Method for formulating a biofertilizer and biopesticide

Abstract

A composition based on three strains of microorganisms to promote plant growth and serve as a plant protection product to protect plants from damaging influences including plant disease, pests or insects. The composition is comprised of isolated strains of Paenibacillus polymyxa M.sub.10, Azospirillum canadense B.sub.2, and Bacillus pumilus L.sub.13. A mineral mix is used to maintain the microorganism viable for a long time, maintaining high efficacy as a biopesticide and biofertilizer. The mineral mix combined with the fermented microorganisms is then further combined with a nutritional additive to enhance the biofertilizing and biopesticidal features of the composition. The resulting composition can be applied to plants in liquid form or to the soil in solid form.

Claims

1. A composition for a bio-pesticide and biofertilizer consisting essentially of equal parts of: a) An isolated strain of Paenibacillus polymyxa L.sub.13; b) An isolated strain of Azospirillum canadense B.sub.2; c) An isolated strain of Bacillus pumilus M.sub.10.

2. A method for formulating a dry composition form for applying to soil where plants will be grown and for applying this composition, the method comprising the steps of: a) isolating a strain of Paenibacillus polymyxa L.sub.13, isolating a strain of Azospirillum canadense B.sub.2, isolating a strain of Bacillus pumilus M.sub.13; b) growing each strain in a fermentor to make a culture and then combining the three cultures; c) impregnating a mineral mix with a nutritional solution to create a wet mixture; d) impregnating said wet mixture with said fermented microorganisms; e) drying the resulting composition; and f) applying said resulting composition to the soil where plants will be grown.

3. The method of claim 2 wherein said mineral mix includes feldspar, phosphate rock, clay, and leonardite.

4. The method of claim 2 wherein said nutritional solution includes a protein hydrolyzer, soy flour, yeast extract, sodium chloride, and water.

5. The method of claim 4 wherein said nutritional solution includes 5 g of the protein hydrolyzer, 5 g of soy flour, 1 g of yeast extract, and 2 g of sodium chloride, per liter of water.

6. The method of claim 2 wherein said composition is dried at no more than 45 degrees Celsius until the moisture level is no higher than 10%.

7. The method of claim 2 wherein 50 mL of culture of each strain are used for one kilogram of said mineral mix.

8. A method for applying the composition of claim 1 in liquid form to seeds or plants comprising the steps of: a) isolating a strain of Paenibacillus polymyxa L.sub.13, isolating a strain of Azospirillum canadense B.sub.2, isolating a strain of Bacillus pumilus M.sub.13; b) growing each strain in a fermentor to make a culture and then combining the three cultures; c) mixing said combination of fermented microorganisms with sodium metabisulfite and polyvinyl alcohol to create a homogenous mix; and d) applying said composition in liquid form to seeds or directly to said plants.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) With the above and other related objects in view, the invention consists in the details of construction and combination of parts as will be more fully understood from the following description, when read in conjunction with the accompanying drawings in which:

(2) FIG. 1 represents a flow chart showing the steps involved in the fermentation process of each microorganism used in the composition.

(3) FIG. 2 represents a flow chart illustrating the steps involved in the method used to produce an embodiment of the present invention in solid form.

(4) FIG. 3 shows a flow chart illustrating the steps involved in the method used to produce an embodiment of the present invention in liquid form.

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION

(5) The present invention includes a composition comprising of three strains of microorganisms, namely, Bacillus pumilus strain M.sub.10, Azospirillum canadense strain B.sub.2, and Paenibacillus polymyxa strain L.sub.13.

(6) The composition acts as a biofertilizer that is a solubilizer of phosphates and potassium. In addition, as a biofertilizer the composition is capable of nitrogen fixing, produces phytohormones, namely cytokinins and Gibberellin. It also produces siderophores and reduces and can potentially eliminate the need for chemical-based fertilizers. The phytohormones are used to stimulate the growth of vegetables and their roots.

(7) As seen in FIG. 1, fermentation process 20 undergoes several steps. The fermentation process for each strain beings with conserving each strain in glycerol at −20° C. until needed as shown in step 22. The fermentation process then undergoes several stages of inoculation to increase the cellular count of each strain in the composition. The first step in the inoculation is to activate the three strains. This is done in a Petri dish with an Agar solid medium and is incubated at 30 degrees Celsius for 72 hours as shown in step 24. The second step in the inoculation process is referred to as pre-inoculation, as shown in step 26, and is generally done in a laboratory. A working volume of the three strains is combined in sufficient nutritional solution, M9, so as to fill approximately 60-90% of a bioreactor vessel. The strains remain in the nutritional solution within the bioreactor vessel for 24 hours. The nutritional solution, M9, includes (quantities are per liter of water): 5 g of protein hydrolyzer, 5 g of soy flour, 1 g of yeasts extract, and 2 g of sodium chloride. Pre-inoculation step 26 is used to further reproduce the cellular count of each strain. The resulting product from pre-inoculation step 26 is used to inoculate an even larger bioreactor vessel that can further reproduce the cellular count of each strain, as shown in step 27. Step 27 is repeated using the same proportion and time requirements described above until the desired cellular count for each strain is achieved. In a preferred embodiment, step 27 is performed until the cellular count for each strain is at least 10.sup.9 UFC×mL.sup.−1 for each strain. The bioreactor vessel that ends up with the desired cellular count is known as the industrial fermenter as shown in step 28. The fermentation broth with the desired cellular count obtained in step 28 is then kept refrigerated at 2-6 degrees Celsius until formulation into solid or liquid form as shown in step 29. The cultures should not be maintained for longer than 30 days. The same process is repeated for the microorganism strain, Paenibacillus polymyxa L.sub.13 and Azospirillum canadense B.sub.2. The Azospirillum canadense B.sub.2 strain ends up slightly less concentrated than the previous two strains and also ends up with a slightly elevated pH.

(8) The composition can be formulated in liquid or solid form, however, in both cases the composition is made up of each microorganism in equal parts. As seen in FIG. 2, to produce the composition in solid form 40 each microorganism undergoes fermentation process 20 so that each microorganism can reproduce. A mineral mix, as shown in step 42, is used when formulating the composition in 25 solid form to maintain the microorganisms viable, stimulate reproduction and promote a slow release of the composition. The mineral mix, which has a slow release function, includes in a preferred embodiment, 5% leornadite, 2% feldspar, 2% phosphate rock, and 91% clay by weight.

(9) The mineral mix provides a minimum of basic nutrients to plants and crops. It also has a slow release function and preserves the viability of the microorganisms during the time between manufacturing and application. The purpose of the leornadite as organic matter is to provide nutrition to plants. The purpose of the feldspar is provide a source of potassium in case the soil does not have sufficient potassium to allow plant growth. The purpose of the phosphate rock is to provide a source of phosphate in case the soil does not have sufficient phosphate to stimulate plant growth. The purpose of the clay is to release cells slowly. Once the cells are released they begin to solubilize potassium from the feldspar and phosphate from the phosphate rock. In addition to potassium and phosphate, plants require nitrogen, which is fixed in the composition using the three microorganisms that make up the composition. The granular size of each mineral is preferably not to exceed approximately 1.5 mm. However, is has been found that a range for this diameter could be from 0.5 mm to 2.5 mm.

(10) The minerals are all mixed to create a homogenous mix that is used as a carrier for the absorption of the microorganisms. As seen in step 44 the homogenous mix is then impregnated with a nutritional solution, also referred to as M9, in the proportion of 150 mL of nutritional solution, M9, per 1 kg of mineral mix, also known as a carrier, to create a wet mixture. The wet mixture previously obtained is then impregnated with 50 mL of the fermentation broth of each strain, as seen in step 46. The resulting product is then dried at no more than 45 degrees Celsius until the moisture level is no higher than 10%, as shown in step 47. The resulting product is then packaged and stored as seen in step 48 and applied to the soil where the plants are grown as shown in step 49. Finally, the resulting composition in solid form is applied to the soil where the plants are grown.

(11) As seen in FIG. 3, the method to apply the composition in liquid form 60 consists of undergoing the three fermentations done using fermentation process 20, one for each microorganism so that each microorganism can reproduce. The fermentation process is the same for liquid form as it is for solid form. The fermented broth with microorganisms is then homogenously mixed, as shown in step 62, with polyvinyl alcohol as shown in step 64, and sodium metabisulfite, as shown in step 66, to create a homogenous mix using the following proportions, in a preferred embodiment:

(12) a) 315 mL of Paenibacillus polymyxa L.sub.13 culture;

(13) b) 315 mL of Azospirillum canadense B.sub.2 culture;

(14) c) 315 mL of Bacillus pumilus M.sub.10 culture;

(15) d) 50 mL of polyvinyl alcohol; and

(16) e) 0.15 g of sodium metabisulfite

(17) The final product in liquid form is then bottled and stored as shown in step 68 and added to seeds or the soil where plants are grown, as shown in step 69.

(18) 1. The Name and Address of the Depository:

(19) NRRL. AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION

(20) National Center for Agricultural Utilization Research

(21) Agricultural Research Service, U.S. Department of Agriculture

(22) 1815 North University Street, Peoria, Ill. 61604 U.S.A.

(23) 2. The Accession Number Given to the Deposit:

(24) Paenibacillus polymyxa L.sub.13 accession number B-50890

(25) Azospirillum canadense B.sub.2 accession number B-50891

(26) Bacillus pumilis M.sub.10 accession number B-50892

(27) 3. The Date of Deposit:

(28) Dec. 12, 2013

(29) The foregoing description conveys the best understanding of the objectives and advantages of the present invention. Different embodiments may be made of the inventive concept of this invention. It is to be understood that all matter disclosed herein is to be interpreted merely as illustrative, and not in a limiting sense.