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WO1996039799A1 - A method of preparing a culture using a magnetic field and a compositon containing the same - Google Patents

A method of preparing a culture using a magnetic field and a compositon containing the same Download PDF

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Publication number
WO1996039799A1
WO1996039799A1 PCT/US1996/009574 US9609574W WO9639799A1 WO 1996039799 A1 WO1996039799 A1 WO 1996039799A1 US 9609574 W US9609574 W US 9609574W WO 9639799 A1 WO9639799 A1 WO 9639799A1
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WO
WIPO (PCT)
Prior art keywords
culture
microorganisms
composition
resulting
odor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1996/009574
Other languages
French (fr)
Inventor
Craig Jones
D. Michael Bitz
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EKMA Inc
Original Assignee
EKMA Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/476,374 external-priority patent/US5866112A/en
Priority claimed from US08/569,769 external-priority patent/US5843427A/en
Application filed by EKMA Inc filed Critical EKMA Inc
Priority to AU62625/96A priority Critical patent/AU6262596A/en
Publication of WO1996039799A1 publication Critical patent/WO1996039799A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L9/00—Disinfection, sterilisation or deodorisation of air
    • A61L9/01—Deodorant compositions
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F5/00—Fertilisers from distillery wastes, molasses, vinasses, sugar plant or similar wastes or residues, e.g. from waste originating from industrial processing of raw material of agricultural origin or derived products thereof
    • C05F5/006—Waste from chemical processing of material, e.g. diestillation, roasting, cooking
    • C05F5/008—Waste from biochemical processing of material, e.g. fermentation, breweries
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F9/00—Fertilisers from household or town refuse
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G5/00—Fertilisers characterised by their form
    • C05G5/20—Liquid fertilisers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses

Definitions

  • the present invention relates, in general, to a biocatalyst and to a method of stimulating biological activity using same.
  • the present invention relates to a composition capable of 5 stimulating plant growth and to a method of preparing such a composition.
  • the invention also relates to a composition capable of reducing or eliminating odors and to a method of odor reduction or elimination based on same.
  • the invention further relates to a method of 0 effecting bioremediation.
  • compositions capable of stimulating biological activity, including microbial activity have application in a number of areas. Such compositions can be used, for example, to stimulate plant growth and thus are useful in farming and commercial and residential landscape maintenance.
  • Various plant growth stimulating compositions are available that are derived from natural or from synthetic sources.
  • the compositions of the present invention have constituents from both sources and have the advantage of superior growth stimulatory properties.
  • the compositions of the present invention have the further advantage that they can be tailored so as to be optimum for a particular plant type growing under particular soil conditions.
  • compositions that stimulate biological activity can also be used to reduce or eliminate odors .
  • odors are sufficiently offensive to be problematic for individuals having to work in proximity with the odor producing material. Examples of such situations include cattle feed lots, swine barns, poultry houses and the like. In these settings, the ability to reduce or eliminate odor production would greatly enhance the quality of the working environment.
  • the present invention provides a composition suitable for use in such settings.
  • compositions that stimulate biological activity can also be used to effect bioremediation.
  • the present invention relates to a method of preparing a culture of microorganisms .
  • the method comprises : i) obtaining a starting culture sample of microorganisms from the gastrointestinal track of a mammal; ii) culturing the sample in a medium comprising sodium, potassium, calcium, magnesium, inorganic phosphorus and chlorine or salts thereof; iii) culturing the sample resulting from step (ii) in the presence of a food source comprising a grain or a grass; and iv) separating the culture of microorganisms resulting from step (iii) from the food source.
  • the culture resulting from step (iv) is exposed to a magnetic field.
  • microorganisms are removed from the culture resulting from step (iv) , with or without exposure to a magnetic field.
  • the present invention relates to a biological activity stimulatory composition
  • a biological activity stimulatory composition comprising a component produced by the above method and a formulation comprising Na, Cl, P, Mg, Ca, S, Zn, Cu, Co, I, Se, Fe, K, Mn, Mo, Si, B, Ni and Rb.
  • the present invention relates to such a formulation.
  • the present invention relates to method of stimulating the growth of a plant.
  • the method comprises administering to the plant the above composition under conditions such that the stimulation is effected.
  • the present invention relates to a method of preparing a culture of microorganisms for use as a constituent of a plant growth stimulating composition comprising exposing the culture to a magnetic field under conditions such that thickening of the cell walls of the microorganisms, as determined by light microscopy, is effected.
  • the present invention relates to an odor-reducing composition
  • an odor-reducing composition comprising an acid component, an iron component and a nitrogen componen .
  • the present invention relates to a method of reducing or eliminating an offensive odor comprising contacting the compound responsible for the odor with the above odor-reducing composition under conditions such that the odor is eliminated or reduced.
  • the present invention relates to a method of inhibiting odor production at a source comprising contacting the source with the above odor-reducing composition under conditions such that the inhibition is effected.
  • the present invention relates to a method of effecting bioremediation.
  • the method comprises applying an amount the above-described biological activity stimulatory composition to a site in need of bioremediation sufficient to effect bioremediation.
  • Figure 1 Diagram of fermentation tanks for preparation of microbial cultures.
  • Figure 2 Diagram of orientation of magnets relative to recirculation tube.
  • the present invention relates to a method of processing microbial cultures for use as constituents of compositions that stimulate biological activity, for example, of microorganisms present at a particular site.
  • the invention relates to plant growth stimulating compositions.
  • the invention also relates to nutrient formulations to be used in combination with such processed cultures in the stimulating compositions.
  • the cultures and formulations of the invention can be used to stimulate the growth of a variety of plant types including sugar cane, vegetables, fruits, grasses and tropical plants.
  • the cultures and formulations can also be used to advantage on ornamental plants.
  • the present invention also relates to a method of reducing or eliminating odors and inhibiting the production thereof, and to compositions suitable for use in effecting that reduction/elimination or inhibition.
  • the odor-reducing compositions of the invention can be used to reduce or eliminate odor production in a variety of settings, including barns, poultry houses, kennels and other animal holding areas, feed lots, and areas where water is contained, such as lagoons. These compositions can also be used in waste control area such as landfills, trash transfer centers, leachate reservoirs and animal disposal areas.
  • the composition of the present invention comprises an acid component or salt thereof such as citric acid, acetic acid, ascorbic acid, malic acid or tartaric acid, an iron component, such as a ferric salt, for example, ferric chloride, and a nitrogen source, such as urea.
  • the composition can further comprise one or more of the following: a molybdenum component, for example, molybdic acid or salt thereof, a copper component, for example, copper sulfate, aloe vera and a gum component, such as xanthan gum or guar gum.
  • composition of the invention can include an enzyme component derived from a microbial culture supernatant or prepared from polysaccharide hydrolases such as starch hydrolases, including ⁇ -amylase and glucoamylase, and galactomannan hydrolases, such as hemicellulase.
  • polysaccharide hydrolases such as starch hydrolases, including ⁇ -amylase and glucoamylase, and galactomannan hydrolases, such as hemicellulase.
  • Enzyme components of each of the compositions of the present invention can be derived from a microbial culture or supernatant thereof using the following preparative procedure.
  • Starting cultures suitable for use in preparing microbial cultures appropriate for various aspects of the invention can be obtained by combining isolates of specific microbial strains or by obtaining a mixed culture from an animal, for example, from the gastrointestinal track of an animal, preferably, a mammal, more preferably a herbivore, most preferably a cow (e.g., a lactating cow) .
  • Starting cultures can be obtained, for example, from a sample aspirated from the stomach of an animal (e.g. from the rumen of a herbivore) or from a fecal sample taken from the intestinal track of an animal.
  • the starting culture is cultured for an initial period (e.g.
  • a medium that can be prepared from natural sources (e.g. from the saliva of a herbivore (e.g. a cow)) or from chemicals (culturing can be carried out in a container such as Tank 1 of Figure 1) .
  • a bolus e.g. about 1 liter
  • a filter e.g. about an 80 micron filter
  • the bolus is washed with warm water (about 10 liters of water per liter of bolus) and the filtrate (pH preferably about 6.3 to 6.8) is obtained and used as the initial culture medium.
  • warm water about 10 liters of water per liter of bolus
  • the filtrate pH preferably about 6.3 to 6.8
  • synthetic medium it is preferably formulated so as to contain the following:
  • a culture medium containing the following can be used:
  • concentrations of the culture medium components can vary depending on the starting culture and on the target, however, typically concentrations vary, for example, by plus or minus 45%, preferably, plus or minus 20% from the above.
  • the starting culture sample is incubated in the culture medium, preferably, at a pH in the range of 6.9 to 7.3. Adjustments in pH can be made at this point and throughout the process using a variety of acids and bases, sulfuric, hydrochloric and citric being the preferred acids, citric being more preferred, and potassium hydroxide, calcium hydroxide and sodium bicarbonate being the preferred bases, sodium bicarbonate being most preferred.
  • air is introduced (e.g. by compressed air injection) to maintain an oxygen content in the range of 3 to 5 ppm (oxygen, nitrogen and argon being major components of compressed air) .
  • oxygen, nitrogen and argon being major components of compressed air
  • Cells increase in number, nutrient content and in cell-wall content.
  • a food source (substrate) is subsequently (e.g. after about 23 to 28 hrs, preferably 24 hrs) added to the medium/starting culture sample mixture.
  • the food source can comprise a mixture of feed grains and grasses. Preferably, at least three of the following are added in approximately equal parts by weight:
  • a mixture of crushed corn, oats, alfalfa and whole peanuts is preferred. The same is advantageous for tropical plants.
  • grasses are the target plant (e.g. in the case of golf course maintenance)
  • a mixture of crushed corn, oats, alfalfa and flax is preferred.
  • alfalfa, wheat soy beans and barley are preferred.
  • the food source is typically added to the medium/starting culture sample together with a further volume of liquid (i.e., culture medium and water (e.g., in a ratio of about 1:5 to 1:4)) in a ratio of about 1 kg of dry matter to about 7-8 liters of liquid. Multiple additions of food source and liquid to the original medium/starting culture sample can be made, 2 additions at approximately 24 hour intervals being preferred.
  • an approximately neutral pH is maintained, a pH in the range of about 6.9 to about 7.3 being preferred.
  • the temperature is maintained, preferably, in the range of about 34° to 41°C, 37° to 40°C being preferred.
  • the resulting broth is well mixed, for example, by recirculating the broth in a recirculation tank.
  • the recirculation is typically for a period of about 24 hours, after which time the broth is allowed to stand for a period sufficient to allow the particulate matter to settle out.
  • An aliquot of the broth supernatant is then removed and placed in a second container (e.g., a tank such as Tank 2 in Figure 1) .
  • a second container e.g., a tank such as Tank 2 in Figure 1 .
  • the pH of the transferred aliquot is slowly reduced (e.g., over a period of several hours) to about 4.5 to about 6.3, 5.1/5.8 to 6.3 being preferred, 5.1 to 6.1 being more preferred (the pH can in fact range from 3.4 to 9.0) .
  • the temperature is maintained in the range of about 34°C to 41°, 37° to 41°C being preferred.
  • a minimal amount of a second food source is added (e.g., about l%-3% v/v of the aliquot, 3% being preferred) .
  • the second food source is, for example, molasses (e.g., sugar cane or citrus molasses) , aloe vera, papaya juice, stearate or glycogen.
  • Sugar cane molasses is preferred when sugar cane or grass is the target plant, citric molasses being preferred in the case of citrus and vegetable crops as well as tropical plants (papaya juice can also be advantageous in the case of tropical plants) .
  • Glycogen, aloe and citrus molasses are preferred when the composition is to be used for odor reduction or elimination.
  • the number of cells per ml is, advantageously, in the range of 700,000 to 1.5 million per ml, about 850,000 cells/ml to 900,000 cells/ml being preferred, around 890,000 cells/ml being most preferred.
  • the cell count can be increased by delaying the transfer of the aliquot from the first tank to the second.
  • an aliquot of the culture e.g., transferred to a tank such as Tank 3 of Figure 1
  • a magnetic field preferably twice
  • the field is created using an electromagnet or permanent magnets, for example, rare earth magnets.
  • an appropriate field can be created, for example, by two opposing magnets.
  • Magnets suitable for use in the present invention have a strength in the range of 1200 to 4500 gauss, about 3500 gauss magnets being preferred.
  • Figure 2 includes a diagram of a preferred orientation of such magnets. While, in the Figure, like poles (i.e., north poles) are shown to face either side of the recirculation tube, such need not be the case (e.g. opposite poles can also face the tube) .
  • magnetic fields can also be generated by particle movement.
  • the exposure of the microorganisms to the magnetic field results in an increase in the thickness of the microbial cell wall and an increase in cell mobility, as viewed under light microscopy.
  • the invention contemplates the use of magnetic fields that can achieve these ends.
  • the temperature is, advantageously, maintained between 18° and 42°C.
  • the cell concentration is, preferably, held between 100,000 and 150,000 cells/ml.
  • a formulation of nutrients is added that can include Na, Cl, P, Mg, Ca, S, Zn, Cu, Fe, K, Mn, Mo, Si, B, Ni, and Rb, preferably, also Co, I, or Se.
  • the formulation has the following composition and the concentration ranges listed (g/1) reflect the increase in concentration of the components in the culture upon addition of the formulation to the culture:
  • Chloroacetic acid .0001-.04 .0005-.03
  • Formulations advantageous for tropical plants, vegetables and grass are as follows (expressed in g/1 of culture, the form in which each is added being as indicated above) (see Example for sugar cane values) : Tropical Golf Course
  • the values for tropical plants can vary, for example, by plus or minus 59%, preferably, plus or minus 28%; the values for vegetables by plus or minus 430%, preferably, plus or minus 22%; the values for grass by plus or minus 450%, preferably, plus or minus 20%; and the values for sugar cane by plus or minus 45%, preferably, 20%.
  • the resulting composition can be processed (e.g., filtered or centrifuged) so as to remove microorganisms, used immediately or stored, for example, for as long as two years.
  • Stored compositions containing the microorganisms can be processed to remove the microorganisms prior to use.
  • the pH is maintained, preferably, at about 5 (e.g., 4.9 to 5.2) , however, a pH range of 5.5 and 6.5 can also be used.
  • the temperature can be held between 5°C and 45°C, a temperature in the range of 34-41°C being preferred. Storage in the absence of ultra violet light is preferred.
  • aliquots of the composition can be centrifuged, for example, first at a low speed (e.g., about 3500 rpm) and for a short duration (e.g., about 5-10 min) .
  • the resulting supernatant can then be recentrifuged (preferably under refrigeration) at a greater speed (e.g., about 20,000 rpm or up to about 45,000 g) for a longer time (e.g., about an hour or longer) to pellet cell walls and other cellular debris that may be present.
  • a greater speed e.g., about 20,000 rpm or up to about 45,000 g
  • a longer time e.g., about an hour or longer
  • a variety of filtration processes can be used to remove the microorganisms.
  • a series of Thompson E1300 series stainless steel filters ranging from 30 down to 150 mesh can be used first to effect filtration, followed, for example, by a second filtration using the same filter with a 5 ⁇ filter paper and then a third filtration using the same filter with a I ⁇ filter paper.
  • the resulting supernatant can be assayed to ensure that appropriate concentrations of enzymes (e.g., amylase, and hemicellulase and/or glucanase) are present.
  • enzymes e.g., amylase, and hemicellulase and/or glucanase
  • amylase activity is assayed using a colorimetric visual endpoint determination. Kits are commercially available for conducting such assays, one such kit being available from Sigma Chemical Company (e.g., catalog numbers 577-250 and 577-M) .
  • the time required for sucrose-starch mixtures, when treated with iodine solution, to change from blue to reddish-brown is inversely proportional to amylase activity.
  • the amylase activity of the supernatant (filtrate) is preferably at least 45,000-45,4000 ct/ml .
  • Amylase levels can be adjusted by combining aliquots of the composition resulting from the culture process described above, processed to remove intact microorganisms and cellular debris.
  • Hemicellulase levels in the resulting supernatant (filtrate) are advantageously at least 2,000 ct/mg.
  • Hemicellulase levels can be measured using a ⁇ galactase dehydrogenase system (locust bean gum substrate) .
  • a commercial kit for such purpose can be used (e.g., Sigma H 0771) .
  • Table 1 below includes preferred components of the composition of the invention and concentration ranges at which those components can be present in the composition.
  • Table II includes a specific odor-reducing composition suitable for use in the present invention.
  • Citric Acid Food grade
  • HMIS Anhydrous .002%
  • Ferric Chloride Anhydrous .00011%
  • Viscosity can be measured using a viscometric tube, stop watch and temperature using carboxy methylcellulose as the standard.
  • the composition of the invention can be specifically formulated to suit a specific application need.
  • the composition when an odor-producing source is exposed to UV light, the composition can be formulated to include a UV protectant (e.g., sodium alginate, a microbial gum) .
  • a UV protectant e.g., sodium alginate, a microbial gum
  • the composition when the odor-producing source is exposed to drying conditions, the composition can be formulated (e.g., with a gum) so as to render it capable of retaining its moisture content.
  • the composition of the present invention can be used alone or in combination with disinfectants and/or perfumes, depending on the odor problem to be addressed. When such disinfectants, perfumes, etc are used, they can be applied separately or co-formulated with the composition of the invention using known formulating techniques .
  • composition of the present invention can be carried out in a variety of ways as convenient in view of the odor-producing source.
  • the composition can be provided as an aerosol into the atmosphere surrounding the odor-producing source, thereby effecting direct contact with the odor-producing molecules in the air and, upon settling to the ground, with the odor-producing source.
  • the composition can likewise be directly applied to the odor-producing source by, for example, pouring, spraying, etc, the composition onto the source. It will be appreciated that the number of applications of the compositions will vary with the particular odor problem being addressed. In the case of water containment situations such as lagoons, where short carbons chains are involved, the application can be, for example, 1 oz/1000 gallons per week.
  • the odor-producing sources to which the present method and composition have applicability are contaminated with microbes (e.g., anaerobes and aerobes) , the metabolism of which is believed to result in the release of aromatic compounds into the environment that are offensive to the olfactory system. It is also believed that the composition, on contact with the airborne odiferous aromatic compounds, chemically reacts therewith in a manner that results in decomposition of the aromatic compounds to products having an acceptable odor.
  • microbes e.g., anaerobes and aerobes
  • composition of the present invention upon contact with the odor-producing source (e.g., microbially contaminated animals, feces, feed and the like) , is believed to alter the microbial population thereof in favor of aerobic organisms (or metabolism in the case of facilitative anaerobes) . Aerobic metabolism of the source by the microbes present therein is believed to result in the release of more acceptable (from an odor standpoint) products into the environmen .
  • the odor-producing source e.g., microbially contaminated animals, feces, feed and the like
  • the regimen used to apply the composition can be optimized for any particular plant.
  • 1.5 gallons of the composition can be applied per acre to a sugar cane crop per year in approximately four equal applications; about two gallons can be applied per acre of citrus grove per year in two equal applications; about 3 gallons can be applied per acre of golf course grass in two equal applications; and for vegetable crops, about 2-2.5 gallons can be applied per acre per year in two equal applications.
  • about 1 gallon can be applied per acre per month.
  • the composition is, advantageously, diluted about 20:1 with water and applied by the spraying of the diluted composition, however, other modes of application (e.g., irrigation) can also be used.
  • composition results in a significant stimulation of plant growth.
  • the composition can also be applied directly to seed and it can be used as a root drench to decrease transplantation shock and to increase root production
  • the composition can be.applied alone or with other agents, eg an insecticide, herbicide or fungicide.
  • the present invention relates to a composition suitable for enhancing sucrose accumulation in plants (e.g., fruits and vegetables, particularly sugar cane and pineapple) .
  • the composition comprises a culture prepared essentially as described above. During the process of recirculation, for example, through the magnetic field, however, a different formulation is added than that described above.
  • molybdic acid (Mn0 3 ) (0.00001 g/1) ; sulphur (0.00006 g/1) ; iron (Fe3/2 H 2 0 4 S) (0.00006 g/1) ; manganese (MnS0 4 H 2 0) (.030 g/1); boric acid (H 3 Bo 3 ) (.0000002 g/1); nickel carbonate (NiC0 3 ) (.0000005 g/1); cobalt (CoCCH 3 COO) 2 4H 2 0 ( .00000005/gl) ; copper (CuS0 4 ) (0.0000002 g/1); zinc stearate (Zn(C 18 H 35 0 2 ) 2 (0.0004 g/1); silica (Si) (0.004 g/1) and selenium (4(HN0 3 )Se) (0.00000003 g/1).
  • the bacteria can be removed from the composition by centrifferrite, a stearate (Zn(C 18 H 35 0
  • the invention is described herein primarily in the context of a plant growth stimulator or odor- reducer, it is, in a broader sense, a microflora accelerator. It is in this context that the invention further relates to a method of effecting bioremediation and composition suitable for use therein.
  • the composition of the invention targets the sulphur links of short and long chain carbon structures present at a bioremediation site.
  • bioremediation sites include animal waste sites, animal feedlots, lagoons, enclosed facilities (such as dairy parlors, swine barns, chicken houses, aviaries, kennels, etc), human waste treatment plants, land fills, and leachate water runoffs from sanitary facilities.
  • Bioremediation sites can exist in fresh or salt water marine environments. Bioremediation is particularly important when fuel oils, coal tars, napthas or benzene are present in the soil or water (or in the atmosphere) . The present invention is applicable to all such situations.
  • the composition used to effect bioremediation is prepared essentially as described above. Concentrations of specific components, however, will vary depending on the target site.
  • composition invention to a site in need of bioremediation can be made by any suitable application protocol, spraying and fogging being examples.
  • the composition can be simply poured onto and mixed into the contaminated soil or water at a rate that will vary with the nature of the contamination.
  • 5 to 7 oz of the composition of the invention can be applied per 1,000 gallon of leachate; 5 to 15 gallons can be applied per acre foot of soil to be remediated; about 3 gallons of the present composition can be applied per area foot of liquid state petroleum reservoir; and about 5 oz can be applied per 5,000 cubic feet of air.
  • the optimum application protocol for a particular remediation site can be readily determined, as can the optimum composition.
  • the advantages of the present invention result, at least in part, from the effects of components of the present composition on nitrofication which in turn enhances sulphur metabolism.
  • alcohols, aldehydes, organic acids, esters, ketones, phenols and sulphur compounds are believed to be produced.
  • the sulphur compounds are of particular importance.
  • boron and magnesium are expected to stimulate the fermentation process, along with oxygen, nitrogen, and argon (the major components of compressed atmospheric air) . When these constituents are added, they are believed to facilitate the nitrogen cycle of yeast present in the culture.
  • yeast convert sugar in the food substrate and the second food source, alcohols, esters and gums are formed (gums can include alginate, microbial gums, plant exudate and bean gum) .
  • Groups of carbohydrates of particular interest include such gums and cellulose compounds, for example, those derived from the food substrate.
  • the ability of yeast to uptake nitrogen is augmented by certain aerobic bacteria, azotabactors and cyanobactors, along with other nitrogen fixers. These bacteria are believed to be stimulated by molybdenum, boron and magnesium, which elements are believed to be important to the production of gums which, in turn, are important in sulfur metabolism. The importance of molybdenum, boron and magnesium is believed to result from the role played by these elements in the following enzymatic processes.
  • the process of nitrogen fixation requires the nitrogenase complex which consists of a reductase (which provides electrons with high reducing power) and a nitrogenase (which uses these electrons to reduce N 2 to NH4+) .
  • Each component is an iron-sulfur protein in which iron is bonded to the sulfur atom of a cysteine residue and to inorganic sulfide.
  • the nitrogenase component of the complex also contains one or two molybdenum atoms.
  • the conversion of N 2 into NH 4+ by the nitrogenase complex requires ATP and a powerful reductant .
  • the source of high potential electrons in this six-electron reduction is reduced ferredoxin.
  • ATP binds to the reductase and shifts the redox potential of the enzyme from -0.29V to -0.40V by altering its conformation.
  • ATP is hydrolyzed and the reductase dissociates from the nitrogenase component.
  • N 2 bound to the nitrogenase component of the complex is reduced to NH + .
  • a kinase catalyzes the transfer of a phosphoryl groups from ATP to an acceptor.
  • Hexokinase catalyzes the transfer of a phosphoryl group from ATP to a variety of six-carbon sugars.
  • Hexokinase requires Mg 2+ (or another divalent metal ion such as Mn 2+ ) for activity. The divalent metal ion forms a complex with ATP.
  • Acetylcholinesterase is an enzyme that catalyzes the hydrolysis of the ester bond in acetylcholine.
  • Acetate and choline are two important substances in the formation of gums and waxes .
  • Sulfation is defined as any process of introducing an S0 4 group into an organic compound in which the reaction product (sulfate) exhibits the characteristic -OSO 3 - molecular configuration. Sulfation involves the reaction wherein a -COS- linkage is formed by the action of a sulfating agent on an alkene, alcohol, or phenol.
  • the alcohol sulfates are readily susceptible to hydrolysis in acidic media. Sulfation of fatty alcohols and polyalkoxy reductases occurs in the present process and the sulfation products lend themselves to detergent action as emulsifiers . Gums that are produced by the present process can store and stabilize products of microbial sulphur metabolism. Gums also serve generally to stabilize the fermentation components and thus facilitate storage of the product of the present method. The stabilization of the sulphur metabolism components allows immediate reaction with hydrocarbon chains in the environment by removing the sulfur link from those hydrocarbon chains for detergent reaction with associated alcohols.
  • Magnetism can alter the end-product production.
  • bio ⁇ catalysts e.g., proteins, vitamins, trace elements
  • desired sulfur containing compounds are enriched, including ⁇ -linked purines, biotin, sulfinated carbohydrates, etc, and the levels of undesirable gases can be decreased.
  • Magnetics when combined with the present formulation, apparently provide for a more rapid conversion through the nitrogen and sulphur cycles to end complexes of sulphur metabolism.
  • a one liter sample is aspirated from the rumen of a 7.5 year old lactating Holstein cow using a rumen aspirator (Johnson and Johnson) .
  • the sample is taken about 12-14 hours after feeding. Observed microscopically, the sample includes Clostridia, Bacillus, Azotobacter and protozoa (at least 100 cells of each per ml of sample) .
  • the one liter sample is added to a culture medium (169 liters) that includes:
  • the sample and the culture medium (Mixture A) are maintained in Tank 1 (see Figure 1) at a temperature of 37°C and at a pH in the range of 6.9-7.3 for a first 24 hour period. At this stage, and throughout the process, pH adjustments are made using citric acid or sodium bicarbonate, as appropriate. During this first 24 hr period, Mixture (A) is agitated by the injection of compressed air which results in the presence in Mixture (A) of about 3-5 ppm oxygen.
  • Mixture (B) is added to Mixture (A) in Tank 1.
  • Mixture (B) comprises 140 liters of water, pH 7.0-7.1, and 30 liters of the culture medium described above into which air has been injected to achieve an oxygen content of 3-5 ppm.
  • Mixture (B) also includes approximately 20 kg of a substrate comprising the following in approximately equal parts by weight:
  • Substrate crushed corn oats alfalfa whole peanuts
  • the pH of Mixture (B) is maintained at about 7.1 to 7.2, the temperature at about 34-40°C, and the oxygen content at about 3-5 ppm (by injection of compressed air) . These same conditions are maintained after the addition of Mixture (B) to Mixture (A) to form
  • Mixture (C) is maintained in Tank 1 at about 37°C for a second 24 hr period with agitation by compressed air injection.
  • Mixture (D) is added to Mixture (C) in Tank 1 to yield Mixture (E) .
  • Mixture (D) like Mixture (B) , comprises 140 liters of water, pH 7.1 to 7.2, and 30 liters of culture medium. Mixture (D) also includes 20 kg of the substrate described above.
  • Mixture (E) is maintained in Tank 1 for a third 24 hr period with agitation by compressed air injection (temperature 40°C; pH 7.1; oxygen content 3-5 ppm) .
  • Mixture (E) is recirculated for ten minute periods.
  • Tank 1, and the recirculation system associated therewith, is designed such that complete recirculation of Mixture (E) can be effected in the ten minute period. That recirculation is carried out at two hour intervals for a fourth 24 hour period.
  • Mixture (E) is then allowed to stand for a time sufficient to permit particulate matter to settle out.
  • E-T2 (175 liters) is transferred to a further recirculation tank, Tank 3 (see Figure 1) and a "micronutrient" package is added.
  • the contents of the package is formulated so that the addition thereof to the 175 liters results in the following concentrations, expressed as g/1 of Mixture (E-T2) :
  • the content of Tank 3 (Mixture (E-T3)) is recirculated, and, during recirculation, is passed though a magnetic field (an 80 gal/min pump is used in the recirculation process) .
  • the field is generated by six 3500 gauss rare earth magnets oriented as shown in Figure 2 with respect to a 1W diameter PVP 80 gauge recirculation tube, a 1/32" phenolic band being located between the magnets and the tube.
  • the pH is maintained at between 5.5 and 6.5 and the temperature at about 37°C.
  • Compressed air is injected during recirculation to maintain an oxygen content of 3-5 ppm.
  • the composition resulting after 10 minutes of recirculation is stored for about 24 hours at a temperature of 35-38°C.
  • composition resulting from the foregoing process is applied to sugar cane by spraying four times per year for a total annual application of 1.5 gallons per acre.

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Abstract

The present invention relates, in general, to a method of stimulating microbial activity and composition suitable for use therein and, in particular, to a composition capable of stimulating plant growth and to a method of preparing such a composition. The invention further relates to a method of effecting bioremediation.

Description

A METHOD OF PREPARING A CULTURE USING A MAGNETIC FIELD AND A COMPOSITION CONTAINING THE SAME
This is a continuation-in-part of Application No. 08/476,374, filed June 7, 1995, Application No. 08/569,769, filed December 8, 1995, and Application 5 No. 08/476,109, filed June 7, 1995, all of which applications are continuations-in-part of Application No. 08/376,553, filed January 20, 1995, the entire contents of all four applications being incorporated herein by reference.
0 TECHNICAL FIELD
The present invention relates, in general, to a biocatalyst and to a method of stimulating biological activity using same. In particular, the present invention relates to a composition capable of 5 stimulating plant growth and to a method of preparing such a composition. The invention also relates to a composition capable of reducing or eliminating odors and to a method of odor reduction or elimination based on same. The invention further relates to a method of 0 effecting bioremediation.
BACKGROUND
Compositions capable of stimulating biological activity, including microbial activity, have application in a number of areas. Such compositions can be used, for example, to stimulate plant growth and thus are useful in farming and commercial and residential landscape maintenance. Various plant growth stimulating compositions are available that are derived from natural or from synthetic sources. The compositions of the present invention have constituents from both sources and have the advantage of superior growth stimulatory properties. The compositions of the present invention have the further advantage that they can be tailored so as to be optimum for a particular plant type growing under particular soil conditions.
Compositions that stimulate biological activity can also be used to reduce or eliminate odors . Of particular importance are those situations where the odors are sufficiently offensive to be problematic for individuals having to work in proximity with the odor producing material. Examples of such situations include cattle feed lots, swine barns, poultry houses and the like. In these settings, the ability to reduce or eliminate odor production would greatly enhance the quality of the working environment. The present invention provides a composition suitable for use in such settings.
Compositions that stimulate biological activity can also be used to effect bioremediation. OBJECTS AND SUMMARY OF THE INVENTION
It is a general object of the present invention to provide a method of stimulating biological activity, including microbial activity. It is a specific object of the present invention to provide a composition capable of stimulating the growth of plants.
It is another object of the invention to provide a method of processing a microbial culture so as to render it suitable for use as a constituent of a plant growth stimulating composition.
It is a further object of the invention to provide a nutrient formulation suitable for use as a constituent of a plant growth stimulating composition. It is a further object of the present invention to provide a composition capable of reducing or eliminating odors .
It is another object of the invention to provide a method of treating an odor producing source so as to reduce or eliminate odor production therefrom.
It is a further object of the invention to provide a method of effecting bioremediation.
The foregoing objects are met by the present invention. In one embodiment, the present invention relates to a method of preparing a culture of microorganisms . The method comprises : i) obtaining a starting culture sample of microorganisms from the gastrointestinal track of a mammal; ii) culturing the sample in a medium comprising sodium, potassium, calcium, magnesium, inorganic phosphorus and chlorine or salts thereof; iii) culturing the sample resulting from step (ii) in the presence of a food source comprising a grain or a grass; and iv) separating the culture of microorganisms resulting from step (iii) from the food source. In a further embodiment, the culture resulting from step (iv) is exposed to a magnetic field. In yet another embodiment, microorganisms are removed from the culture resulting from step (iv) , with or without exposure to a magnetic field.
In a further embodiment, the present invention relates to a biological activity stimulatory composition comprising a component produced by the above method and a formulation comprising Na, Cl, P, Mg, Ca, S, Zn, Cu, Co, I, Se, Fe, K, Mn, Mo, Si, B, Ni and Rb. In yet a further embodiment, the present invention relates to such a formulation.
In still another embodiment, the present invention relates to method of stimulating the growth of a plant. The method comprises administering to the plant the above composition under conditions such that the stimulation is effected. In yet another embodiment, the present invention relates to a method of preparing a culture of microorganisms for use as a constituent of a plant growth stimulating composition comprising exposing the culture to a magnetic field under conditions such that thickening of the cell walls of the microorganisms, as determined by light microscopy, is effected.
In another embodiment, the present invention relates to an odor-reducing composition comprising an acid component, an iron component and a nitrogen componen .
In a further embodiment, the present invention relates to a method of reducing or eliminating an offensive odor comprising contacting the compound responsible for the odor with the above odor-reducing composition under conditions such that the odor is eliminated or reduced.
In another embodiment, the present invention relates to a method of inhibiting odor production at a source comprising contacting the source with the above odor-reducing composition under conditions such that the inhibition is effected.
In a further embodiment, the present invention relates to a method of effecting bioremediation. The method comprises applying an amount the above-described biological activity stimulatory composition to a site in need of bioremediation sufficient to effect bioremediation. Further objects and advantages of the present invention will be clear from the description that follows .
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Diagram of fermentation tanks for preparation of microbial cultures.
Figure 2. Diagram of orientation of magnets relative to recirculation tube.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of processing microbial cultures for use as constituents of compositions that stimulate biological activity, for example, of microorganisms present at a particular site. In one embodiment, the invention relates to plant growth stimulating compositions. The invention also relates to nutrient formulations to be used in combination with such processed cultures in the stimulating compositions. The cultures and formulations of the invention can be used to stimulate the growth of a variety of plant types including sugar cane, vegetables, fruits, grasses and tropical plants. The cultures and formulations can also be used to advantage on ornamental plants.
The present invention also relates to a method of reducing or eliminating odors and inhibiting the production thereof, and to compositions suitable for use in effecting that reduction/elimination or inhibition. The odor-reducing compositions of the invention can be used to reduce or eliminate odor production in a variety of settings, including barns, poultry houses, kennels and other animal holding areas, feed lots, and areas where water is contained, such as lagoons. These compositions can also be used in waste control area such as landfills, trash transfer centers, leachate reservoirs and animal disposal areas.
In the context of odor reduction/elimination, the composition of the present invention comprises an acid component or salt thereof such as citric acid, acetic acid, ascorbic acid, malic acid or tartaric acid, an iron component, such as a ferric salt, for example, ferric chloride, and a nitrogen source, such as urea. The composition can further comprise one or more of the following: a molybdenum component, for example, molybdic acid or salt thereof, a copper component, for example, copper sulfate, aloe vera and a gum component, such as xanthan gum or guar gum. In addition, the composition of the invention can include an enzyme component derived from a microbial culture supernatant or prepared from polysaccharide hydrolases such as starch hydrolases, including α-amylase and glucoamylase, and galactomannan hydrolases, such as hemicellulase.
Enzyme components of each of the compositions of the present invention can be derived from a microbial culture or supernatant thereof using the following preparative procedure.
Starting cultures suitable for use in preparing microbial cultures appropriate for various aspects of the invention can be obtained by combining isolates of specific microbial strains or by obtaining a mixed culture from an animal, for example, from the gastrointestinal track of an animal, preferably, a mammal, more preferably a herbivore, most preferably a cow (e.g., a lactating cow) . Starting cultures can be obtained, for example, from a sample aspirated from the stomach of an animal (e.g. from the rumen of a herbivore) or from a fecal sample taken from the intestinal track of an animal. The starting culture, whether obtained from a natural source or prepared from isolates, is cultured for an initial period (e.g. 21 to 31 hours, preferably, 24 hours) in the presence of a medium that can be prepared from natural sources (e.g. from the saliva of a herbivore (e.g. a cow)) or from chemicals (culturing can be carried out in a container such as Tank 1 of Figure 1) . When prepared from saliva, the following procedure can be used. A bolus (e.g. about 1 liter) is taken from the mouth of the animal (e.g. a cow) and placed on a filter (e.g. about an 80 micron filter) .
The bolus is washed with warm water (about 10 liters of water per liter of bolus) and the filtrate (pH preferably about 6.3 to 6.8) is obtained and used as the initial culture medium. When synthetic medium is used, it is preferably formulated so as to contain the following:
sodium potassium calcium magnesium inorganic phosphorus chloride
As an example, a culture medium containing the following can be used:
Sodium bicarbonate .0225 g/liter
Potassium bicarbonate .00125 g/liter
Calcium carbonate .000025 g/liter
Magnesium carbonate (anhydrous) .0000375 g/liter Phosphoric acid .003375 g/liter
Chloroacetic acid .002125 g/liter
The concentrations of the culture medium components can vary depending on the starting culture and on the target, however, typically concentrations vary, for example, by plus or minus 45%, preferably, plus or minus 20% from the above.
The starting culture sample is incubated in the culture medium, preferably, at a pH in the range of 6.9 to 7.3. Adjustments in pH can be made at this point and throughout the process using a variety of acids and bases, sulfuric, hydrochloric and citric being the preferred acids, citric being more preferred, and potassium hydroxide, calcium hydroxide and sodium bicarbonate being the preferred bases, sodium bicarbonate being most preferred. During this initial incubation period, and throughout the process, air is introduced (e.g. by compressed air injection) to maintain an oxygen content in the range of 3 to 5 ppm (oxygen, nitrogen and argon being major components of compressed air) . During this initial period, nitrogen, sugar and oxygen uptake occurs. Cells increase in number, nutrient content and in cell-wall content.
A food source (substrate) is subsequently (e.g. after about 23 to 28 hrs, preferably 24 hrs) added to the medium/starting culture sample mixture. The food source can comprise a mixture of feed grains and grasses. Preferably, at least three of the following are added in approximately equal parts by weight:
crushed corn oats milo alfalfa sunflower seeds peanuts (whole) wheat soybeans barley rice flax.
For sugar cane and vegetable crops, a mixture of crushed corn, oats, alfalfa and whole peanuts is preferred. The same is advantageous for tropical plants. When grasses are the target plant (e.g. in the case of golf course maintenance) , a mixture of crushed corn, oats, alfalfa and flax is preferred. For odor reducing compositions, alfalfa, wheat soy beans and barley are preferred. The food source is typically added to the medium/starting culture sample together with a further volume of liquid (i.e., culture medium and water (e.g., in a ratio of about 1:5 to 1:4)) in a ratio of about 1 kg of dry matter to about 7-8 liters of liquid. Multiple additions of food source and liquid to the original medium/starting culture sample can be made, 2 additions at approximately 24 hour intervals being preferred.
Throughout this period of incubation, an approximately neutral pH is maintained, a pH in the range of about 6.9 to about 7.3 being preferred. The temperature is maintained, preferably, in the range of about 34° to 41°C, 37° to 40°C being preferred.
After the final addition of food substrate, the resulting broth is well mixed, for example, by recirculating the broth in a recirculation tank. The recirculation is typically for a period of about 24 hours, after which time the broth is allowed to stand for a period sufficient to allow the particulate matter to settle out.
An aliquot of the broth supernatant is then removed and placed in a second container (e.g., a tank such as Tank 2 in Figure 1) . By separating the supernatant aliquot from the particulate matter, the microorganisms present in the aliquot are separated from their food source (thereby causing a "secondary shunt metabolism" to be effected) . The pH of the transferred aliquot is slowly reduced (e.g., over a period of several hours) to about 4.5 to about 6.3, 5.1/5.8 to 6.3 being preferred, 5.1 to 6.1 being more preferred (the pH can in fact range from 3.4 to 9.0) . The temperature is maintained in the range of about 34°C to 41°, 37° to 41°C being preferred. A minimal amount of a second food source is added (e.g., about l%-3% v/v of the aliquot, 3% being preferred) . The second food source is, for example, molasses (e.g., sugar cane or citrus molasses) , aloe vera, papaya juice, stearate or glycogen. Sugar cane molasses is preferred when sugar cane or grass is the target plant, citric molasses being preferred in the case of citrus and vegetable crops as well as tropical plants (papaya juice can also be advantageous in the case of tropical plants) . Glycogen, aloe and citrus molasses are preferred when the composition is to be used for odor reduction or elimination.
At this point in the process, the number of cells per ml is, advantageously, in the range of 700,000 to 1.5 million per ml, about 850,000 cells/ml to 900,000 cells/ml being preferred, around 890,000 cells/ml being most preferred. The cell count can be increased by delaying the transfer of the aliquot from the first tank to the second. After the pH has been reduced and the second food source added, an aliquot of the culture (e.g., transferred to a tank such as Tank 3 of Figure 1) can be recirculated through a magnetic field (preferably twice) . The field is created using an electromagnet or permanent magnets, for example, rare earth magnets. In the case of permanent magnets, an appropriate field can be created, for example, by two opposing magnets. Magnets suitable for use in the present invention have a strength in the range of 1200 to 4500 gauss, about 3500 gauss magnets being preferred. Figure 2 includes a diagram of a preferred orientation of such magnets. While, in the Figure, like poles (i.e., north poles) are shown to face either side of the recirculation tube, such need not be the case (e.g. opposite poles can also face the tube) . In addition to the use of magnets as described above, magnetic fields can also be generated by particle movement.
The exposure of the microorganisms to the magnetic field results in an increase in the thickness of the microbial cell wall and an increase in cell mobility, as viewed under light microscopy. The invention contemplates the use of magnetic fields that can achieve these ends. When magnetic field exposure is not used, the temperature is, advantageously, maintained between 18° and 42°C. Further, the cell concentration is, preferably, held between 100,000 and 150,000 cells/ml. When recirculation through the magnetic field is carried out, a formulation of nutrients is added that can include Na, Cl, P, Mg, Ca, S, Zn, Cu, Fe, K, Mn, Mo, Si, B, Ni, and Rb, preferably, also Co, I, or Se. Generally, molybdenum, boron and magnesium are important to fermentation stimulation in the present process (together with the compressed air components) . Preferably, the formulation has the following composition and the concentration ranges listed (g/1) reflect the increase in concentration of the components in the culture upon addition of the formulation to the culture:
Broad range(g/l) Preferred Range (g/l)
Sodium bicarbonate .0001-.10 .0005-.090
Chloroacetic acid .0001-.04 .0005-.03
Phosphoric acid (liquid) .001-.05 .002-.02 Magnesium carbonate (anhydrous) .000075-.05 .001-.004
Calcium carbonate .000250-.300 .001-.004
Sulfur (from sulfates in composition) .000075-.04 .002-.006
Zinc stearate .00004-.008 .0003-.005
Copper sulfate .00000001-.06 .00000001-.00001 Cobalt acetate tetrahydrate .0000005-.0000000006 .00000004-.000000003
Iodine (liquid) .000000003-.000006 .00000005-.000000008
Se (plasma grade std (liquid)) .000000003-.00001 .00000002-.000001
Iron sulfate .0000002-.00009 .000005-00006
Potassium bicarbonate .05-.0006 .04-.006 Manganese sulfate monohydrate .000005-.0045 .00045-.00003
Molybdic acid 85% (powder) .00000001-.00004 .00000019-.000005
Silicon (reference std solution (1000 ppm)) .0000005-.0005 .00005-.0001
Boric acid .000002-.0000000003 .00000003-.0000002
Nickel carbonate .000000005-.00005 .0000005-.000003 Rubidium chloride .000000009-.0000095 .00000006-.00000055
Other forms of the indicated elements can also be used so long as they are acceptable to the microorganisms. When recirculation through a magnetic field is not carried out, the formulation of nutrients is added subsequent to the reduction of the pH.
Formulations advantageous for tropical plants, vegetables and grass (e.g., golf course grass) are as follows (expressed in g/1 of culture, the form in which each is added being as indicated above) (see Example for sugar cane values) : Tropical Golf Course
Plants Veσetables Grass
Na .018 .018 .018
Cl .003 .003 .003
P .002 .002 .002
Mg .004 .0031 .0035
Ca .004 .004 .004
S .006 .006 .006
Zn .00003 .00003 .000038
Cu .00002 .00002 .00002
Co .00000008 .00000005 .000000009
I .0000008 .0000008 .0000008
Se .0000006 .00000045 .00000064
Fe .00006 .00006 .00006
K .006 .006 .006
Mn .00001 .00001 .00001
Mo .000003 .000004 .000009
Si .00001 .00001 .00001
B .000005 .000005 .000005
Ni .0000003 .0000003 .0000003
Rb .000007 .000007 .000007
These advantageous values (and those for sugar cane) can vary. The values for tropical plants can vary, for example, by plus or minus 59%, preferably, plus or minus 28%; the values for vegetables by plus or minus 430%, preferably, plus or minus 22%; the values for grass by plus or minus 450%, preferably, plus or minus 20%; and the values for sugar cane by plus or minus 45%, preferably, 20%.
Upon completion of the magnetic field exposure and/or nutrient formulation addition, the resulting composition can be processed (e.g., filtered or centrifuged) so as to remove microorganisms, used immediately or stored, for example, for as long as two years. Stored compositions containing the microorganisms can be processed to remove the microorganisms prior to use. During storage, the pH is maintained, preferably, at about 5 (e.g., 4.9 to 5.2) , however, a pH range of 5.5 and 6.5 can also be used. The temperature can be held between 5°C and 45°C, a temperature in the range of 34-41°C being preferred. Storage in the absence of ultra violet light is preferred.
To remove microorganisms from the composition resulting from the process described above, aliquots of the composition can be centrifuged, for example, first at a low speed (e.g., about 3500 rpm) and for a short duration (e.g., about 5-10 min) . The resulting supernatant can then be recentrifuged (preferably under refrigeration) at a greater speed (e.g., about 20,000 rpm or up to about 45,000 g) for a longer time (e.g., about an hour or longer) to pellet cell walls and other cellular debris that may be present. One skilled in the art will appreciate that any of a number of centrifuges can be used, including, for example, a Beckman J-25 centrifuge. One skilled in the art will also appreciate that a variety of filtration processes can be used to remove the microorganisms. For example, a series of Thompson E1300 series stainless steel filters ranging from 30 down to 150 mesh can be used first to effect filtration, followed, for example, by a second filtration using the same filter with a 5μ filter paper and then a third filtration using the same filter with a Iμ filter paper.
ODOR REDUCTION
When cells and cellular debris are removed, the resulting supernatant (filtrate) can be assayed to ensure that appropriate concentrations of enzymes (e.g., amylase, and hemicellulase and/or glucanase) are present. Typically, amylase activity is assayed using a colorimetric visual endpoint determination. Kits are commercially available for conducting such assays, one such kit being available from Sigma Chemical Company (e.g., catalog numbers 577-250 and 577-M) . The time required for sucrose-starch mixtures, when treated with iodine solution, to change from blue to reddish-brown is inversely proportional to amylase activity. The amylase activity of the supernatant (filtrate) is preferably at least 45,000-45,4000 ct/ml . Amylase levels can be adjusted by combining aliquots of the composition resulting from the culture process described above, processed to remove intact microorganisms and cellular debris. Hemicellulase levels in the resulting supernatant (filtrate) are advantageously at least 2,000 ct/mg. Hemicellulase levels can be measured using a βgalactase dehydrogenase system (locust bean gum substrate) . A commercial kit for such purpose can be used (e.g., Sigma H 0771) .
Table 1 below includes preferred components of the composition of the invention and concentration ranges at which those components can be present in the composition.
TABLE I
Preferred Range Range mg/ml mg/ml
Citric Acid .001 .020 .0001 .100
Ferric .0008 .000011 .008 .0000011 Chloride
Molybdenum .0008 .000011 .008 .0000011
Copper Sulfate .0008 .000011 .008 .0000011
Urea .100 . .003 1.000 .0003
Aloe Vera 2.100 .600 3.000 .003
Xanthan Gum 2.100 .600 3.000 .003
Guar Gum 2.100 .600 3.000 .003
Culture super¬ 3.00 .600 5.000 .003 natant or filtrate pH of Water 3.5 4.5 3.0 5.0 base Table II includes a specific odor-reducing composition suitable for use in the present invention.
ABLE II
1. Citric Acid (Food grade) (HMIS Anhydrous) .002% 2. Ferric Chloride (Anhydrous) .00011%
3. Molybdenum (Molybdic Acid 85% Powder) .00011%
4. Copper (Copper Sulphate) .00011%
5. Urea (Feed Grade) 45% .05%
6. Aloe Vera 10/50% Viscosity1 1.6% 7. Xanthan Gum 30/50% Viscosity 1.6%
8. Guar Gum 30/50% Viscosity 1.6%
9. Culture supernatant 20/50% Viscosity 1.0%
Viscosity can be measured using a viscometric tube, stop watch and temperature using carboxy methylcellulose as the standard.
1 Poise (P) = 0.1 Pa'S mPa's (=CP) CP = Centi Poise
% Concentration £C£I %Vol/Sol
Aloe Vera 50% 2500 CP 1.6%
Xanthan Gum 1% 600 CP 1.6%
Guar Gum 1.5% 15000 CP 1.6%
Culture 50% 4300 CP 1.6%
The composition of the invention can be specifically formulated to suit a specific application need. For example, when an odor-producing source is exposed to UV light, the composition can be formulated to include a UV protectant (e.g., sodium alginate, a microbial gum) . Further, when the odor-producing source is exposed to drying conditions, the composition can be formulated (e.g., with a gum) so as to render it capable of retaining its moisture content. The composition of the present invention can be used alone or in combination with disinfectants and/or perfumes, depending on the odor problem to be addressed. When such disinfectants, perfumes, etc are used, they can be applied separately or co-formulated with the composition of the invention using known formulating techniques .
Application of the composition of the present invention can be carried out in a variety of ways as convenient in view of the odor-producing source. For example, the composition can be provided as an aerosol into the atmosphere surrounding the odor-producing source, thereby effecting direct contact with the odor-producing molecules in the air and, upon settling to the ground, with the odor-producing source. The composition can likewise be directly applied to the odor-producing source by, for example, pouring, spraying, etc, the composition onto the source. It will be appreciated that the number of applications of the compositions will vary with the particular odor problem being addressed. In the case of water containment situations such as lagoons, where short carbons chains are involved, the application can be, for example, 1 oz/1000 gallons per week. While not wishing to be bound by any particular theory, it is believed that the odor-producing sources to which the present method and composition have applicability are contaminated with microbes (e.g., anaerobes and aerobes) , the metabolism of which is believed to result in the release of aromatic compounds into the environment that are offensive to the olfactory system. It is also believed that the composition, on contact with the airborne odiferous aromatic compounds, chemically reacts therewith in a manner that results in decomposition of the aromatic compounds to products having an acceptable odor. The composition of the present invention, upon contact with the odor-producing source (e.g., microbially contaminated animals, feces, feed and the like) , is believed to alter the microbial population thereof in favor of aerobic organisms (or metabolism in the case of facilitative anaerobes) . Aerobic metabolism of the source by the microbes present therein is believed to result in the release of more acceptable (from an odor standpoint) products into the environmen .
Plant Growth Simulation
The regimen used to apply the composition can be optimized for any particular plant. By way of example, 1.5 gallons of the composition can be applied per acre to a sugar cane crop per year in approximately four equal applications; about two gallons can be applied per acre of citrus grove per year in two equal applications; about 3 gallons can be applied per acre of golf course grass in two equal applications; and for vegetable crops, about 2-2.5 gallons can be applied per acre per year in two equal applications. In the case of tropical plants, about 1 gallon can be applied per acre per month. The composition is, advantageously, diluted about 20:1 with water and applied by the spraying of the diluted composition, however, other modes of application (e.g., irrigation) can also be used. Foliar spraying is preferred in the case of tropical plants. Application of the composition results in a significant stimulation of plant growth. The composition can also be applied directly to seed and it can be used as a root drench to decrease transplantation shock and to increase root production The composition can be.applied alone or with other agents, eg an insecticide, herbicide or fungicide.
In a specific embodiment, the present invention relates to a composition suitable for enhancing sucrose accumulation in plants (e.g., fruits and vegetables, particularly sugar cane and pineapple) . The composition comprises a culture prepared essentially as described above. During the process of recirculation, for example, through the magnetic field, however, a different formulation is added than that described above. In this embodiment, the following components are added (preferred concentrations in g/1 of culture) : molybdic acid (Mn03) (0.00001 g/1) ; sulphur (0.00006 g/1) ; iron (Fe3/2 H204S) (0.00006 g/1) ; manganese (MnS04H20) (.030 g/1); boric acid (H3Bo3) (.0000002 g/1); nickel carbonate (NiC03) (.0000005 g/1); cobalt (CoCCH3COO)2 4H20 ( .00000005/gl) ; copper (CuS04) (0.0000002 g/1); zinc stearate (Zn(C18H3502)2 (0.0004 g/1); silica (Si) (0.004 g/1) and selenium (4(HN03)Se) (0.00000003 g/1). The bacteria can be removed from the composition by centrifugation or filtration and the resulting composition applied to plants, e.g., at a 1:20 dilution. Application regimens vary with the plant and growth conditions.
Bioremediation,
While the invention is described herein primarily in the context of a plant growth stimulator or odor- reducer, it is, in a broader sense, a microflora accelerator. It is in this context that the invention further relates to a method of effecting bioremediation and composition suitable for use therein.
In serving as a bioremediator, the composition of the invention targets the sulphur links of short and long chain carbon structures present at a bioremediation site. Examples of such sites include animal waste sites, animal feedlots, lagoons, enclosed facilities (such as dairy parlors, swine barns, chicken houses, aviaries, kennels, etc), human waste treatment plants, land fills, and leachate water runoffs from sanitary facilities. Bioremediation sites can exist in fresh or salt water marine environments. Bioremediation is particularly important when fuel oils, coal tars, napthas or benzene are present in the soil or water (or in the atmosphere) . The present invention is applicable to all such situations. The composition used to effect bioremediation is prepared essentially as described above. Concentrations of specific components, however, will vary depending on the target site.
Application of the composition invention to a site in need of bioremediation can be made by any suitable application protocol, spraying and fogging being examples. Where conditions at the site dictate, the composition can be simply poured onto and mixed into the contaminated soil or water at a rate that will vary with the nature of the contamination. By way of example, however, 5 to 7 oz of the composition of the invention can be applied per 1,000 gallon of leachate; 5 to 15 gallons can be applied per acre foot of soil to be remediated; about 3 gallons of the present composition can be applied per area foot of liquid state petroleum reservoir; and about 5 oz can be applied per 5,000 cubic feet of air. The optimum application protocol for a particular remediation site can be readily determined, as can the optimum composition.
While not wishing to be bound by theory, it is believed that the advantages of the present invention result, at least in part, from the effects of components of the present composition on nitrofication which in turn enhances sulphur metabolism. In the fermentation process of the present invention, alcohols, aldehydes, organic acids, esters, ketones, phenols and sulphur compounds are believed to be produced. As it is understood, the sulphur compounds are of particular importance. As indicated above, boron and magnesium are expected to stimulate the fermentation process, along with oxygen, nitrogen, and argon (the major components of compressed atmospheric air) . When these constituents are added, they are believed to facilitate the nitrogen cycle of yeast present in the culture. As yeast convert sugar in the food substrate and the second food source, alcohols, esters and gums are formed (gums can include alginate, microbial gums, plant exudate and bean gum) . Groups of carbohydrates of particular interest include such gums and cellulose compounds, for example, those derived from the food substrate. The ability of yeast to uptake nitrogen is augmented by certain aerobic bacteria, azotabactors and cyanobactors, along with other nitrogen fixers. These bacteria are believed to be stimulated by molybdenum, boron and magnesium, which elements are believed to be important to the production of gums which, in turn, are important in sulfur metabolism. The importance of molybdenum, boron and magnesium is believed to result from the role played by these elements in the following enzymatic processes.
The process of nitrogen fixation requires the nitrogenase complex which consists of a reductase (which provides electrons with high reducing power) and a nitrogenase (which uses these electrons to reduce N2 to NH4+) . Each component is an iron-sulfur protein in which iron is bonded to the sulfur atom of a cysteine residue and to inorganic sulfide. The nitrogenase component of the complex also contains one or two molybdenum atoms. The conversion of N2 into NH4+ by the nitrogenase complex requires ATP and a powerful reductant . In most nitrogen-fixing micro-organisms, the source of high potential electrons in this six-electron reduction is reduced ferredoxin. ATP binds to the reductase and shifts the redox potential of the enzyme from -0.29V to -0.40V by altering its conformation. ATP is hydrolyzed and the reductase dissociates from the nitrogenase component. Finally, N2 bound to the nitrogenase component of the complex is reduced to NH+. In relation to the phosphoryl transfer, a kinase catalyzes the transfer of a phosphoryl groups from ATP to an acceptor. Hexokinase catalyzes the transfer of a phosphoryl group from ATP to a variety of six-carbon sugars. Hexokinase requires Mg2+ (or another divalent metal ion such as Mn2+) for activity. The divalent metal ion forms a complex with ATP.
Boron acids are another kind of transition state analog for enzymes that form acyl-enzyme intermediates. Acetylcholinesterase is an enzyme that catalyzes the hydrolysis of the ester bond in acetylcholine. Acetate and choline are two important substances in the formation of gums and waxes . Sulfation is defined as any process of introducing an S04 group into an organic compound in which the reaction product (sulfate) exhibits the characteristic -OSO3- molecular configuration. Sulfation involves the reaction wherein a -COS- linkage is formed by the action of a sulfating agent on an alkene, alcohol, or phenol. Unlike the sulfonates, which exhibit excellent hydrolytic stability, the alcohol sulfates are readily susceptible to hydrolysis in acidic media. Sulfation of fatty alcohols and polyalkoxy reductases occurs in the present process and the sulfation products lend themselves to detergent action as emulsifiers . Gums that are produced by the present process can store and stabilize products of microbial sulphur metabolism. Gums also serve generally to stabilize the fermentation components and thus facilitate storage of the product of the present method. The stabilization of the sulphur metabolism components allows immediate reaction with hydrocarbon chains in the environment by removing the sulfur link from those hydrocarbon chains for detergent reaction with associated alcohols. Basic fermentation processes of alcohols, aldehydes etc, break down components of plant cellulose and short chain carbon structures . As indicated above, it is advantageous to maintain a pH of 5.1 to 6.1 during fermentation. As the fermentation process moves into the acid cycle, gums are formed. Once the gums begin to form, the pH ranges, for example, between 6.1 and 6.8 (this range can be broader, for example, 5.2 and 6.8) . Gums are anionic which makes them advantageous in storing the acidic fermentation products as well as by products of sulphur metabolism. The product of the present invention is believed to permit a more rapid conversion of the nitrogen cycle through ATP conversion to the sulphur complexes in anaerobic microbial production by the use gum and wax production phenomena. The chemistry of the present process is believed to divert sulfur metabolism so as to lessen the production of sulfites and mercaptans.
Hence, the production of end-products such as hydrogen sulfite and methane, which are formed under anaerobic conditions where energy sources are apparently involved with hydrogen via dehydrogenase systems, are believed to be reduced.
Magnetism can alter the end-product production. By adjusting fields and field flux and making them permanent and/or oscillating along with altering bio¬ catalysts (e.g., proteins, vitamins, trace elements) and using the proper gas mixture of oxygen, nitrogen and argon, desired sulfur containing compounds are enriched, including α-linked purines, biotin, sulfinated carbohydrates, etc, and the levels of undesirable gases can be decreased. Magnetics, when combined with the present formulation, apparently provide for a more rapid conversion through the nitrogen and sulphur cycles to end complexes of sulphur metabolism.
Certain aspects of the invention are described in greater detail in the non-limiting Example that follows. EXAMPLE
Preparation of Composition for Stimulating Growth of Sugar Cane
Approximately a one liter sample is aspirated from the rumen of a 7.5 year old lactating Holstein cow using a rumen aspirator (Johnson and Johnson) . The sample is taken about 12-14 hours after feeding. Observed microscopically, the sample includes Clostridia, Bacillus, Azotobacter and protozoa (at least 100 cells of each per ml of sample) . The one liter sample is added to a culture medium (169 liters) that includes:
Culture medium:
Sodium bicarbonate 3.80 g
Potassium bicarbonate 0.21125 g Calcium carbonate 0.004225 g
Magnesium carbonate (anhydrous) 0.0063375 g
Phosphoric acid 0.570375 g
Chloroacetic acid 0.359125 g
The sample and the culture medium (Mixture A) are maintained in Tank 1 (see Figure 1) at a temperature of 37°C and at a pH in the range of 6.9-7.3 for a first 24 hour period. At this stage, and throughout the process, pH adjustments are made using citric acid or sodium bicarbonate, as appropriate. During this first 24 hr period, Mixture (A) is agitated by the injection of compressed air which results in the presence in Mixture (A) of about 3-5 ppm oxygen.
At the end of the first 24 hour period, Mixture (B) is added to Mixture (A) in Tank 1. Mixture (B) comprises 140 liters of water, pH 7.0-7.1, and 30 liters of the culture medium described above into which air has been injected to achieve an oxygen content of 3-5 ppm. Mixture (B) also includes approximately 20 kg of a substrate comprising the following in approximately equal parts by weight:
Substrate: crushed corn oats alfalfa whole peanuts
The pH of Mixture (B) is maintained at about 7.1 to 7.2, the temperature at about 34-40°C, and the oxygen content at about 3-5 ppm (by injection of compressed air) . These same conditions are maintained after the addition of Mixture (B) to Mixture (A) to form
Mixture (C) . Mixture (C) is maintained in Tank 1 at about 37°C for a second 24 hr period with agitation by compressed air injection.
At the end of this second 24 hour period, Mixture (D) is added to Mixture (C) in Tank 1 to yield Mixture (E) . Mixture (D) , like Mixture (B) , comprises 140 liters of water, pH 7.1 to 7.2, and 30 liters of culture medium. Mixture (D) also includes 20 kg of the substrate described above. Mixture (E) is maintained in Tank 1 for a third 24 hr period with agitation by compressed air injection (temperature 40°C; pH 7.1; oxygen content 3-5 ppm) .
At the completion of the third 24 hour period, Mixture (E) is recirculated for ten minute periods. Tank 1, and the recirculation system associated therewith, is designed such that complete recirculation of Mixture (E) can be effected in the ten minute period. That recirculation is carried out at two hour intervals for a fourth 24 hour period. Mixture (E) is then allowed to stand for a time sufficient to permit particulate matter to settle out. At the end of that fourth 24 hour period,
170 liters of Mixture (E) supernatant is transferred to Tank 2 (see Figure 1) . Compressed air is injected into Tank 2 to effect agitation of the aliquot of Mixture (E) present therein (Mixture (E-T2) ) and to maintain an oxygen content of 3-5 ppm. Mixture (E-T2) is maintained at a temperature of 37°-41°C and the pH is slowly reduced to 5.8-6.3 (i.e., over about a 3 hour period) and 3% (v/v) sugar cane molasses is added. The number of microorganisms present in Mixture (E-T2) is about 890,000 cells per ml.
Mixture (E-T2) (175 liters) is transferred to a further recirculation tank, Tank 3 (see Figure 1) and a "micronutrient" package is added. The contents of the package is formulated so that the addition thereof to the 175 liters results in the following concentrations, expressed as g/1 of Mixture (E-T2) :
Sodium bicarbonate .018
Chloroacetic acid .003 Phosphoric acid (liquid) .002
Magnesium carbonate (anhydrous) .002
Calcium carbonate .004
Sulfur (from sulfates in composition) .006
Zinc stearate .00003 Copper sulfate .00002
Cobalt acetate tetrahydrate .00000004
Iodine (liquid) .0000008
Se (plasma grade std (liquid)) .000001
Iron sulfate .00006 Potassium bicarbonate .006
Manganese sulfate monohydrate .00001
Molybdic acid 85% (powder) .000001 Silicon (reference std solution (1000 ppm) .00001
Boric acid .000005 Nickel carbonate .00000005
Rubidium chloride .000007
The content of Tank 3 (Mixture (E-T3)) is recirculated, and, during recirculation, is passed though a magnetic field (an 80 gal/min pump is used in the recirculation process) . The field is generated by six 3500 gauss rare earth magnets oriented as shown in Figure 2 with respect to a 1W diameter PVP 80 gauge recirculation tube, a 1/32" phenolic band being located between the magnets and the tube. During recirculation, the pH is maintained at between 5.5 and 6.5 and the temperature at about 37°C. Compressed air is injected during recirculation to maintain an oxygen content of 3-5 ppm. The composition resulting after 10 minutes of recirculation is stored for about 24 hours at a temperature of 35-38°C.
The composition resulting from the foregoing process is applied to sugar cane by spraying four times per year for a total annual application of 1.5 gallons per acre.
* * *
All documents cited above are hereby incorporated in their entirety by reference. One skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.

Claims

WHAT IS CLAIMED TS :
1. A method of preparing a culture comprising: i) obtaining a starting culture sample of microorganisms from the gastrointestinal track of a mammal; ii) culturing said sample in a medium comprising sodium, potassium, calcium, magnesium, inorganic phosphorus and chlorine or salts thereof; iii) culturing the sample resulting from step (ii) in the presence of a food source comprising a grain or a grass; iv) separating the culture of microorganisms resulting from step (iii) from said food source; v) exposing said culture resulting from step (iv) to a magnetic field.
2. The method according to claim 1 wherein said starting culture sample is obtained from a herbivore.
3. The method according to claim 1 wherein said food source comprises at least one of corn, oats, milo, alfalfa, sunflower seeds, peanuts, wheat, soy beans, barley, rice or flax.
4. The method according to claim 1 wherein said magnetic field is that generated by at least two rare earth magnets each of 1200 to 4500 gauss.
5. The method according to claim 1 wherein said exposure to said magnetic field is effected by passing the culture resulting from step (iv) between two magnets wherein like poles of said magnets face the flow path of said culture resulting from step (iv) .
6. A culture produced by the method of claim 1.
7. A composition comprising the culture according to claim 6 and a formulation comprising Na, Cl, P, Mg, Ca, S, Zn, Cu, Co, I, Se, Fe, K, Mn, Mo, Si, B, Ni and Rb.
8. A formulation comprising Na, Cl, P, Mg, Ca, S, Zn, Cu, Fe, K, Mn, Mo, Si, B, Ni and Rb.
9. A method of stimulating the growth of a plant comprising administering to said plant the composition according to claim 7 under conditions such that said stimulation is effected.
10. A method of preparing a culture of microorganisms for use as a constituent of a plant growth stimulating composition comprising exposing a culture of microorganisms to a magnetic field under conditions such that thickening of the cell walls of said microorganisms, as determined by light microscopy, is effected.
11. A method of preparing a culture comprising: i) obtaining a starting culture sample of microorganisms corresponding to that of the gastrointestinal track of a mammal; ii) culturing said sample in a medium comprising sodium, potassium, calcium, magnesium, inorganic phosphorus and chlorine or salts thereof; iii) culturing the sample resulting from step (ii) in the presence of a food source; and iv) separating the culture of microorganisms resulting from step (iii) from said food source.
12. A culture produced by the method of claim 11.
13. A composition comprising the culture according to claim 12 and a formulation comprising Mg, Mo, and B.
14. A method of stimulating the growth of a plant comprising administering to said plant the composition according to claim 13 under conditions such that said stimulation is effected.
15. The culture according to claim 12 wherein said culture of microorganisms resulting from step (iv) comprises gums or waxes .
16. The method according to claim 11 wherein sulphur metabolites present in said culture of microorganisms resulting from step (iv) are stored or stabilized with gums or waxes.
17. The method according to claim 11 further comprising exposing said culture resulting from step (iv) to a magnetic field.
18. The method according to claim 11 further comprising exposing said culture resulting from step (iv) to a magnetic field under conditions such that the off-gasing of products deleterious to plants is increased or the rate of nitrogen or sulphur cycle occurring in said culture resulting from step (iv) is increased.
19. The method according to claim 11 further comprising introducing argon, oxygen and nitrogen during steps (ii) , (iii) or (iv) .
20. The method according to claim 11 further comprising removing microorganisms from said culture resulting the step (iv) .
21. The method according to claim 11 further comprising adding to said culture of microorganisms resulting from step (iv) a formulation of nutrients comprising Na, Cl, P, Mg, Ca, S, Zn, Cu, Fe, K, Mn, Mo, Si, B, Ni and Rb.
22. A method of effecting bioremediation at a site in need of such remediation comprising contacting said site with an amount of the culture according to claim 12 or the composition according to claim 13 sufficient to effect said remediation.
23. The method according to claim 22 wherein said site is water, soil or air.
24. A method of stimulation the activity of microorganisms in si tu comprising applying to said microorganisms the culture according to claim 12 or the composition according to claim 13 in an amount sufficient to effect said stimulation.
25. The method of claim 24 wherein said microorganisms are present at a site in need of bioremediation.
26. The method of claim 24 wherein said microorganisms are present on a plant or soil surrounding roots of said plant .
27. The method of claim 24 wherein said microorganisms are present at an odor producing site.
28. A composition suitable for use in reducing or eliminating an offensive odor comprising an acid component, or salt thereof, an iron component and a nitrogen component.
29. The composition according to claim 28 further comprising a polysaccharide hydrolase.
30. The composition according to claim 29 wherein said hydrolase is a starch hydrolase, -amylase, glucoamylase, galactomannan hydrolase, or hemicellulase.
31. The composition according to claim 28 further comprising at least one of a molybdenum component, a copper component and a gum component.
32. The composition according to claim 28 wherein said acid is ascorbic, citric or acetic acid, or salt thereof.
33. A method of reducing or eliminating an offensive odor comprising contacting a compound responsible for said odor with the composition according to claim 28 under conditions such that said odor is eliminated or reduced.
34. A method of inhibiting odor production at a source comprising contacting said source with the composition according to claim 28 under conditions such that said inhibition is effected.
PCT/US1996/009574 1995-06-07 1996-06-05 A method of preparing a culture using a magnetic field and a compositon containing the same Ceased WO1996039799A1 (en)

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US08/476,374 US5866112A (en) 1995-01-20 1995-06-07 Methods of odor treatment
US08/569,769 1995-12-08
US08/569,769 US5843427A (en) 1995-01-20 1995-12-08 Method of using a plant growth stimulating composition

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CN102972124A (en) * 2012-12-15 2013-03-20 安徽科技学院 Indoor production method of radish sprouting vegetables by adopting rare-earth plant growth lamp
RU2479494C2 (en) * 2008-09-22 2013-04-20 Вильям Стивен ЛОПЕС Unit of treatment by magnetic field for conditioning fluids
CN103086793A (en) * 2013-01-10 2013-05-08 山西昌鑫生物农业科技有限公司 Amino-acid water-soluble bio-fertilizer and preparation method thereof
CN105944827A (en) * 2016-04-21 2016-09-21 洛南县恒丰非金属矿业有限公司 Method for extracting rare earth tailings discarded by preconcentration from molybdenum floatation tailings

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