Microbial inoculant formulations

Patent metadata

Field	Value
Publication number	US-10757946-B2
Application number	US-201514856020-A
Country	US
Kind code	B2
Filing date	Sep 16, 2015
Priority date	Sep 16, 2014
Publication date	Sep 1, 2020
Grant date	Sep 1, 2020

Abstract

Compositions comprising dried formulations of viable Methylobacterium as well as methods for making and using the formulations are provided. In particular the compositions provide for Methylobacterium formulations with improved viability, shelf-life, and plant or seed treatment characteristics.

First claim

What is claimed is:

1. A dried composition that is substantially free of water, essentially free of water, or essentially dry, wherein said composition comprises a mono-culture of Methylobacterium at a titer of at least 5×10 8 colony forming units per gram of the dried composition and sucrose, wherein the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), and NLS0089 (NRRL B-50933) and wherein said composition is prepared by drying a biphasic mixture by lyophilization or spray drying, wherein said biphasic mixture comprises:
(i) a mono-culture of Methylobacterium adhered to an insoluble or partially soluble particulate solid substance comprising a plurality of particles of about 1 micron to about 1000 microns in average length or average diameter by having been axenically grown on the solid substance, wherein said mono-culture-of Methylobacterium is essentially free of contaminating microorganisms and can colonize a plant and/or a plant part, and
(ii) sucrose at a concentration of about 4.6% (w/v) to 7% (w/v), and wherein at least about 50%, 60%, or 70% of Methylobacterium cells in the dried composition are viable after at least 75 days at room temperature.

2. The composition of claim 1 , wherein the solid substance has a Methylobacterium titer of 5×10 8 , 1×10 9 , or 1×10 10 colony-forming units per gram of the solid substance to about 5×10 13 colony-forming units of Methylobacterium per gram of the solid sub stance.

3. The composition of claim 1 , wherein said composition further comprises skim milk.

4. The composition of claim 1 , wherein the composition further comprises an agriculturally acceptable adjuvant, agriculturally acceptable excipient, a pesticide, a bacteriostatic agent, a biopesticidal microorganism, or a plant beneficial microorganism.

5. The composition of claim 1 , wherein the solid substance is a polymer or a salt crystal.

6. The composition of claim 1 , wherein the Methylobacterium adhered thereto cannot be substantially removed from the solid substance by washing in growth media.

7. The composition of claim 1 , wherein the biphasic mixture is prepared by resuspending said Methylobacterium adhered to a solid substance in an aqueous solution containing the sucrose after being harvested from a fermentation broth but before being dried.

8. The dried composition of claim 1 wherein the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), and NLS0089 (NRRL B-50933).

9. The composition of claim 8 , wherein the composition is substantially free of water and has a residual moisture content of 5% to 0.2%.

10. The composition of claim 8 , wherein the composition is essentially free of water and has a residual moisture content of 0.2% to 0.001%.

11. The composition of claim 8 , wherein the composition is essentially dry and has a residual moisture content of less than 0.001%.

12. The composition of claim 1 , wherein the dried composition has a Methylobacterium titer of 1×10 9 , or 1×10 10 colony-forming units per gram of the dried composition to about 5×10 12 , 1×10 13 , or 5×10 13 colony-forming units per gram of the dried composition.

13. The composition of claim 1 , wherein at least 70% of the Methylobacterium cells in the dried preparation are viable after at least 311 days at room temperature.

14. The composition of claim 1 , wherein the solid substance provides for increased yield of the Methylobacterium relative to yield obtained by growing the Methylobacterium in liquid media alone.

15. The composition of claim 1 , wherein said composition is a dry powder formulation.

16. The composition of claim 8 , wherein the solid substance is a polymer or a salt crystal.

17. The composition of claim 8 , wherein said composition is a dry powder formulation.

18. The composition of claim 1 wherein said solid substance comprises a plurality of particles of 1 micron to 1000 microns in average length or average diameter.

19. The composition of claim 8 wherein said solid substance comprises a plurality of particles of 2 to 500 microns in average length or average diameter.

20. The composition of claim 4 , wherein the adjuvant comprises polyvinyl acetate, polyvinyl acetate copolymer, hydrolyzed polyvinyl acetate, polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohol, polyvinyl alcohol copolymer, polyvinyl methyl ether, polyvinyl methyl ether-maleic anhydride copolymer, wax, latex polymer, a cellulose, ethylcelluloses, methylcellulose, hydroxy methylcellulose, hydroxypropylcellulose, hydroxymethylpropylcellulose, polyvinyl pyrrolidone, alginate, dextrin, malto-dextrin, polysaccharides, fat, oil, protein, karaya gum, jaguar gum, tragacanth gum, polysaccharide gum, mucilage, gum arabic, shellac, vinylidene chloride polymer and copolymers soybean-based protein polymer and copolymer, lignosulfonate, acrylic copolymers, starches, polyvinylacrylate, zein, gelatin, carboxymethylcellulose, chitosan, polyethylene oxide, acrylimide polymer, and acrylimide copolymer, polyhydroxyethyl acrylate, methylacrylimide monomer, ethylcellulose, polychloroprene, or a mixture thereof.

21. The composition of claim 4 , wherein the pesticide is selected from the group consisting of an insecticide, a fungicide, a nematocide, and a bacteriocide.

Assignees

• Newleaf Symbiotics Inc
• Newleaf Symbiotic Inc

Inventors

• Allen Kimberly
• Bogosian Gregg

Classifications

• A01N63/20 Primary

  Bacteria; Substances produced thereby or obtained therefrom · CPC title

• C12N1/04

  Preserving or maintaining viable microorganisms (immobilised microorganisms C12N11/00) · CPC title

• C05F11/08

  Organic fertilisers containing added bacterial cultures, mycelia or the like · CPC title

• A01N63/00

  Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates (containing compounds of determined constitution A01N27/00 - A01N59/00; unicellular algae A01N65/03) · CPC title

• A01N63/10 Primary

  Animals; Substances produced thereby or obtained therefrom · CPC title