# US 10,368,548 B2 · Biological inoculant for promotion of growth in forest species and method for obtaining the same

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- Number: US 10,368,548 B2
- Document ID: US10368548B2
- Status: Granted
- Kind: B2
- Application number: 14/783,393
- Filed: 2014-04-10
- Published: 2016-03-10
- Granted: 2019-08-06
- Earliest claimed: 2013-04-11
- Assignees: Pontificia Universidad Javeriana
- Applicants: Pontificia Universidad Javeriana
- Inventors: Lucía Ana Diaz; Lina Marcela Morales Palencia; Juan Sebastian P. Beltran Acosta
- Web page: https://patentgrep.com/patent/US10368548B2

## CPC

- C05F 11/08: Organic fertilisers containing added bacterial cultures, mycelia or the like
- A01N 63/20: Bacteria; Substances produced thereby or obtained therefrom
- A01N 63/27: Pseudomonas
- C05B 17/00: Other phosphatic fertilisers, e.g. soft rock phosphates, bone meal
- C12N 1/20: Bacteria; Culture media therefor
- C05F: ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM 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
- C05B: PHOSPHATIC FERTILISERS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to the production and application of a biological inoculant for use in plant material sown in nurseries. The invention also relates to a method for the immobilization of bacterial strains on a support and application to plant materials. The invention further relates to the use of native teak tree rhizosphere bacteria as a means for promoting plant growth. The invention consists in developing a biological product based on applying rhizosphere bacteria to a carbonized teak sawdust support, which can be used to obtain plants with improved characteristics in less time.

## Claims

### Claim 1

An immobilizing process for obtaining a biological inoculant that stimulates plant growth characterized in that it comprises the steps of:
a) forming a support medium from teak wood sawdust at a size between 400-1000 μm subjected to a heat treatment such that the sawdust burns homogeneously, does not generate ash, possesses a bulk density of 0.1 g/l to 0.2 g/l, where its color is between the 7.5R2/4 and 10YR2/4 categories with particles present in 10Y1/2 color according to the Munsell color chart and having the following composition:
i) total organic carbon from 20% to 40%,
ii) total nitrogen between 0.1% and 0.5%,
iii) carbon/nitrogen >90,
iv) cation exchange capacity >20 meq/100 g,
v) calcium between 8 and 15 meq/100 g,
vi) magnesium 6 to 12 meq/100 g,
vii) potassium 4 to 8 meq/100 g,
viii) sodium 1 to 5 meq/100 g,
ix) available phosphorus between 400 and 750 ppm;
b) culturing one or more plant growth promoting strains of the negative Gram group of bacteria in a fermentation system until the end of their exponential or logarithmic phase, when they reach maximum cell biomass production and generate secondary metabolites that increase the solubilization of nutrients in the substrates for sowing plants and increase the plant growth, wherein the secondary metabolites are acids and compounds regulating plant growth, removing the supernatant and re-suspending it in 0.85% concentration saline to a final cell concentration of 1012 CFU/mL;
c) mixing each solution resulting from step b) with the teak sawdust support obtained in step a) in proportion of inoculum: support from 2:5 to 2:7; and
d) cultivating the mixture obtained in the step c) for a period of time between 24 and 72 hours at a temperature between 25° C. and 42° C. and optionally including excipients, to obtain the biological inoculant.

### Claim 2 (depends on 1)

An immobilizing process for obtaining a biological inoculant that stimulates plant growth according to claim 1, wherein the strains are Gram negative bacteria belonging to the genera Pseudomonas, Burkholderia, Stenotrophomonas, Rhizobium, Bradyrhizobium, Sinorhizobium, Azospirillum, Azotobacter, Klebsiella, Enterobacter or Sphingomonas.

### Claim 3 (depends on 1)

An immobilizing process for obtaining a biological inoculant that stimulates plant growth according to claim 1, wherein the bacterial strain is a rhizosphere strain selected from the group consisting of at least one of strain TgLGBR285 Stenotrophomonas sp. and strain TgLGBR250 Enterobacter sp.

### Claim 4 (depends on 1)

An immobilizing process for obtaining a biological inoculant that stimulates plant growth according to claim 1, wherein the temperature for growing the mixture is between 25° C. and 42° C.

### Claim 5 (depends on 1)

An immobilizing process for obtaining a biological inoculant that stimulates plant growth according to claim 1, wherein the bacteria solubilize phosphorus.

### Claim 6 (depends on 1)

An immobilizing process for obtaining a biological inoculant that stimulates plant growth according to claim 1, characterized in that the treated teak sawdust support has a pH between 6.8 and 7.8.

### Claim 7 (depends on 1)

An immobilizing process for obtaining a biological inoculant that stimulates plant growth according to claim 1, wherein the treated teak sawdust support has a cation exchange capacity between 20 and 30 meq/100 g.

### Claim 8 (depends on 1)

An immobilizing process for obtaining a biological inoculant that stimulates plant growth according to claim 1, characterized in that the treated teak sawdust support has a total count of heterotrophic, actinomycetes and fungi of less than 10 CFU/g.

### Claim 9 (depends on 1)

An immobilizing process for obtaining a biological inoculant that stimulates plant growth according to claim 1, wherein the sawdust possesses a bulk density of 0.167 g/l.

### Claim 10 (depends on 1)

A teak support characterized in that it is obtained according to the process in claim 1.

### Claim 11 (depends on 10)

A teak support according to claim 10 wherein the teak sawdust support has a pH between 6.8 and 7.8.

### Claim 12 (depends on 10)

A teak support according to claim 10 wherein the teak sawdust support has a cation exchange capacity between 20 and 30 meq/100 g.

### Claim 13 (depends on 10)

A teak support according to claim 10 wherein the treated teak sawdust support has total heterotrophic counts, actinomycetes and fungi less 10 CFU/g.

### Claim 14 (depends on 1)

A biological inoculant comprising a teak sawdust support that stimulates growth characterized in that it is obtained according to the process of claim 1.

### Claim 15 (depends on 14)

A biological inoculant according to claim 14 characterized in that it is used to decrease a plant's time in a nursery.

### Claim 16 (depends on 14)

A biological inoculant according to claim 14, wherein the biological strain is selected from the group consisting of at least one of strain TgLGBR285 Stenotrophomonas sp. and strain TgLGBR250 Enterobacter sp.

### Claim 17 (depends on 2)

A biological inoculant comprising a teak sawdust support that stimulates growth characterized in that it is obtained according to the process of claim 2.

### Claim 18 (depends on 3)

A biological inoculant comprising a teak sawdust support that stimulates growth characterized in that it is obtained according to the process of claim 3.

### Claim 19 (depends on 4)

A biological inoculant comprising a teak sawdust support that stimulates growth characterized in that it is obtained according to the process of claim 4.

### Claim 20 (depends on 5)

A biological inoculant comprising a teak sawdust support that stimulates growth characterized in that it is obtained according to the process of claim 5.