an electromagnetic field having a frequency in the range
of 250 to 260 MHz and an amplitude in the range of 1000 to 5000 mV
for a period of time sufficient to cause said plurality of yeast
cells to adapt to soil.
42. The method of claim 30, further comprising: preparing a mixture
comprising said plurality of yeast cells and soil; culturing said
mixture in an electromagnetic field having a frequency in the range
of 1087 to 1097 MHz and an amplitude in the range of 1000 to 5000
mV for a period of time sufficient to cause said plurality of yeast
cells to adapt to soil.
43. The method of claim 32, further comprising: preparing a mixture
comprising said plurality of yeast cells and soil; culturing said
mixture in an electromagnetic field having a frequency in the range
of 1382 to 1392 MHz and an amplitude in the range of 1000 to 5000
mV for a period of time sufficient to cause said plurality of yeast
cells to adapt to soil.
44. The method of claim 35, further comprising: preparing a mixture
comprising said plurality of yeast cells and soil; culturing said
mixture in an electromagnetic field having a frequency in the range
of 1690 to 1700 MHz and an amplitude in the range of 1000 to 5000
mV for a period of time sufficient to cause said plurality of yeast
cells to adapt to soil.
45-50. (canceled)
51. A method for enhancing plant growth comprising growing the
plant in the presence of a biological fertilizer composition of any
one of the claims 1-13 and 21-23.
Description
1. FIELD OF THE INVENTION
[0001] 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
[0002] 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.sub.3.sup.- or NH.sub.4.sup.+), phosphorus (taken up as
H.sub.2PO.sub.4.sup.-), and potassium (taken up as K.sup.+)
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.)
[0003] 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 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.
[0004] 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.
[0005] Biological fertilizers utilizing microorganisms have been
proposed as alternatives to mineral fertilizers. Naturally
occurring nitrogen fixing microorganisms including bacteria, such
as Rhizobium, Azotobacter, and Azospirillum, (See for example, U.S.
Pat. No. 5,071,462) and fungi, such as Aspergillus flavus-oryzae,
(See, for example, U.S. Pat. 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. Pat. No. 5,912,398) or in
combination with nitrogen fixing microorganisms (e.g., U.S. Pat.
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, see, for example, U.S. Pat. No. 5,578,486; PCT
publication WO 95/09814; Chinese patent publication: CN 1081662A;
CN 1082016A; CN 1082017A; CN 1103060A; and CN 1109595A.
[0006] 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.
[0007] 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
[0008] 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 and/or 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.
[0009] 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 or 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 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.
[0010] 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.
[0011] 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.
[0012] 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
[0013] FIG. 1. Activation of yeast cells. 1 yeast culture; 2
container; 3 electromagnetic field source.
[0014] FIG. 2. Formation of symbiosis-like relationships among
yeast strains. 4 electromagnetic field source for nitrogen-fixing
yeast; 5 electromagnetic field source for P-decomposing yeast; 6
electromagnetic field source for K-decomposing yeast; 7
electromagnetic field source for C-decomposing yeast; 8 yeast
culture; 9 container.
[0015] 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.
[0016] FIG. 4. Organic material grinding process. 16 organic raw
material; 17 crusher; 18 grinder; 19 organic material in powder
form.
[0017] FIG. 5. Inorganic material grinding process. 20 inorganic
raw material; 21 crusher, 22 grinder, 23 inorganic material in
powder form.
[0018] FIG. 6. Yeast fermentation process. 24 activated yeast
cells; 25 tank for culturing yeast cells, starch:water (35.degree.
C.)=1:2.5, semi-aerobic fermentation at 28 to 30.degree. C. for 48
to 72 hours; 26 harvested culture.
[0019] 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.
[0020] 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.
[0021] FIG. 9. Fertilizer production process. 39 mixture of yeast;
40 mixture of organic and inorganic materials; 41 granulizer; 42
fertilizer granules.
[0022] FIG. 10. Drying process. 43 fertilizer granules; 44 first
dryer; 45 second dryer; 46 dried fertilizer.
[0023] 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
[0024] 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.
[0025] The biological fertilizer compositions of the invention can
replace chemical/mineral fertilizers in supplying nitrogen (N),
phosphorus (P), and potassium (K) to plants, 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 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.
[0026] 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 of potassium 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.
[0027] 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 micronutrients, such as but not limited
to boron, copper, iron, manganese, molybdenum, and zinc.
[0028] 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 limited
to, ammonia, ammonium salts, urea, and nitrates.
[0029] As used herein, the phrase "decomposition of phosphorus
minerals or compounds" refers 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 compound(s) which can be more
readily 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.
[0030] As used herein, the phrase "decomposition of potassium
minerals or compounds" refers biological processes which convert
potassium (K) compounds, such as but not limited to those
water-insoluble potassium compounds present in potassium mica, into
one or more different potassium compound(s) 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.
[0031] 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 molecule(s) 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.
[0032] 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.
[0033] 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.
[0034] 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-CM.sub.61 can be used with
environmental and organic wastes, such as waste and manure from
urban areas and animal manure.
[0035] 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.
[0036] 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, in 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 1000 l, one electrode wire having a diameter of between 3-30
mm can be used for each 1000 l 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, Endomycopsis; Geotrichum, Hansenula, 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 11001, 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 1311, 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 chevalieri
Guillermond, AS2.131, AS2.213; Saccharomyces delbrueckii Lindner,
AS2.285; Saccharomyces delbrueckii Lindner ver. mongolicus Lodder,
AS2.209, AS2.11157; 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 oviformis Osterwalder, AS2.100;
Saccharomyces rosei Lodder et kreger van Rij, AS2.287;
Saccharomyces rouxi Boutroux, AS2.178, AS2.180, AS2370, AS2.371;
Saccharomyces sake Yabe, ACCC2045; Saccharomyces uvarum Beyer, 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;
Saccharomyces sp., AS2.311; Saccharomyces ludwigii Hansen,
ACCC2044, AS2.243, AS2.508; Saccharomyces sinenses Yue, AS2.1395;
Schizosaccharomyces octosporus Beijerinck, ACCC 2046, AS2.1148;
Schizosaccharomyces pombe 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, IFFI.1056; 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 fluo{overscore (r)}esens (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 Krusei (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; Candida pseudotropicalis (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, AS2.511, 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(C.sub.2)a, ID 17 b(C.sub.2)b, ID 17
d(C.sub.2)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 (y.sub.2);
Cryptococcus neoformans (Sanfelice) Vuillemin, ID 25 u(D.sub.2)u,
ID 25 v(D.sub.2)v, ID 25 w(D.sub.2)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 sp., ACCC2011, 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; Hansenula 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
(Demme) Lodder, ACCC2031, AS2.21, AS2.22, AS2.103, AS2.105,
AS2.108, AS2.140, AS2.166, AS2.272, AS2.279, AS2.282; Rhodotorula
sinesis Lee, AS2.1391; Saccharomyces bailii 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.
[0037] 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 albicans (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(C.sub.2)a, ID 17 b(C.sub.2)b, ID 17 d(C.sub.2)d; Citeromyces
matritensis (Santa Maria) Santa Maria, AS2.1401; Crebrothecium
ashbyii (Guillermond) Routein, ACCC2013, ACCC2014; Cryptococcus
laurentii (Kufferath) Skinner, ACCC2007, AS2.114, ID 95 (y.sub.2);
Cryptococcua neoformans (Sanfelice) Vuillemin, ID 25 u(D.sub.2)u,
ID 25 v(D.sub.2)v, ID 25 w(D.sub.2)vw, Debaryomyces hansenii (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.
[0038] 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.
[0039] 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.
[0040] 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. Pat. 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-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).
[0041] 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.
[0042] 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 one 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.
[0043] In another embodiment of the invention, where the yeast
cells of the various 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 for
this optional process are described in Section 5.7.
[0044] 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.
[0045] 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.
[0046] 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
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.
[0047] Described respectively in Sections 5.1-5.6 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 5.7 describes the methods for establishing a
symbiosis-like relationship among yeast strains in a fertilizer
composition of the invention. Section 5.8 describes methods for
adapting yeast cells of the invention to a particular type of soil.
Section 5.9 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 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.
[0048] 5.1. Nitrogen-Fixing Yeast Cell Component
[0049] 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.
[0050] 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.
[0051] 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
can be tested for their ability to fix nitrogen by methods well
known in the art.
[0052] 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 0.1% 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.
[0053] 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.sub.3,
KH.sub.2PO.sub.4, MgSO.sub.4, NaCl, and CaSO.sub.4.
1TABLE I Composition for a culture medium for nitrogen-fixing yeast
Medium Composition Quantity KH.sub.2PO.sub.4 0.2 g K.sub.2HPO.sub.4
0.2 g MgSO.sub.4.7H.sub.2O 0.25 g CaCO.sub.3.5H.sub.2O 3.5 g
CaSO.sub.4.2H.sub.2O 0.5 g NaCl 0.25 g Yeast extract paste 0.3 g
Sucrose 12.0 g Distilled water or autoclaved water 1000 ml
[0054] 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.
[0055] The process is initiated by inoculating each 100 ml of
medium with 1 ml of an inoculum of the selected yeast strain(s) at
a cell density of 10.sup.2-10.sup.5 cell/ml, preferably
3.times.10.sup.2-10.sup.4 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 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. The amplitude of the field is in the range of 1000-2000 mV,
preferably at about 1250 mV. 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 FIG. 1. The process of the invention is
carried out at temperatures ranging-from about 25.degree. to
30.degree. C.; however, it is preferable to conduct the process at
28.degree. 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/m.sup.3, preferably 0.4 mol/m.sup.3. 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.
[0056] 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.degree.
C. to 4.degree. C. The nitrogen-fixing yeast cells may also be
dried and stored in powder form.
[0057] 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. Pat.
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.degree. 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.
5.2. Phosphorus-Decomposing Yeast Cell Component
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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 CaCO.sub.3, MgSO.sub.4,
NaCl, and CaSO.sub.4. Insoluble phosphorus-containing substances in
a suitable form are also included in the media Non-limiting
examples include powder of rock phosphate of .gtoreq.200 mesh.
Other insoluble phosphorus-containing substances can also be used
either separately or in combination.
2TABLE II Composition for a culture medium for P-decomposing yeast
Medium Composition Quantity Sucrose 15 g NaCl 1.2 g
MgSO.sub.4.7H.sub.2O 0.2 g CaCO.sub.3.5H.sub.2O 3.0 g
CaSO.sub.4.2H.sub.2O 0.3 g KNO.sub.3 0.3 g Yeast extract paste 0.5
g Rock phosphate 1.2 g; Powder of >200 mesh Autoclaved water
1000 ml
[0063] 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.
[0064] The process is initiated by inoculating each 100 ml of
medium with 1 ml of an inoculum of the selected yeast strain(s) at
a cell density of 10.sup.2-10.sup.5 cell/ml, preferably
3.times.10.sup.2-10.sup.4 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 360 to 370 MHz, preferably at about 366 MHz, more preferably in
the range of 366.199 to 366.287 MHz, and most preferably at 366.243
MHz. The amplitude of the field is in the range of 1000 to 2000 mV,
preferably at about 1230 mV. 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 FIG. 1. The process of the invention
is carried out at temperatures ranging from about 25.degree. to
30.degree. C.; however, it is preferable to conduct the process at
28.degree. 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/m.sup.3, preferably 0.4 mol/m.sup.3. 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.
[0065] 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.degree. C. to
4.degree. C. The P-decomposing yeast cells may also be dried and
stored in powder form.
[0066] 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.degree. C. for 24-56 hours. The amount of
biologically available phosphorus in the form of PO.sub.4.sup.3- 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 PO.sub.4.sup.3- in the culture is increased by at least 10 mg
for each gram of yeast dry weight. For example, after activation,
the amount of PO.sub.4.sup.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
[0067] 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.
[0068] 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.
[0069] 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-300 MHz 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.
[0070] 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 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.
[0071] 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 (NH.sub.4).sub.2HPO.sub.4,
CaCO.sub.3, MgSO.sub.4, NaCl, and CaSO.sub.4. Insoluble
potassium-containing substances in a suitable form are also
included in the media. Non-limiting examples include powder of
potassium mica of .gtoreq.200 mesh. Other insoluble
potassium-containing substances can also be used either separately
or combined.
3TABLE III Composition for a culture medium for K-decomposing yeast
Medium Composition Quantity Sucrose 15 g NaCl 1.2 g
MgSO.sub.4.7H.sub.2O 0.2 g CaCO.sub.3.5H.sub.2O 3.0 g
CaSO.sub.4.2H.sub.2O 0.3 g (NH.sub.4).sub.2HPO.sub.4 0.3 g Yeast
extract paste 0.3 g Potassium mica 1.2 g, Powder of >200 mesh
Autoclaved water 1000 ml
[0072] 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 of culture and local supply of media components.
[0073] The process is initiated by inoculating each 100 ml of
medium with 1 ml of an inoculum of the selected yeast stain(s) at a
cell density of 10.sup.2-10.sup.5 cell/ml, preferably
3.times.10.sup.2-10.sup.4 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 250-260 MHz, preferably at about 255 MHz, 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 1000-2000 mV,
preferably at About 1340 mV. 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 4850 mV. An exemplary set-up of the culture
process is depicted in FIG. 1. The process of the invention is
carried out at temperatures ranging from about 25.degree. to
30.degree. C.; however, it is preferable to conduct the process at
28.degree. 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/m.sup.3, preferably 0.4 mol/m.sup.3. 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.
[0074] 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.degree. C. The
K-decomposing yeast cells may also be dried and stored in powder
form.
[0075] 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.degree. C. for 24-56 hours. The amount of
biologically available potassium in the form of K.sup.+ 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.sup.+ 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.sup.+ in a culture of Saccharomyces cerevisiae Hansen
strain AS2.441 can reach about 4050 mg/g.
5.4. Complex Carbon-Decomposing Yeast Cell Component
[0076] The carbon-decomposing (C-decomposing) yeast of the
invention converts complex, usually high molecular weight, carbon
compounds and material, 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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 (NH.sub.4).sub.2HPO.sub.4,
CaCO.sub.3, MgSO.sub.4, NaCl, and CaSO.sub.4.
4TABLE IV Composition for a culture medium for C-decomposing yeast
Medium Composition Quantity Cellulose 5.0 g; Powder of >100 mesh
NaCl 0.6 g MgSO.sub.4.7H.sub.2O 0.3 g CaCO.sub.3.5H.sub.2O 1.5 g
CaSO.sub.4.2H.sub.2O 0.4 g (NH.sub.4).sub.2HPO.sub.4 0.3 g Yeast
extract paste 0.5 g K.sub.2HPO.sub.4 0.5 g Autoclaved water 1000
ml
[0081] 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.
[0082] The process is initiated by inoculating each 100 ml of
medium with 1 ml of an inoculum of the selected yeast strain(s) at
a cell density of 10.sup.2-10.sup.5 cell/ml, preferably
3.times.10.sup.2-10.sup.4 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 1087-1097 MHz, preferably about at 1092, more preferably in the
range of 1092.346 to 1092.428 MHz, and most preferably at 1092.387
MHz. The amplitude used can be in the range of 1000-200 mV,
preferably at about 1530 mV. 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 FIG. 1. The process of the invention is
carried out at temperatures ranging from about 25.degree. to
30.degree. C.; however, it is preferable to conduct the process at
28.degree. 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/m.sup.3, preferably 0.4 mol/m.sup.3. 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.
[0083] 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.degree. C. The
C-decomposing yeast cells may also be dried and stored in powder
form.
[0084] 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.degree. 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 of simple carbohydrates in a culture of Saccharomyces
cerevisiae Hansen AS2.406 can reach 27200 mg/g.
5.5. Growth Factors Producing Yeast Cell Component
[0085] The growth factors producing (GP-producing) yeast of the
present invention produces vitamins and other nutrients, such as
but not limited to, 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, inositol, in such
amounts that can support the growth of other yeast strains. Such
growth factors are produced by yeast during the fermentation
process.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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 NH.sub.4NO.sub.3,
K.sub.2HPO.sub.4, CaCO.sub.3, MgSO.sub.4, NaCl, and CaSO.sub.4.
5TABLE V Composition for a culture medium for GP-producing yeast
Medium Composition Quantity Starch 8.0 g; Powder of >120 mesh
NaCl 0.3 g MgSO.sub.4.7H.sub.2O 0.2 g CaCO.sub.3.5H.sub.2O 0.5 g
CaSO.sub.4.2H.sub.2O 0.2 g NH.sub.4NO.sub.3 0.3 g K.sub.2HPO.sub.4
0.8 g Autoclaved water 1000 ml
[0090] 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.
[0091] The process is initiated by inoculating each 100 ml of
medium with 1 ml of an inoculum of the selected yeast strain(s) at
a cell density of 10.sup.2-10.sup.5 cell/ml, preferably
3'10.sup.2-10.sup.4 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 1382-1392 MHz, preferably at about 1387 MHz, more preferably in
the range of 1387.517 to 1387.595 MHz, and most preferably at
1387.556 MHz. The amplitude used can be in the range of 1000-2000
mV, preferably at about 1620 mV. 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 FIG. 1. The process of the
invention is carried out at temperatures ranging from about
25.degree. to 30.degree. C.; however, it is preferable to conduct
the process at 28.degree. 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/m.sup.3, preferably
0.4 mol/m.sup.3. 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.
[0092] 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.degree. C. The
GP-producing yeast cells may also be dried and stored in powder
form.
[0093] 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.degree. C. for
32-48 hours. The amount of growth factors as represented by the
total amount of vitamin B1, B2, B6, and B12 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 B1, B2, B6, and B12 in a culture
of Saccharomyces cerevisiae Hansen AS2.382 can reach an aggregate
of 6120 mg/g.
5.6. ATP-Producing Yeast Cell Component
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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 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.
[0098] 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 (NH.sub.4).sub.2HPO.sub.4,
CaCO.sub.3, MgSO.sub.4, NaCl, and CaSO.sub.4.
6TABLE VI Composition for a culture medium for ATP-producing yeast
Medium Composition Quantity Starch 10.0 g NaCl 0.2 g
MgSO.sub.4.7H.sub.2O 0.2 g CaCO.sub.3.5H.sub.2O 0.8 g
CaSO.sub.4.2H.sub.2O 0.2 g NH.sub.4NO.sub.3 0.2 g K.sub.2HPO.sub.4
0.5 g Autoclaved water 1000 ml
[0099] 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.
[0100] The process is initiated by inoculating each 100 ml of
medium with 1 ml of an inoculum of the selected yeast strain(s) at
a cell density of 10.sup.2-10.sup.5 cell/ml, preferably
3.times.10.sup.2-10.sup.4 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 1690-1700 MHz, preferably at about 1694 MHz, 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
1000-2000 mV, preferably at about 1470 mV. 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 4780 mV. An
exemplary set-up of the culture process is depicted in FIG. 1. The
process of the invention is carried out at temperatures ranging
from about 25.degree. to 30.degree. C.; however, it is preferable
to conduct the process at 28.degree. 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/m.sup.3, preferably
0.4 mol/m.sup.3. 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.
[0101] 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.degree. C. The
ATP-producing yeast cells may also be dried and stored in powder
form.
[0102] 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.degree. 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 Saccharomyces cerevisiae
Hansen strain AS2.16 can reach about 3320 mg/g.
5.7. Formation of Symbiosis-Like Relationships
[0103] 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.
[0104] 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 CaCO.sub.3,
MgSO.sub.4, NaCl, and CaSO.sub.4.
7TABLE VII Composition for a culture medium for formation of
symbiosis-like relation Medium Composition Quantity NaCl 0.5 g
MgSO.sub.4.7H.sub.2O 0.4 g CaCO.sub.3.5H.sub.2O 3.0 g
CaSO.sub.4.2H.sub.2O 0.3 g Yeast extract paste 0.3 g Potassium mica
1.2 g; Powder of >200 mesh Rock phosphate 1.2 g; Powder of
>200 mesh Cellulose 5.0 g; Powder of >200 mesh Autoclaved
water 1000 ml
[0105] 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.
[0106] The culturing process may preferably be conducted under
conditions in which the concentration of dissolved oxygen is
between 0.025 to 0.8 mol/m.sup.3, preferably 0.4 mol/m.sup.3. The
oxygen level can be controlled by any conventional means known to
one skilled in the art, including but not limited to
stirring-and/or bubbling. The process of the invention is carried
out at temperatures ranging from about 25.degree. to 30.degree. C.;
however, it is preferable to conduct the process at 28.degree. C.
The process is initiated in sterilized medium by inoculating
typically about 20 ml of each inoculum of the four strains of yeast
cells, each at a cell density of about 10.sup.8 cell/ml. The
optional process can be scaled up or down according to needs.
[0107] 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:
(1) 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
360-370 MHz, preferably at about 366 MHz, more preferably in the
range of 366.199 to 366.287 MHz, and most preferably at 366.243
MHz, for phosphorus-decomposing; (3) in the range of 250-260 MHz,
preferably at about 255 MHz, 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 1087-1097 MHz,
preferably about at 1092, more preferably in the range of 1092.346
to 1092.428 MHz, and most preferably at 1092.387 MHz, for complex
carbon-decomposing. The amplitude of each electromagnetic field is
repeatedly cycled between 0-3000 mV, preferably between 20-1800 mV,
in steps of 1 mV at a rate of 18-23 minutes per complete cycle. An
exemplary set-up of the culture process is depicted in FIG. 2.
5.8. Soil Adaptation
[0108] 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.
[0109] 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.
[0110] A suspension containing 10 ml of yeasts at a density of
10.sup.6 cell/ml is mixed with a 1000 cm.sup.3 of the soil medium.
The process can be scaled up or down according to 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
100-3000 mV, preferably 2100 mV, can be used. The culture is
incubated at temperatures that cycle between about 3.degree. C. to
about 48.degree. C. For example, in a typical cycle, the
temperature of the culture may start at 35-48.degree. C. and be
kept at this temperature for about 1-2 hours, then adjusted up to
42-45.degree. C. and kept at this temperature for 1-2 hours, then
adjusted to 26-30.degree. C. and kept at this temperature for about
2-4 hours, and then brought down to 5-10.degree. C. and kept at
this temperature for about 1-2 hours, and then the temperature may
be raised again to 35-45.degree. 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.degree. 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.degree.
C. An exemplary set-up of the culture process is depicted in FIG.
3.
5.9. Separation or Enrichment of Yeast Cells
[0111] 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. Pat. 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 separated yeast cells can then be dried, and
stored.
5.10. Manufacture of the Biological Fertilizers
[0112] 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
[0113] 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 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.
[0114] 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 sufficient potassium minerals.
[0115] 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.
[0116] 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 .ltoreq.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 .gtoreq.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 5.
5.10.2. Fermentation Process Using Growth Factor-Producing
Yeast
[0117] In the present invention, the preparation of GP-producing
yeast is carried 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.
[0118] 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, is used to prepare the
fermentation medium. The fermentation is carried out at a
temperature between 20-30.degree. C., preferably between
25-28.degree. 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 2.times.10.sup.10
cells/ml. The fermentation broth is kept at a temperature in the
range of 15-28.degree. 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
[0119] 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.
[0120] 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.degree. C., preferably
between 25-28.degree. 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-concentrtion. 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
2.times.10.sup.10 cells/ml. The fermentation broth is kept at a
temperature in the range of 15-28.degree. C. and must be used
within 24 hours. Alternatively, the ATP-producing yeasts can be
drained, dried and stored in powder form.
5.10.4. Preparation of Mixture of Raw Materials
[0121] 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.
8TABLE VIII Ratio of raw materials Material Percentage Requirement
Powder of organic materials 60-71% .gtoreq.150 mesh, water content
.ltoreq.5% Powder of inorganic 15-20% .gtoreq.150 mesh, water
content materials .ltoreq.3% Starch 10-15% regular starch powder,
water content .ltoreq.8%
5.10.5. Preparation of Yeast Mixture
[0122] 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.degree. 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.
9TABLE IX Ratio of microorganisms Percentage Yeast Quantity (dry
weight) Note Nitrogen-fixing yeast 1.0-2.0 kg 0.1-0.2% Dry yeast
powder Phosphorus-decomposing 1.0-2.0 kg 0.1-0.2% Dry yeast powder
yeast Potassium-decomposing 1.0-2.0 kg 0.1-0.2% Dry yeast powder
yeast Carbon-decomposing 1.0-2.0 kg 0.1-0.2% Dry yeast powder yeast
Growth factor-producing 25 L 1% Yeast yeast fermentation broth
ATP-producing yeast 75 L 3% Yeast fermentation broth
5.10.6. Manufacture of Biological Fertilizer
[0123] 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.degree. 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.degree.
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.
[0124] 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.
10TABLE X Composition of the biological fertilizer (for one metric
ton of fertilizer) Percentage (dry Quantity weight) Note Mixture of
raw materials 952-956 kg 95.2-95.4% Dry weight Mixture of yeasts
100 L 4.4-4.8% Dry weight
6. EXAMPLE
[0125] The following example demonstrates the manufacture of a
biological fertilizer composition of the present invention. This
example is a preferred embodiment of the present invention.
[0126] 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.
[0127] 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 P.sub.2O.sub.5.
Coal mine waste and phosphate rock were prepared according to
Sections 5.10.1.
[0128] 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 10 ml of seed solution per liter
of medium. The fermentation was carried out at a temperature of
28.+-.1.degree. C. and an oxygen concentration of 0.4 mol/m.sup.3
in a clean environment where there were no sources of
electromagnetic fields for about 48 hours. After fermentation, the
cell counts reached about 2.times.10.sup.10 cells/ml.
[0129] 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 10 ml of seed solution per liter of medium.
The fermentation was carried out at a temperature of
28.+-.1.degree. C. and an oxygen concentration of 0.4 mol/m.sup.3
in a clean environment where there were no sources of
electromagnetic fields for about 56 hours. After fermentation, the
cell counts reached about 2.times.10.sup.10 cells/ml.
[0130] The mixture of raw materials was prepared according to Table
XI and the procedure in Section 5.10.4.
11TABLE XI Ratio of raw materials Material Percentage Requirement
Powder of coal mine waste 65% .gtoreq.150 mesh, water content
.ltoreq.5% Powder of phosphate rock 20% .gtoreq.150 mesh, water
content .ltoreq.3% Starch 15% regular starch powder, water content
.ltoreq.8%
[0131] The yeast mixture was prepared according to Table XII and
the procedure described in Section 5.10.5.
12TABLE XII Ratio of yeasts (for 1 metric ton of fertilizer)
Percentage Yeast Quantity (dry weight) Note Nitrogen-fixing yeast
2.0 kg 0.2% Dry yeast powder AS2.502 Phosphorus-decomposing 2.0 kg
0.2% Dry yeast powder yeast AS2.535 Potassium-decomposing 2.0 kg
0.2% Dry yeast powder yeast AS2.441
* * * * *