# US 11,432,551 B2 · Microbial fermentation methods and compositions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,432,551 B2 - Document ID: US11432551B2 - Status: Granted - Kind: B2 - Application number: 16/906,554 - Filed: 2020-06-19 - Published: 2020-10-08 - Granted: 2022-09-06 - Earliest claimed: 2012-06-01 - Assignees: NewLeaf Symbiotics, Inc. - Applicants: NewLeaf Symbiotics, Inc. - Inventors: Gregg Bogosian - Web page: https://patentgrep.com/patent/US11432551B2 ## 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 obtaining a Methylobacterium preparation comprising: preparing a culture medium comprising a mono-culture or co-culture of Methylobacterium, wherein said medium comprises a liquid phase and a solid phase, wherein the liquid and solid phase are provided as sterile components, wherein the solid phase comprises a plurality of particles which are suspended in the liquid phase, wherein the particles are about 1 micron to about 1000 microns in average length or average diameter, and wherein the solid phase does not comprise viable photosynthetic microorganisms; (ii) growing said Methylobacterium from an initial titer in said culture medium, wherein said culture medium is essentially free of microorganisms other than Methylobacterium, wherein said particles provide for a decrease in doubling time of said Methylobacterium grown in said culture medium relative to the doubling time of the Methylobacterium grown in liquid media alone, and wherein the Methylobacterium titer in said culture medium is increased at least ten-fold relative to the initial titer of the Methylobacterium in the culture medium; (iii) producing a fermentation broth with a Methylobacterium titer of at least 5×108 colony-forming units per milliliter; and (iv) harvesting the Methylobacterium from said fermentation broth, thereby obtaining the Methylobacterium preparation. ### Claim 2 (depends on 1) The method of claim 1, wherein said method comprises the further step of drying the harvested Methylobacterium. ### Claim 3 (depends on 2) The method of claim 2, wherein drying is effected by spray drying, lyophilization, desiccation, heating, and combinations thereof. ### Claim 4 (depends on 1) The method of claim 1, wherein the titer of said Methylobacterium strain is increased at least 100-fold relative to the initial titer of the Methylobacterium in the culture medium. ### Claim 5 (depends on 1) The method of claim 1, wherein the particles are about 2 microns to about 1000 microns in average length or average diameter. ### Claim 6 (depends on 1) The method of claim 1, wherein said solid phase comprises about 0.02% to about 20% of said medium by mass. ### Claim 7 (depends on 1) The method of claim 1, wherein said solid phase comprises about 0.02% to about 0.5% of said medium by mass. ### Claim 8 (depends on 1) The method of claim 1, wherein at least 99.9% of the microorganisms present in the fermentation broth are Methylobacterium. ### Claim 9 (depends on 1) The method of claim 1, wherein said solid phase provides for adherent growth of said Methylobacterium and/or wherein said solid phase does not serve as a carbon source for said Methylobacterium. ### Claim 10 (depends on 1) The method of claim 1, wherein said solid phase comprises salt crystals. ### Claim 11 (depends on 10) The method of claim 10, wherein said salt crystals are insoluble or only partially soluble carbonates, chromates, sulfites, phosphates, hydroxides, oxides, and sulfides. ### Claim 12 (depends on 1) The method of claim 1, wherein said solid phase comprises a polysaccharide. ### Claim 13 (depends on 1) 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. ### Claim 14 (depends on 1) The method of claim 1, wherein the Methylobacterium titer in said culture medium is increased at least 1,000-fold relative to the initial titer of the Methylobacterium in the culture medium. ### Claim 15 (depends on 10) The method of claim 10, wherein said salt crystals are magnesium phosphate and/or calcium phosphate. ### Claim 16 (depends on 1) The method of claim 1, wherein said solid phase comprises diatomaceous earth. ### Claim 17 (depends on 12) The method of claim 12, wherein said polysaccharide is selected from the group consisting of a cellulosic polysaccharide, a chitinous polysaccharide, and a galactan polysaccharide. ### Claim 18 (depends on 1) The method of claim 1, wherein said liquid phase further comprises a carbon source supporting the growth of said Methylobacterium.