# US 11,998,015 B2 · Microbial fermentation methods and compositions

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- Number: US 11,998,015 B2
- Document ID: US11998015B2
- Status: Granted
- Kind: B2
- Application number: 17/814,739
- Filed: 2022-07-25
- Published: 2023-03-16
- Granted: 2024-06-04
- Earliest claimed: 2012-06-01
- Assignees: NewLeaf Symbiotics, Inc.
- Applicants: NewLeaf Symbiotics, Inc.
- Inventors: Gregg Bogosian
- Web page: https://patentgrep.com/patent/US11998015B2

## CPC

- A01N 63/20: Bacteria; Substances produced thereby or obtained therefrom
- C12N 1/20: Bacteria; Culture media therefor
- C12N 11/02: Enzymes or microbial cells immobilised on or in an organic carrier
- C12N 11/14: Enzymes or microbial cells immobilised on or in an inorganic carrier
- C12N 11/16: Enzymes or microbial cells immobilised on or in a biological cell
- Y02E 50/30: Fuel from waste, e.g. synthetic alcohol or diesel
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## 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.

## Claims

### Claim 1

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 a fermentation product comprising a solid substance wherein a mono-culture or co-culture of Methylobacterium is adhered thereto by having been grown on said solid substance, wherein said solid substance comprises a plurality of particles of about 1 micron to about 1000 microns in average length or average diameter and is not a photosynthetic microorganism, and wherein said fermentation product is essentially free of contaminating microorganisms other than the Methylobacterium, and wherein said composition (i) has a Methylobacterium titer of at least 5×108 colony-forming units per ml of said composition for a fermentation broth, or (ii) has a Methylobacterium titer of at least 5×108 colony-forming units per gram of said composition for a dry composition.

### Claim 2 (depends on 1)

The method of claim 1, wherein said plant is a cereal plant and said composition is applied in an amount sufficient to provide for an increase in nodal root growth of said cereal plant.

### Claim 3 (depends on 1)

The method of claim 1, wherein said composition is applied to a cereal plant seed.

### Claim 4 (depends on 3)

The method of claim 3, wherein said cereal plant seed is a corn, barley, millet, oat, rice, rye, sorghum, triticale, or wheat seed.

### Claim 5 (depends on 1)

The method of claim 1, wherein said composition further comprises at least one agriculturally acceptable adjuvant or agriculturally acceptable excipient.

### Claim 6 (depends on 5)

The method of claim 5, 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.

### Claim 7 (depends on 6)

The method of claim 6, wherein clay or inorganic solid is calcium bentonite, kaolin, china clay, talc, perlite, mica, vermiculite, silica-s, quartz powder, montmorillonite or a mixture thereof.

### Claim 8 (depends on 5)

The method of claim 5, wherein said agriculturally acceptable adjuvant is a polysaccharide.

### Claim 9 (depends on 1)

The method of claim 1, wherein said composition further comprises at least one pesticide.

### Claim 10 (depends on 9)

The method of claim 9, wherein said pesticide is selected from the group consisting of an insecticide, a fungicide, a nematocide, and a bacteriocide.

### Claim 11 (depends on 1)

The method of claim 1, wherein said fermentation product is a dry composition obtained by lyophilization or spray drying.

### Claim 12

A plant or plant part that is at least partially coated with a composition comprising a fermentation product that comprises: (i) an exogenous solid substance having a mono-culture or co-culture of Methylobacterium adhered thereto by having been grown on said solid substance as an axenic culture that is essentially free of contaminating microorganisms; and (ii) an agriculturally acceptable adjuvant, wherein said adjuvant is a sticker, extender, humectant, drift-reducer, water conditioner, coating promoter, or combination thereof; an agriculturally acceptable excipient; or a combination thereof.

### Claim 13 (depends on 12)

The plant or plant part of claim 12, wherein:
(i) said agriculturally acceptable excipient is selected from the group consisting of woodflours, clays, activated carbon, diatomaceous earth, fine-grain inorganic solids and calcium carbonate; and/or
(ii) said agriculturally acceptable adjuvant is a polysaccharide.

### Claim 14 (depends on 12)

The plant or plant part of claim 12, wherein said plant or plant part is selected from the group consisting of 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, and conifer plant or plant part.

### Claim 15 (depends on 12)

The plant part of claim 12, wherein said plant part is a cereal seed.

### Claim 16 (depends on 15)

The cereal seed of claim 15, wherein said composition (i) has a Methylobacterium titer of at least 5×108 colony-forming units per ml of said composition for a liquid fermentation broth, or (ii) has a Methylobacterium titer of at least 5×108 colony-forming units per gram of said composition for a dry composition.

### Claim 17 (depends on 15)

The cereal plant seed of claim 15, wherein said seed is a corn, barley, millet, oat, rice, rye, sorghum, triticale, or wheat seed.

### Claim 18 (depends on 12)

The plant or plant part of claim 12, wherein said composition further comprises at least one pesticide.

### Claim 19 (depends on 18)

The plant or plant part of claim 18, wherein said pesticide is selected from the group consisting of an insecticide, a fungicide, a nematocide, and a bacteriocide.

### Claim 20 (depends on 12)

The plant or plant part of claim 12, wherein said fermentation product is a dry composition obtained by lyophilization or spray drying.

### Claim 21 (depends on 5)

The method of claim 5, wherein said agriculturally acceptable adjuvant is a fat, oil, or wax.

### Claim 22 (depends on 5)

The method of claim 5, wherein said agriculturally acceptable adjuvant is an acrylic or acrylamide polymer or copolymer.

### Claim 23 (depends on 5)

The method of claim 5, wherein said agriculturally acceptable adjuvant is a cellulose.

### Claim 24 (depends on 8)

The method of claim 8, wherein said polysaccharide is a dextrin, maltodextrin, starch, chitosan, alginate, or gum.

### Claim 25 (depends on 5)

The method of claim 5, wherein said agriculturally acceptable adjuvant is a protein.

### Claim 26 (depends on 25)

The method of claim 25, wherein said protein is a soybean-based protein polymer, a zein or a gelatin.

### Claim 27 (depends on 23)

The method of claim 23, wherein said cellulose is ethylcellulose, methylcellulose, hydroxy methylcellulose, hydroxypropylcellulose, hydroxymethylpropylcellulose, or carboxymethylcellulose.

### Claim 28 (depends on 13)

The plant or plant part of claim 13, wherein said polysaccharide is a dextrin, maltodextrin, starch, chitosan, alginate, or gum.

### Claim 29 (depends on 12)

The plant or plant part of claim 12, wherein said agriculturally acceptable adjuvant is a protein.

### Claim 30 (depends on 29)

The plant or plant part of claim 29, wherein said protein is a soybean-based protein polymer, a zein, or a gelatin.

### Claim 31 (depends on 5)

The method of claim 5, wherein said agriculturally acceptable adjuvant is a polymer or copolymer.

### Claim 32 (depends on 31)

The method of claim 31, wherein said polymer or copolymer is polyvinyl acetate, a polyvinyl acetate polymer or copolymer, hydrolyzed polyvinyl acetate, a polyvinylpyrrolidone, a polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohol, a polyvinyl alcohol copolymer, polyvinyl methyl ether, a polyvinyl methyl ether-maleic anhydride copolymer, a polyvinylacrylate, a vinylidene chloride polymer or copolymer, polychloroprene, or a polyvinyl pyrrolidone polymer.

### Claim 33 (depends on 31)

The method of claim 31, wherein said polymer or copolymer is an acrylic or acrylamide polymer or copolymer.

### Claim 34 (depends on 31)

The method of claim 31, wherein said polymer or copolymer is polyethylene oxide.

### Claim 35 (depends on 31)

The method of claim 31, wherein said polymer or copolymer is a lignosulfate polymer.

### Claim 36 (depends on 31)

The method of claim 31, wherein said polymer or copolymer is shellac.

### Claim 37 (depends on 31)

The method of claim 31, wherein said polymer or copolymer is a latex polymer.