Bacterial Fermentation Methods and Compositions

Abstract

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.

What is claimed is: 
     
         1 . A method for obtaining a  Methylobacterium  preparation comprising growing  Methylobacterium  in an emulsion comprising a continuous phase and a dispersed phase that is immiscible or only partially miscible in the continuous phase, thereby obtaining a  Methylobacterium  preparation. 
     
     
         2 . The method of  claim 1 , wherein said  Methylobacterium  is a mono-culture or co-culture of  Methylobacterium.    
     
     
         3 . The method of  claim 1 , wherein (a) the dispersed phase comprises a non-aqueous liquid and the continuous phase comprises an aqueous liquid or (b) the dispersed phase comprises an aqueous liquid and the continuous phase comprise a non-aqueous liquid. 
     
     
         4 . The method of  claim 3 , wherein the non-aqueous liquid has a miscibility in water that is equal to or less than that of n-pentanol at 25° C. 
     
     
         5 . The method of  claim 1 , wherein dispersed phase provides for an increased yield of said  Methylobacterium  relative to a yield obtained by growing the  Methylobacterium  under identical conditions except for being grown in a non-emulsion that comprises a liquid corresponding to that of the continuous phase. 
     
     
         6 . The method of  claim 1 , further comprising harvesting  Methylobacterium  grown in the media. 
     
     
         7 . The method of  claim 1 , wherein the emulsion further comprises an emulsifier in an amount sufficient to stabilize the emulsion. 
     
     
         8 . The method of  claim 7 , wherein the emulsifier is selected from the group consisting of thickeners, surfactants, and combinations thereof. 
     
     
         9 . The method of  claim 3 , wherein the non-aqueous liquid comprises an alcohol, an aldehyde, a ketone, a fatty acid, a phospholipid, or any combination thereof. 
     
     
         10 . The method of  claim 9 , wherein the alcohol is selected from the group consisting of aliphatic alcohols containing at least 5 carbons and sterols. 
     
     
         11 . The method of  claim 3 , wherein the non-aqueous liquid comprises one or more animal, microbial, synthetic, or plant oils. 
     
     
         12 . The method of  claim 11 , wherein the plant oil is selected from the group consisting of corn, soybean, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame seed, safflower, and combinations thereof. 
     
     
         13 . The method of  claim 1 , wherein said emulsion does not contain a photosynthetic microorganism or wherein the emulsion further comprises one or more non-photosynthetic microorganisms of pre-determined identity other than  Methylobacterium.    
     
     
         14 . The method of  claim 1 , wherein the dispersed phase comprises at least about 0.02% to about 20% of said emulsion by mass. 
     
     
         15 . The method of  claim 3 , wherein the non-aqueous liquid is an agriculturally acceptable adjuvant or agriculturally acceptable excipient. 
     
     
         16 . The method of  claim 1 , wherein said growing comprises the steps of inoculating said emulsion with said  Methylobacterium  and incubating said inoculated emulsion under conditions sufficient to provide for growth of said  Methylobacterium.    
     
     
         17 . The method 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 LMG6378,  M. phyllosphaerae, M. oryzae, M. platani, M. populi , and  M. zatmanii.    
     
     
         18 . The method of  claim 1 , wherein said emulsion is essentially free of contaminating microorganisms. 
     
     
         19 . The method of  claim 6 , wherein said harvesting comprises recovering all or a portion of the  Methylobacterium  from the emulsion. 
     
     
         20 . The method of  claim 19 , said method further comprising dehydrating the recovered portion of the  Methylobacterium.    
     
     
         21 . A  Methylobacterium  preparation obtained by the method of any one of  claims 1 - 20 , wherein either the dispersed phase or the continuous phase comprises a non-aqueous liquid that has a miscibility in water that is equal to or less than that of n-pentanol at 25° C. 
     
     
         22 . A method for treating a plant or a plant part with  Methylobacterium  comprising the step of applying to said plant or plant part a composition comprising the  Methylobacterium  preparation of  claim 21 . 
     
     
         23 . The method of  claim 22 , wherein said composition further comprises an agriculturally acceptable adjuvant or an agriculturally acceptable excipient. 
     
     
         24 . The method of  claim 23 , wherein the composition lacks a solid substance. 
     
     
         25 . The method of  claim 22 , wherein said plant part is a seed and said composition has a  Methylobacterium  titer of at least about 5×10 8  colony-forming units per gram of said composition to about 5×10 13  colony-forming units per gram of said composition. 
     
     
         26 . The method of  claim 22 , wherein said plant part is a seed, stem, root, flower, cotyledon, a coleoptile, fruit, or a leaf. 
     
     
         27 . The method of  claim 22 , wherein said plant or plant part is a 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, or conifer plant or plant part. 
     
     
         28 . A plant or plant part obtained by the method of any one of  claims 22 - 27 , wherein the plant or plant part is at least partially coated with the  Methylobacterium  preparation. 
     
     
         29 . A processed plant product obtained from the plant or plant part of  claim 28 , wherein said processed product contains the emulsion. 
     
     
         30 . The processed plant product of  claim 29 , wherein said product is a meal, paste, flour, flake, or feed. 
     
     
         31 . The processed plant product of any one of  claims 29 - 30 , wherein said processed product is non-regenerable. 
     
     
         32 . A fermentation product that comprises an emulsion comprising a continuous phase and a dispersed phase that is immiscible or only partially miscible in the continuous phase, and a mono-culture or co-culture of  Methylobacterium.    
     
     
         33 . The fermentation product of  claim 32 , wherein (a) the dispersed phase comprises a non-aqueous liquid and the continuous phase comprises an aqueous liquid or (b) the dispersed phase comprises an aqueous liquid and the continuous phase comprise a non-aqueous liquid 
     
     
         34 . The fermentation product of  claim 33 , wherein the non-aqueous liquid has a miscibility in water that is equal to or less than that of n-pentanol at 25° C. 
     
     
         35 . The fermentation product of  claim 32 , wherein said fermentation product is essentially free of contaminating microorganisms. 
     
     
         36 . The fermentation product of  claim 32 , wherein said fermentation product further comprises one or more microorganisms of pre-determined identity other than  Methylobacterium.    
     
     
         37 . The fermentation product of  claim 32 , wherein said fermentation product lacks a solid substance. 
     
     
         38 . The fermentation product of  claim 32 , wherein said product does not contain a photosynthetic microorganism. 
     
     
         39 . A composition that comprises an emulsion comprising a continuous phase and a dispersed phase, and a mono-culture or co-culture of  Methylobacterium , wherein (a) the dispersed phase comprises a non-aqueous liquid and the continuous phase comprises an aqueous liquid or (b) the dispersed phase comprises an aqueous liquid and the continuous phase comprise a non-aqueous liquid, and wherein the non-aqueous liquid has a miscibility in water that is equal to or less than that of n-pentanol at 25° C. 
     
     
         40 . The composition of  claim 39 , wherein the composition is essentially free of contaminating microorganisms. 
     
     
         41 . The composition of  claim 39  wherein the composition further comprises at least one of an agriculturally acceptable adjuvant and/or an agriculturally acceptable excipient. 
     
     
         42 . The composition of  claim 39 , wherein the composition lacks a solid substance. 
     
     
         43 . The composition of  claim 39 , wherein said second liquid comprises an alcohol, an aldehyde, a ketone, a fatty acid, a phospholipid, or any combination thereof. 
     
     
         44 . The composition of  claim 39 , wherein the alcohol is selected from the group consisting of aliphatic alcohols containing at least 5 carbons and sterols. 
     
     
         45 . The composition of  claim 39 , wherein the second liquid comprises one or more animal, microbial, synthetic, or plant oils. 
     
     
         46 . The composition of  claim 45 , wherein the plant oil is selected from the group consisting of corn, soybean, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame seed, safflower, and combinations thereof. 
     
     
         47 . The composition of  claim 39 , wherein said solid substance further comprises an agriculturally acceptable adjuvant or an agriculturally acceptable excipient. 
     
     
         48 . The composition of  claim 39 , wherein the composition does not contain a photosynthetic microorganism. 
     
     
         49 . The composition of  claim 39 , wherein said composition further comprises at least one pesticide and/or at least one bacteriostatic agent. 
     
     
         50 . The composition of  claim 49 , wherein said pesticide is selected from the group consisting of an insecticide, a fungicide, a nematocide, and a bacteriocide, wherein said pesticide does not substantially inhibit growth of said  Methylobacterium.    
     
     
         51 . A method for treating a plant or a plant part with  Methylobacterium  comprising the step of applying to said plant or plant part a fermentation product of any one of  claims 32 - 38  or a composition of any one of  claims 39 - 50 . 
     
     
         52 . The method of  claim 51 , wherein said plant part is a seed, stem, root, flower, cotyledon, a coleoptile, fruit, or a leaf. 
     
     
         53 . The method of  claim 51 , wherein said plant or plant part is a 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, or conifer plant or plant part. 
     
     
         54 . A plant obtained by the method of  claim 51 , wherein the plant is at least partially coated with the fermentation product of the composition. 
     
     
         55 . A plant part obtained by the method of  claim 51 , wherein the plant is at least partially coated with the fermentation product of the composition. 
     
     
         56 . A plant or plant part, wherein the plant or plant part is at least partially coated with an emulsion comprising a first aqueous liquid, a second liquid having a miscibility in water that equal to or less than that of n-pentanol at 25° C., and a mono-culture or co-culture of an exogenous  Methylobacterium.