# US 12,364,267 B2 · Whole cell methanotroph based biostimulant compositions, methods and applications thereof

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- Number: US 12,364,267 B2
- Document ID: US12364267B2
- Status: Granted
- Kind: B2
- Application number: 17/928,096
- Filed: 2021-05-28
- Published: 2023-07-13
- Granted: 2025-07-22
- Earliest claimed: 2020-05-28
- Assignees: String Bio Private Limited
- Applicants: String Bio Private Limited
- Inventors: Rajeev Kumar Sarma; Uday Kashinath Avalakki; Ravindra Babu Bondalakunta; Prashanth Muralidhar Udagatti; Vinod Munisanjeevaiah Lakshmi Kumar; Ezhilkani Subbian; Pavithra GJ
- Web page: https://patentgrep.com/patent/US12364267B2

## CPC

- A01N 63/20: Bacteria; Substances produced thereby or obtained therefrom
- A01P 21/00: Plant growth regulators
- C05F 11/08: Organic fertilisers containing added bacterial cultures, mycelia or the like
- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/205: Bacterial isolates
- C12R 2001/01: Bacteria or Actinomycetales {; using bacteria or Actinomycetales}
- Y02E 50/30: Fuel from waste, e.g. synthetic alcohol or diesel
- Y02W 30/40: Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
- 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
- A01P: BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- C05F: ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02W: CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT

## Abstract

Whole cell based biostimulant compositions and methods for improving agricultural productivity the compositions including: a microbial consortium having gammaproteobacterial methanotroph. As a result of these methanotrophs, the biostimulant composition enables plant performance improvement, utilization of methane, and facilitates improved nitrogen fixation in plants. The composition also helps in reducing the need of external chemical fertilizers for plant growth, development, performance and/or survival.

## Claims

### Claim 1

A biostimulant composition comprising a microbial consortium of whole cells, wherein the consortium comprises at least 50% whole cells of gammaproteobacterial methanotroph, wherein the composition:
a. improves or enhances performance of a plant,
b. increases availability or efficient utilization of at least one of nitrogen, phosphorus and potassium by the plant,
c. reduces need for external addition of at least one nutrient selected from nitrogen, phosphorus and potassium, either individually or as part of a fertilizer for the plant, or
d. any combination thereof compared to a plant where the composition has not been applied.

### Claim 2 (depends on 1)

The biostimulant composition as claimed in claim 1, wherein the microbial consortium comprises at least about 60% to about 100% whole cells of gammaproteobacterial methanotroph.

### Claim 3 (depends on 1)

The biostimulant composition as claimed in claim 1, wherein the gammaproteobacterial methanotroph is a type I or type X methanotroph belonging to genus selected from a group comprising Methylococcus, Methylomonas, Methylobacter, Methyloglobulus, Methylovulum, Methylomicrobium, Methylosarcina, Methylosphaera, Methyloprofundus, Methylosoma, Methylocucumis, Methylocaldum, Methyloparacoccus, Methylogaea, Methylomagnum, Methyloterricola, Methylothermus, Methylohalobius, Methylomarinovum, Methylomarinum and Crenothrix, or any combination thereof.

### Claim 4 (depends on 1)

The biostimulant composition as claimed in claim 1, wherein the gammaproteobacterial methanotroph is Methylococcus capsulatus.

### Claim 5 (depends on 1)

The biostimulant composition as claimed in claim 1, wherein the composition is in a solid form or a liquid form, and comprises at least one metabolite, at least one media derived nutrient and optionally at least one agriculturally acceptable excipient.

### Claim 6 (depends on 5)

The biostimulant composition as claimed in claim 5, wherein the microbial consortium of whole cells comprises about 1×103 cells to about 5×1010 cells per gram or per milliliter of the composition and comprises about 0.1% to about 80% of the composition; with the remainder of the composition comprising about 0.1% to about 10% of at least one metabolite, about 0.1% to about 10% of at least one media derived nutrient and optionally about 0.01% to about 90% of at least one agriculturally acceptable excipient.

### Claim 7 (depends on 5)

The biostimulant composition as claimed in claim 5, wherein the metabolite is selected from a group comprising carbohydrates, lipids, sugars, fatty acids, proteins, peptides, amino acids, nucleic acid, nucleotides, vitamins, organic acids, salts, minerals, osmolytes, extracellular enzymes, bacterial derived components and minerals, or any combination thereof,
wherein the media derived nutrient is selected from a group comprising ions and salts, or a combination thereof, and
wherein the agriculturally acceptable excipient is selected from a group comprising carrier, cell protectant, adjuvant, surfactant, stabilizer, preservative, diluent, suspending agent, dispersing agent and cosolvent, or any combination thereof.

### Claim 8 (depends on 1)

The biostimulant composition as claimed in claim 1, wherein in addition to the gammaproteobacterial methanotroph, the consortium comprises about 1% to about 50% of at least one plant growth-promoting microbe selected from a group comprising nitrogen fixing microorganism, phosphate solubilizing microorganism, mineral solubilizing microorganism, phytohormone secreting microorganism, organic acids secreting bacteria and plant beneficial microbe, or any combination thereof.

### Claim 9 (depends on 1)

A method of:
a) improving or enhancing plant performance, or
b) facilitating simultaneous utilization of methane and nitrogen fixation in a plant, or
c) reducing need of external addition of at least one nutrient or nutrient carrying fertilizer for growth, development, performance or survival of a plant, wherein the nutrient is selected from the group consisting of nitrogen, phosphorus and potassium, or any combination thereof:
the method comprising step of contacting or applying the biostimulant composition of claim 1, to the plant.

### Claim 10 (depends on 9)

The method as claimed in claim 9, wherein the improving or enhancing plant performance comprises stimulating or promoting a quantitative or qualitative plant attribute selected from a group comprising biomass production, yield, photosynthetic activity, nutritional value, secondary metabolites and nutrient use efficiency, or any combination thereof; or
wherein effect of the improved or enhanced plant performance is measured by:
a. increase in number, size or quality of below ground or aerial biomass selected from a group comprising root, shoot, leaf, flowers, anthers, stigma, stamens, fruits and seeds or any combination thereof,
b. increase in photosynthetic activity or chlorophyll content,
c. increase in protein, dietary fibre, β-carotene or essential oil content, or any combination thereof,
d. efficient absorption or utilization of available or externally provided nutrients or minerals, or
e. any combination thereof.

### Claim 11 (depends on 9)

The method as claimed in claim 9, wherein the plant performance is
improved or enhanced through increase in nitrogen fixation in plants resulting in at least one
of increased availability of nitrogen to the plant or efficient utilization of nitrogen by the plant; or
wherein the plant performance is improved or enhanced through increase in expression of genes of nitrogenase cluster selected from a group comprising nifA, nifD, nifH and nifK, or any combination thereof, in the microbial whole cells present in the biostimulant composition; or
wherein the plant performance is improved or enhanced simultaneously along with utilization of methane by the consortium comprising at least 50% whole cells of gammaproteobacterial methanotroph.

### Claim 12 (depends on 9)

The method as claimed in claim 9, wherein the biostimulant composition is in a solid form or a liquid form, and is contacted with or applied to the plant at a concentration ranging from about 1× to 100000× of the dilution of the solid or liquid form of the biostimulant composition; or
wherein the biostimulant composition is contacted with or applied to the plant through its soil, or through aerial or non-aerial parts of the plant selected from a group comprising root, shoot, leaf, flower, anther, stigma, stamen, fruit and seed, or any combination thereof.

### Claim 13 (depends on 9)

The method as claimed in claim 9, wherein the biostimulant composition improves or enhances the plant performance by about 1% to about 500% or by about 1.5 folds to about 10 folds when compared to a respective performance of a plant not contacted with the biostimulant composition comprising a microbial consortium of whole cells, wherein the consortium comprises at least 50% whole cells of gammaproteobacterial methanotroph.

### Claim 14 (depends on 9)

The method as claimed in claim 9, wherein the method increases nitrogen fixation in plants resulting in either increased availability of nitrogen to the plant or efficient utilization of nitrogen by the plant, or both; or
wherein the nitrogen fixation is facilitated by increase in expression of genes of nitrogenase cluster selected from a group comprising nifA, nifD, nifH and nifK, or any combination thereof, in the microbial whole cells present in the biostimulant composition.

### Claim 15 (depends on 9)

The method as claimed in claim 9, wherein the utilization of methane by the consortium comprising at least 50% whole cells of gammaproteobacterial methanotroph, recycles at least about 0.1 kg of methane per kg of biostimulant composition used.

### Claim 16 (depends on 9)

The method as claimed in claim 9, wherein the method reduces need for external addition of at least one of nitrogen, nitrogen carrying fertilizer, phosphorus, phosphorus carrying fertilizer, potassium, and potassium carrying fertilizer for growth, development, performance or survival of the plant, by at least about 10% to about 100%, when compared to the need for addition of nitrogen or nitrogen carrying fertilizer in a plant not contacted with the biostimulant composition comprising a microbial consortium of whole cells, wherein the consortium comprises at least 50% whole cells of gammaproteobacterial methanotroph.

### Claim 17 (depends on 9)

The method as claimed in claim 9, wherein the composition improves or enhances the plant performance by either increasing availability of or efficient utilization of at least one of nitrogen, phosphorus and potassium by the plant, or both.

### Claim 18 (depends on 1)

A process of preparing the biostimulant composition of claim 1, the process comprising combining the consortium comprising at least 50% whole cells of gammaproteobacterial methanotroph, with at least one of metabolite and media derived nutrient, optionally along with at least one agriculturally acceptable excipient.

### Claim 19 (depends on 1)

A biostimulant product comprising:
a. the biostimulant composition of claim 1; and
b. a hydrolysate based biostimulant composition comprising a protein-derived component in an amount of about 30% or less with respect to weight of the composition;
wherein the protein-derived component is obtained from a methanotrophic bacterium.