Microbial fermentation methods and compositions
The present invention provides methods for the cultivation of the Methylobacterium genus of bacteria. In particular the method provides methods for the efficient and inexpensive cultivation of these bacteria. Additionally, the invention provides methods for the utilization of these bacterial cultures to improve plant agriculture.
1 . A fermentation product comprising one or more solid substance and one or more Methylobacterium obtained by fermentation,
a) wherein said fermentation product is essentially free of contaminating microorganisms other than the Methylobacterium,
b) wherein said Methylobacterium has a titer of at least 5×10 8 colony-forming units per gram of solid substance to at least about 5×10 13 colony-forming units of Methylobacterium per gram of solid substance, and
c) wherein said one or more solid substance is an adjuvant.
2 . The fermentation product of claim 1 , wherein the solid substance is an adjuvant selected from the group consisting of: non-ionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant, organo-silicate surfactants, acidified surfactants, latex-based substances, terpene, pyrrolidone-based substance, mineral oil, vegetable oil, esterified vegetable oil, organo-silicate surfactant, acidified surfactant, ammonium sulphate, menthene-based substance, glycerol, propylene glycol, and diethyl glycol.
3 . The fermentation product of claim 1 , wherein the solid substance is an adjuvant selected from the group consisting of: dimethopolysiloxane, ammonium sulphate, polyacrylamides, and polysaccharides.
4 . The fermentation product of claim 1 , wherein the solid substance is an adjuvant selected from the group consisting of: bone meal, gelatin, ground shells, powdered shells, hair, macerated hide, cobs, husks, hulls, leaves, roots, flowers, stems, bark, seeds, bagasse, wheat bran, soy grits, crushed seed cake, stover; wood pulp, sawdust, shavings; ground glass or glass beads, ground ceramic materials, ceramic beads, bentonite, sand, silt, soil, clay, ash, charcoal, latex-based substances, woodflours, clays, activated carbon, china clay, talc, perlite, mica, vermiculite, silicas, quartz powder, montmorillonite, polyvinyl acetates, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetates, polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohols, polyvinyl alcohol copolymers, polyvinyl methyl ether, polyvinyl methyl ether-maleic anhydride copolymer, waxes, latex polymers, ethylcelluloses, methylcelluloses, hydroxy methylcelluloses, hydroxypropylcellulose, hydroxymethylpropylcelluloses, polyvinyl pyrrolidones, karaya gum, jaguar gum, tragacanth gum, polysaccharide gums, mucilage, gum arabics, shellacs, vinylidene chloride polymers, vinylidene chloride copolymers, lignosulfonates, acrylic copolymers, polyvinylacrylates, polyethylene oxide, acrylimide polymers, acrylamide copolymers, polyhydroxyethyl acrylate, methylacrylimide monomers, alginate, polychloroprene, vinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymer.
5 . The fermentation product of claim 1 , wherein the solid substance is an adjuvant selected from the group consisting of: dimethopolysiloxane, agar, polymer or polymeric beads, insoluble or only partially soluble salt crystal, vegetable oil, esterified vegetable oil, inactivated microbial cell, fungal cell, microbial spore, or fungal spore, diatom shell, quiescent microbial cell, fungal cell, microbial spore, or fungal spore, cellular debris of microbial origin, fine-grain inorganic solids, calcium carbonate, calcium bentonite, kaolin, alginates, dextrins, malto-dextrins, fats, oils, proteins, soybean-based protein polymers, soybean-based copolymers, starches, zeins, gelatin, carboxymethylcellulose, chitosan, water-soluble waxes, and anti-foaming agents.
6 . The fermentation product of claim 1 , wherein said one or more solid substance is an adjuvant and comprises particles of about 2 microns to about 750 microns in average length or average diameter.
7 . The fermentation product of claim 1 , wherein said fermentation product is dried.
8 . The fermentation product of claim 1 , wherein said Methylobacterium is selected from the group consisting of: M. aminovorans, M. chloromethanicum, M. dichloromethanicum, M. extorquens, M. fujisawaense, M. mesophilicum, M. organophilum, M. radiotolerans, M. rhodesianum, M. rhodinum, M. thiocyanatum, M. nodulans, M. cerastii, M. gossipiicola, Methylobacterium sp. strain LilIG6378, M. phyllosphaerae, M. oryzae, M. platani, M. populi , and M. zatmanii.
9 . A plant or plant part that is at least partially coated with a composition comprising one or more fermentation product of claim 1 ; and one or more additional composition selected from the group consisting of: sticker, extender, humectant, drift-reducer, water conditioner, and/or coating promoter; and one or more agriculturally acceptable excipient.
10 . The plant of claim 7 , wherein said composition further comprises one or more introduced microorganisms of pre-determined identity other than Methylobacterium.
11 . The plant part of claim 5 , wherein the plant part is a seed.
12 . The plant or plant part of claim 5 , wherein said plant or plant part is selected from the group consisting of corn, Brassica sp., alfalfa, rice, rye, sorghum, pearl millet, proso millet, foxtail millet, finger millet, sunflower, safflower, soybean, tobacco, potato, peanuts, cotton, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, sugar beet, sugarcane, oat, barley, tomato, lettuce, green bean, lima bean, pea, cucurbit, ornamental, and conifer.
13 . A method for obtaining a Methylobacterium preparation comprising:
a) inoculating a monoculture of a Methylobacterium strain into media that comprises a liquid phase and a solid phase to an initial titer of at least 1×10 5 colony-forming units per milliliter of the Methylobacterium in the media, wherein the liquid and solid phase were sterile prior to inoculation of the Methylobacterium strain, wherein said solid phase comprises a plurality of particles which are suspended in the liquid phase,
b) growing the monoculture of a Methylobacterium strain in the media that comprises a liquid phase and a solid phase to provide a fermentation broth, wherein the Methylobacterium titer in the fermentation broth is increased at least 1,000-fold from the initial titer of Methylobacterium in the inoculated media of step (i); and
c) harvesting the Methylobacterium strain grown in the media, thereby obtaining the Methylobacterium preparation;
i. wherein said fermentation product is essentially free of contaminating microorganisms other than the Methylobacterium , and
ii. wherein said Methylobacterium has a titer of at least 5×10 8 colony-forming units per gram of solid substance to at least about 5×10 13 colony-forming units of Methylobacterium per gram of solid substance, and
iii. wherein said one or more solid substance is an adjuvant.
14 . The method of claim 13 , wherein said further comprising lyophilizing and/or spray drying said harvested Methylobacterium.
15 . The method of claim 13 , wherein said solid phase comprises about 0.02% to about 0.5% of said medium by mass.
16 . A method for treating a plant or a plant part with Methylobacterium comprising applying to a plant or plant part a composition comprising a fermentation product of claim 1 and at least one agriculturally acceptable excipient.
17 . The method of claim 16 , wherein said plant part is selected from the group consisting of: seed, stem, root, flower, cotyledon, a coleoptile, fruit, and leaf.
18 . The method of claim 16 , wherein said plant is selected from the group consisting of: corn, Brassica sp., alfalfa, rice, rye, sorghum, pearl millet, proso millet, foxtail millet, finger millet, sunflower, safflower, soybean, tobacco, potato, peanuts, cotton, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, sugar beet, sugarcane, oat, barley, tomato, lettuce, green bean, lima bean, pea, cucurbit, ornamental, and conifer plant.
19 . The method of claim 16 , wherein said agriculturally acceptable excipient is selected from the group consisting of: woodflours, clays, activated carbon, diatomaceous earth, fine-grain inorganic solids, and calcium carbonate.
20 . The method of claim 16 , wherein said agriculturally acceptable adjuvant is selected from the group consisting of: fat, oil, or wax, cellulose, polysaccharide, protein, polymer, and copolymer.
21 . The method of claim 16 , wherein the adjuvant is selected from the group consisting of: non-ionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant, organo-silicate surfactants, acidified surfactants, latex-based substances, terpene, pyrrolidone-based substance, mineral oil, vegetable oil, esterified vegetable oil, organo-silicate surfactant, acidified surfactant, ammonium sulphate, menthene-based substance, glycerol, propylene glycol, and diethyl glycol.
22 . The method of claim 16 , wherein the-adjuvant is selected from the group consisting of: dimethopolysiloxane, ammonium sulphate, polyacrylamides, and polysaccharides.
23 . The method of claim 16 , wherein the adjuvant is selected from the group consisting of: bone meal, gelatin, ground shells, powdered shells, hair, macerated hide, cobs, husks, hulls, leaves, roots, flowers, stems, bark, seeds, bagasse, wheat bran, soy grits, crushed seed cake, stover; wood pulp, sawdust, shavings; ground glass or glass beads, ground ceramic materials, ceramic beads, bentonite, sand, silt, soil, clay, ash, charcoal, latex-based substances, woodflours, clays, activated carbon, china clay, talc, perlite, mica, vermiculite, silicas, quartz powder, montmorillonite, polyvinyl acetates, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetates, polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohols, polyvinyl alcohol copolymers, polyvinyl methyl ether, polyvinyl methyl ether-maleic anhydride copolymer, waxes, latex polymers, ethylcelluloses, methylcelluloses, hydroxy methylcelluloses, hydroxypropylcellulose, hydroxymethylpropylcelluloses, polyvinyl pyrrolidones, karaya gum, jaguar gum, tragacanth gum, polysaccharide gums, mucilage, gum arabics, shellacs, vinylidene chloride polymers, vinylidene chloride copolymers, lignosulfonates, acrylic copolymers, polyvinylacrylates, polyethylene oxide, acrylimide polymers, acrylamide copolymers, polyhydroxyethyl acrylate, methylacrylimide monomers, alginate, polychloroprene, vinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymer.
24 . The method of claim 16 , wherein the adjuvant is selected from the group consisting of: dimethopolysiloxane, agar, polymer or polymeric beads, insoluble or only partially soluble salt crystal, vegetable oil, esterified vegetable oil, inactivated microbial cell, fungal cell, microbial spore, or fungal spore, diatom shells, quiescent microbial cell, fungal cell, microbial spore, or fungal spore, cellular debris of microbial origin, fine-grain inorganic solids, calcium carbonate, calcium bentonite, kaolin, alginates, dextrins, malto-dextrins, fats, oils, proteins, soybean-based protein polymers, soybean-based copolymers, starches, zeins, gelatin, carboxymethylcellulose, chitosan, water-soluble waxes, and anti-foaming agents.