# US 2021/0329925 A1 · Method for enhancing the growth and survival rate of microorganisms

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- Number: US 2021/0329925 A1
- Document ID: US20210329925A1
- Status: Published application
- Kind: A1
- Application number: 17/312,848
- Filed: 2019-12-11
- Published: 2021-10-28
- Earliest claimed: 2018-12-11
- Assignees: Danstar Ferment AG
- Applicants: Danstar Ferment AG
- Inventors: Mike Whiting; Tyler Boa
- Web page: https://patentgrep.com/patent/US20210329925A1

## CPC

- A01N 63/20: Bacteria; Substances produced thereby or obtained therefrom
- A01N 63/22: Bacillus
- A01N 63/25: Paenibacillus
- A01N 63/27: Pseudomonas
- A01N 63/28: Streptomyces
- A01N 63/36: Penicillium
- A01N 63/38: Trichoderma
- Y02A 40/22: Improving land use; Improving water use or availability; Controlling erosion
- Y02P 60/21: Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The present disclosure concerns a method for enhancing or promoting or increasing the fermentative potential of microbial populations and/or the growth rate of microorganisms. The present disclosure also proposes a method for improving survival, viability and/or stability of microorganisms in microbial products, suspensions or of microbial inoculants, during storage.

## Claims

### Claim 1

A method for increasing the yield, growth, growth rate, survival rate and/or viability of a population of microorganisms, the method comprising contacting a protective agent comprising biochar particles, activated carbon and/or charcoal with the population of microorganisms to obtain a mixture.

### Claim 2 (depends on 1)

The method of claim 1, wherein the protective agent comprises or consists essentially of biochar particles.

### Claim 3 (depends on 1)

The method of claim 1, wherein the protective agent comprises or consists essentially of activated carbon.

### Claim 4 (depends on 1, 2, 3)

The method of any one of claims 1 to 3, wherein the protective agent is added at a concentration of 0.01% to 50%, 0.05% to 20%, 0.1% to 15%, 0.1% to 10% or 0.1% to 5% weight/volume of the mixture.

### Claim 5 (depends on 1, 2, 3, 4)

The method of any one of claims 1 to 4, wherein the mixture further comprises a medium.

### Claim 6 (depends on 5)

The method of claim 5, wherein the medium is a liquid medium.

### Claim 7 (depends on 6)

The method of claim 6, wherein the mixture is a liquid inoculant.

### Claim 8 (depends on 5)

The method of claim 5, wherein the medium is a solid substrate.

### Claim 9 (depends on 8)

The method of claim 8, wherein mixture is a microbial inoculant immobilized in the solid substrate.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of any one of claims 1 to 9 further comprising storing the mixture.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The method of any one of claims 1 to 10, wherein the population of microorganisms was grown in the absence of the protective agent.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The method of any one of claims 1 to 10, wherein the population of microorganisms was grown in the presence of the protective agent.

### Claim 13 (depends on 10, 11, 12)

The method of any one of claims 10 to 12, wherein the presence of the protective agent reduces the loss during storage of the population of microorganisms by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or 100% or by no more than 0.1 log CFU, 0.2 log CFU, 0.3 log CFU 0.4 log CFU, 0.5 log CFU, 0.6 log CFU, 0.7 log CFU, 0.8 log CFU, 0.9 log CFU, 1 log CFU, 1.1 log CFU, 1.2 log CFU, 1.3 log CFU, 1.4 log CFU, 1.5 log CFU, 1.6 log CFU, 1.7 log CFU, 1.8 log CFU, 1.9 log CFU or 2 log CFU when compared to a corresponding population of microorganisms in a control mixture which does not comprise the protective agent.

### Claim 14 (depends on 5, 6, 7, 8, 9, 10, 11, 12, 13)

The method of any one of claims 5 to 13, wherein the medium is capable of supporting growth of the population of microorganisms.

### Claim 15 (depends on 14)

The method of claim 14 further comprising fermenting the population of microorganisms.

### Claim 16 (depends on 15)

The method of claim 15, wherein the protective agent is added to the medium prior to or at the beginning of the fermenting step.

### Claim 17 (depends on 15, 16)

The method of claim 15 or 16, wherein the protective agent is added to the medium between the early-log growth phase and the late-log growth phase of the fermenting step.

### Claim 18 (depends on 15, 16, 17)

The method of any one of claims 15 to 17, wherein the protective agent is added to the medium before the stationary phase of the fermenting step.

### Claim 19 (depends on 15, 16, 17, 18)

The method of any one of claims 15 to 18, wherein the protective agent is added to the medium at the stationary phase of the fermenting step.

### Claim 20 (depends on 15, 16, 17, 18, 19)

The method of any one of claims 15 to 19, further comprising, after the fermenting step, adding the protective agent to the population of microorganisms.

### Claim 21 (depends on 20)

The method of claim 20 further comprising storing the population of microorganisms comprising the protective agent.

### Claim 22 (depends on 15, 16, 17, 18, 19, 20, 21)

The method of any one of claims 15 to 21, wherein the yield of the population of microorganism is increased during fermentation by 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300% when compared to a corresponding population of microorganisms fermented in the absence of the protective agent.

### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The method of any one of claims 1 to 22, wherein the protective agent comprises particles having a size less than 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 microns.

### Claim 24 (depends on 23)

The method of claim 23, wherein the size of the particles is less than 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)

The method of any one of claims 1 to 24, wherein the population of microorganisms comprise bacterial or fungal cells.

### Claim 26 (depends on 25)

The method of claim 25, wherein the bacterial or fungal cells are from the genera Achromobacter, Actimomycetes, Agrobacterium, Arthrobacter, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Chromobacterium, Cyanobacteria, Delftia, Enterobacter, Herbaspirillum, Klebsiella, Lactobacillus, Lactococcus, Lysobacter, Methylobacterium, Mitsuaria, Paenibacillus, Pasteuria, Pseudomonas, Rhizobium, Serratia, Sinorhizobium, Streptococcus, Streptomyces, Beauveria, Metarhizium, Isaria, Penicillium, Trichoderma, Chaetomium, Piriformospora, Phlebiopsis or Clonostachys.

### Claim 27 (depends on 25)

The method of claim 25, wherein the bacterial or fungal cells are from the genera Azospirillum, Bradyrhizobium, Delftia, Herbaspirillum, Mesorhizobium, Rhizobium, Sinorhizobium, Piriformospora or Streptomyces.

### Claim 28 (depends on 25)

The method of claim 25, wherein the bacterial or fungal cells comprise Bradyrhizobium elkanii, Bradyrhizobium diazoefficiens, Delftia acidovorans, Bradyrhizobium japonicum, Rhizobium leguminosarum, Rhizobium tropici, Mesorhizobium loti, Azospirillum brazilense, Herbaspirillum huttiense, Streptomyces griseoviridis, Piriformospora indica, or Sinorhizobium meliloti.

### Claim 29 (depends on 25)

The method of claim 25, wherein the bacterial cells comprise Bradyrhizobium elkanii, Delftia acidovorans, Herbaspirillum huttiense, Rhizobium leguminosarum or Azospirillum brazilense.

### Claim 30

A microbial composition comprising (i) a population of microorganisms and (ii) a protective agent comprising biochar particles, activated carbon, and/or charcoal.

### Claim 31 (depends on 30)

The microbial composition of claim 30, wherein the protective agent comprises or consists essentially of biochar particles.

### Claim 32 (depends on 30)

The microbial composition of claim 30, wherein the protective agent comprises or consists essentially of activated carbon.

### Claim 33 (depends on 30, 31, 32)

The microbial composition of any one of claims 30 to 32 further comprising (iii) a medium.

### Claim 34 (depends on 33)

The microbial composition of claim 33, wherein the medium is a liquid medium or a solid substrate.

### Claim 35 (depends on 34)

The microbial composition of claim 34 being a liquid inoculant.

### Claim 36 (depends on 33)

The microbial composition of claim 33, wherein the medium is a solid substrate.

### Claim 37 (depends on 36)

The microbial composition of claim 36 being a microbial inoculant immobilized in the solid substrate.

### Claim 38 (depends on 33, 34, 35, 36, 37)

The microbial composition of any one of claims 33 to 37, wherein the medium is capable of supporting the growth of the population of microorganisms.

### Claim 39 (depends on 30, 31, 32, 33, 34, 35, 36, 37, 38)

The microbial composition of any one of claims 30 to 38, wherein the protective agent is present at a concentration of 0.01% to 50%, 0.05% to 20%, 0.1% to 15%, 0.1% to 10% or 0.1% to 5% weight/volume of the microbial composition.

### Claim 40 (depends on 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)

The microbial composition of any one of claims 30 to 39, wherein the protective agent comprises particles having a size less than 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10.

### Claim 41 (depends on 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)

The microbial composition of any one of claims 30 to 40, wherein the size of the particles is less than 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10.

### Claim 42 (depends on 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)

The microbial composition of any one of claims 30 to 41, wherein the population of microorganisms comprise bacterial or fungal cells.

### Claim 43 (depends on 42)

The microbial composition of claim 42, wherein the bacterial or fungal cells are from the genera Achromobacter, Actimomycetes, Agrobacterium, Arthrobacter, Azospirillum, Azotobacter, Bacillus, Bifidobacterium, Bradyrhizobium, Chromobacterium, Cyanobacteria, Delftia, Enterobacter, Herbaspirillum, Klebsiella, Lactobacillus, Lactococcus, Lysobacter, Methylobacterium, Mitsuaria, Paenibacillus, Pasteuria, Pseudomonas, Rhizobium, Serratia, Sinorhizobium, Streptococcus, Streptomyces, Beauveria, Metarhizium, Isaria, Penicillium, Trichoderma, Chaetomium, Piriformospora, Phlebiopsis or Clonostachys.

### Claim 44 (depends on 42)

The microbial composition of claim 42, wherein the bacterial or fungal cells are from the genera Azospirillum, Bradyrhizobium, Delftia, Herbaspirillum, Mesorhizobium, Rhizobium, Sinorhizobium, Piriformospora or Streptomyces.

### Claim 45 (depends on 42)

The microbial composition of claim 42, wherein the bacterial or fungal cells comprise Bradyrhizobium elkanii, Bradyrhizobium diazoefficiens, Delftia acidovorans, Bradyrhizobium japonicum, Rhizobium leguminosarum, Rhizobium tropici, Mesorhizobium loti, Azospirillum brazilense, Herbaspirillum huttiense, Streptomyces griseoviridis, Piriformospora indica or Sinorhizobium meliloti.

### Claim 46 (depends on 42)

The microbial composition of claim 42, wherein the bacterial cells comprise Bradyrhizobium elkanii, Delftia acidovorans, Herbaspirillum huttiense, Rhizobium leguminosarum or Azospirillum brazilense.