WO2011099024A1

WO2011099024A1 - Preparation of novel bacterial based product for providing nuitrition essential for promoting plant growth

Info

Publication number: WO2011099024A1
Application number: PCT/IN2010/000182
Authority: WO
Inventors: Chetan S. Patel
Original assignee: Patel, Babubhai C.
Priority date: 2010-02-09
Filing date: 2010-03-24
Publication date: 2011-08-18

Abstract

This invention is based upon composition and method of preparing an innovative bio fertilizer prepared from pure culture of bacteria namely Bacillus polymexa or Bacillus subtilis or Pseudomonas putida or Pseudomonas Fluorescens for providing rich nutrients to plant through soil and thereby promoting plant growth.

Description

Preparation of Novel Bacterial Based Product For Providing Nuitrition

Essential For Promoting Plant Growth

PREAMBLE OF INVENTION- This invention is particular described the nature of the invention and the manner in which it is to be performed.

FIELD OF INVENTION- The present invention relates generally to a composition and method of making bacterial containing pure culture of bacteria namely Bacillus polymexa or Bacillus subtilis or Pseudomonas putida or Pseudomonas Fluorescens for promoting plant growth

PRIOR ART-

In the existing system as given in Indian Patent Application No. 0083/CAL/2000 - Granted Patent wherein the invention relates to a process for preparing a novel biocide comprising a consortium of two bacterial and two fungal strains for controlling a severe disease of tea plants caused by pathogenic fungal strains namely Corticium invisum and Corticium theae.. The preparation of the biocide is made by mining the four ingredients strains at the ratio of 4:3:2: 1 and the culture, are prepared in the following manners a) Transferring the Bacillus subtilis and Pseudomanas putida cultures into mother culture scandium, b) allowing the another cultures to grow for 3— 7 day., c) transferring the grown mother cultures to main culture m4dium and allowing the main cultures to grow i

for 3— 7 days, d) transferring the Tnichoderma viridi and Trichoderma harzianum cultures in the mother culture medium, e) allowing the mother cultures to grow for 15 days, (f) transferring the mother cultures 4~ to the main culture, medium and allowing the main cultures to grow for 15 days, g) mixing the said ready cultures— Bacillus subtilis, Pseudomonas putida, Trichoderma viridi and Trichoderma harzianum in a ratio of 4:3 :2: 1 to obtain the biocide.

In the existing system as given in Indian Patent Application No. 85/CAL/2000- Granted Patent wherein the invention relates to a process for preparing a novel biofertiliser strains of nitrogen fixer and phosphate solubiliser to promote soil health in particular for growth of tea plants comprising of nitrogen fixer and phosphate solubiliser in equal proportion wherein the nitrogen fixer is prepared by the steps of a) transferring the stock culture Azotobacter Chroococcum (BAH) into mother culture modified Ashley's medium, b) allowing the mother culture medium to grow for 3-7 days, c) transferring the grown mother culture to main culture medium and d) allowing the main culture medium to grow ge for another 3-7 days to obtain Azotobacter choococcum (BAH) strains and in which the phosphate solubiliser is prepared from as a mixture of Bacillus subtilis (BB15), Pseudomonas Putida (BP 19) Aspergillus awamori (FA 16) and Aspergitlus flavus (FA18) in a ratio of 5:2:2:1 in Pikovskaya's medium wherein Bacillus Subtilis (BB15) and Pseudomonas putida (BP 19) are prepared as in a manner of preparing Azotobacter Choococcum and Aspergillus awamori (FA 16) and Aspergillus flavus (FA 18) (molasses or yeast medium) are prepared by transferring the stock culture to mother culture medium, allowing the mother culture to grow for 15 days, transferring the grown mother culture to main culture medium and allowing it to grow again for another 15 days to obtain Aspergillus awamori (FA 16) and Aspergillus flavus (FA 18) strains. In the existing system as given in Indian Patent Application No. 781/CHENP/2008 - Patent Application, wherein the invention pertains to improved microorganisms and methods for the production of methionine and other sulfur containind the chemicals using the metL geneCO from Bacillus subtilis or a gene related to metL ^' In some embodiments of the present invention, the metl gene or another! gene is integrated in a

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fashion that allows for co-production of a water soluble compound such as methionine or other amino acid and a caortenoid compound.

In the existing system as given in Indian Patent Application No. 3456/DEL/2005 -Patent Application, wherein the invention pertains to the composition B5 prepared from a

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bacterium namely B. subtilis strain B5 has the ability to provide plant growth promotion and control of soil/seed borne pathogens of potato, tegetables and ornamental crop plants. It releases an antibiotic metabolite which is fungicidal, supports immune system, biosynthesizes siderophores, tRNA binder, stimulatory, and decreases toxicity. Apart from fungicidal and bactericidal metabolite, the other metabolite, the other metabolite produced is a flavonoid, which is capable of removing dead cells, acts as a betoxifying agent, keeps cells healthy, increases metabolism and supports immunity. It has a killing effect against pathogenic species of Fusarium, pythium phytophthora Rhizoctonia, Solanacearum, the Alternaria in the form of cell lysis or antibiosis. Inhibits R. solanacearum, the cause of bacterial wilt of potato against whisch no chemical or a resistant variety is effective till date, by antibiosis, competitive inhibition and siderophore production. The effects of mechanisms involved to the reduction of diseases and the enhancement of crop yield reveal the fact that all the effects of interactions i

namely, cell lysis, antibiosis, competition and disease escape, are most obvious in case of attacks by siol-borne pathogens because they are able to operate in the rhizosphere area of the plant. The composition B5 is preparaed in an inexpensive substrate which allows maximum production of viable cells in shortest time with a shelf life of one year at room temperatures (15-37dgC)and gives best results when applied on seed or into soil, safe for persons handing it and non target pathogens with no side, effects on biodiversity.

In the existing system as given in Indian Patent Application! No. 163/MAS/2002 - Granted Patent wherein the invention relates to a method for preparation of a bioformulation comprising selecting the effective bio-control agents from Paecilomyces lalacinus Bainier, Verticillium chlamydosporium Goddard, Trichoderma harzianum Rifai (TH-1) and TH-2, T. viride Pers. Ex. S. F. Grey, Laetisaria arvalis Burdsall, Gliocladium virens Miller, Giddens & Foster and Bacillus subtilis, preparing a medium by mixing thoroughly sourghum flour, river sand and distilled water, sterilizing the medium in an autoclave, shaking the medium to avoid clogging of the substrates, cooling the medium, inoculating the medium with said effective biocontrol agents in a flask, incubating said flask for few days, collecting the biomass of biocontrol agent alongwith media, mixing the said biomass of biocontrol agent and media with a carrier, drying blending the said mixtures air drying the contents and packing them in polythene bags.

In the existing system as given in Indian Patent Application No. 1019/DEL/2007 -Patent Application, wherein the invention relates to a bacterial strain ^of the Bacillus subtilis group, wherein one or more genes involved in the biosynthesis f isovaleric acids have been modified such that the bacterium cannot produce substantial amounts of isovaleric acids.

OBJECT OF THE INVENTION Traditionally, the known bacterial or fungal product for promoting plant growth has different method of its preparation and composition. The present invention is based upon composition and method of making bacterial based product containing pure culture of bacteria namely Bacillus polymexa or Bacillus subtilis or Pseudomonas putida or Pseudomonas Fluorescens for promoting plant growth.

STATEMENT OF INVENTION- The inventor has invented a composition and method of making a novel nutritional and plant growth bacterial product.

DETAILED DESCRIPTION OF INVENTION-

Background of Invention: This invention relates to a method for utilizing bacterial based product containing pure culture of bacteria namely Bacillus polymexa or Bacillus subtilis or Pseudomonas putida or Pseudomonas Fluorescens for providing nutrients and thereby promoting plant growth.

Nutrition is the provision, to cells and organisms, of the materials necessary (in the form of food) to support plant life. A nutrient that is able to limit plant growth is considered an essential plant nutrient if the plant can not complete its full cycle without it. There are

16 essential plant nutrients.

Macronutrients

N = Nitrogen

P = Phosphorous

= Potassium

Ca = Calcium

Mg = Magnesium

S = Sulfur

Si = Silicon Micronutrients (trace level) include:

CI = Chlorine

Fe - Iron

B = Boron

Mn = Manganese

Na = Sodium

Zn - Zinc

Cu = Copper

Ni = Nickel

Mo = Molybdenum

These nutrients are further divided in to the mobile and immobile nutrients. A plant will always supply more nutrients to its younger leaves than its older Ones, so when nutrients are mobile, the lack of nutrients is first visible on older leaves. When nutrient is less mobile, the younger leaves suffer because the nutrient does not move up to them but stays lower in the older leaves. Nitrogen, phosphorus and potassium are mobile nutrients, while the others have varying degrees of mobility.

Plants uptake essential elements from the soil through their roots and from the air (mainly consisting of nitrogen and oxygen) through their leaves.

The present invention is an innovative bio fertilizer prepared from pure culture of bacteria namely Bacillus polymexa or Bacillus subtilis or Pseudomonas putida or Pseudomonas Fluorescens for providing nutrients and thereby promoting plant growth. Material and Method used in preparing the above said innovative nutrient supplier is as under

Step-1 Nucleus culture (Maintain culture)

Pure culture of bacteria {Bacillus polymexa or Bacillus subtilis or Pseudomonas putida or Pseudomonas Fluorescens) is inoculated aseptically on plate; having 20 ml PAC-07 Agar media; such two to three plates are generally inoculated. Such inoculated plate is maintained in BOD incubator at 27 ± 1 °C for 6 to 7 days with 12/12 hr lighting cycle. (Composition for one liter of PAC-07 agar media is 10 gm Beet extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water)

Step-2 Starter culture (In 100 ml flask)

Culture grown on plate is inoculated aseptically in a flask (250 ml capacity) having 100 ml PAC-07 broth media with the help of inoculating loop, and allow to grow at 27 ± 1 °C for at least 3 to 4 days. For superior growth, flask is put on shaker with agitation of media at 150 RPM. Composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water)

Step-3 Growth culture (In 1000 ml flask)

After sufficient growth in 250 ml flask (step-2), 50 ml culture from this grown culture is inoculated aseptically in 500 ml PAC-07 broth media in a 1000 ml capacity flask for further growth at 27 ± 1 °C for at least 3 to 4 days on shaker with 150 RPM, such two flasks is prepared. (Composition for one liter of PAC-07 broth ;media is 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 nil distilled water) Step-4 Seed Fermentor (In small fermentor of 10 lit capacities)

One (1) lit grown culture in step-3 is inoculated aseptically in small fermenter having 10 litter PAC-07 broth media, and allow for further growth at 27 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM. (Composition for one liter of PAC-07 broth media is 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water)

Step-5 Production Fermentor (In Big fermentor of 200 lit capacities)

Ten (10) lit culture grown in step-4 is inoculated aseptically in Big fermenter having 200 litter PAC-07 media (Composition for one liter of PAC-07 broth media is 10 gm Beef extract, 10 gm Peptone, 5 gm NaCi, 20 gm Agar agar and 1000 ml distilled water), and allow for further growth at 27 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM

Step-6

Final out put from big fermentor mix with starch @ 4 to 5 % which act as a stabilizer

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and melto dextrin @ 1 to 2 % which act as a drying agent or with 5 % skim milk powder and agitate continuously by using agitator.

Step-7

Mix culture prepared as per step-6, is than Spray dry at definite -160 °C in let and 70 to 72 °C out let temp in spray dryer unit Step-7 Collecting dry bacterial powder

Material dry in spray dryer unit is than collected in clean stainlesjs steal vassals and store at 5 °C up to further use

Step-8 Mix with suitable carrier

At the time of formulation material previously dry is mix with dextrose @ 10 % ( 10 gm bacterial powder with 90 gm dextrose powder)

Step-9 Packing

Material prepared as above is than pack in 100 gm, 250 gm and 500 gm packing by using pouch packing machine

Step-10 Distribution to farmer, retailer, distributor, etc

The final material prepared has to be used in following form:

Dose in one Acre: In case of Seed treatment add 100 gm per seed required per hector For soil application add 100 gm to 250 gm per hector

Claims

CLAIMS I claim,

1. A composition and method of preparing innovative innovative bio fertilizer prepared from pure culture of bacteria namely Bacillus polymexa or Bacillus subtilis or Pseudomonas putida or Pseudomonas Fluorescens for providing rich nutrients to plant through soil and thereby promoting plant growth, the method comprising steps of nucleus culture, starter culture, growth culture, seed fermentor, production fermentor, mix culture, drying, collecting, mixing wth dextrose and packing the final output.

2. A composition and method of preparing bacterial based product according to claim 1, by inoculating aseptically pure culture of bacteria (Bacillus polymexa or Bacillus subtilis or Pseudomonas putida or Pseudomonas Fluorescens) on two to three plates having 20 ml PAC-07 Agar media and maintaining in BOD incubator at 27 ± 1 °C for 6 to 7 days with 12/12 hr lighting cycle, wherein composition for one liter of PAC-07 agar media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water.

3. A composition and method of preparing bacterial based product according to . claim 1, by inoculating aseptically, culture grown on plate in a flask (250 ml capacity) having 100 ml PAC-07 broth media with the help of inoculating loop, and allowing it to grow at 27 ± 1 °C for at least 3 to 4 days, wherein for superior growth, flask is put on shaker with agitation of media at 150 RPM, wherein composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water.

4. A composition and method of preparing bacterial based product according to claim 1, wherein after sufficient growth in 250 ml flask as per method cited in clam 3, 50 ml culture from this grown culture is inoculated aseptically in 500 ml PAC-07 broth media in a 1000 ml capacity flask for further growth at 27 ± 1 °C for at least 3 to 4 days on shaker with 150 RPM, such two flasks is prepared, wherein composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water.

5. A composition and method of preparing bacterial based product according to claim 1 , wherein, One (1) lit grown culture as per claim 4 is inoculated aseptically in small feanenter having 10 litter PAC-07 broth media, and allowed for further growth at 27 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM,

. wherein, composition for one liter of PAC-07 broth media is 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water.

6. A composition and method of . preparing bacterial based product according to claim 1 , wherein, ten (10) lit culture grown as per method cited in claim 4 is inoculated aseptically in big fermenter having 200 litter PAC-07 media, wherein composition for one liter of PAC-07 broth media contains 10 gm Beef extract, 10 gm Peptone, 5 gm NaCl, 20 gm Agar agar and 1000 ml distilled water, and allowed for further growth at 27 ± 1 °C for at least 3 to 4 days with agitation by motor at 100 RPM.

7. A composition and method of preparing bacterial based product according to claim 1, wherein, final out put from big fermentor is mixed with starch @ 4 to 5 % which act as a stabilizer and melto dextrin @ 1 to 2 % which act as a drying agent or with 5 % skim milk powder and agitate continuously by using agitator.

8. A composition and method of preparing bacterial based product according to claim 1, wherein, mix culture prepared as per method cited in claim 7, is then sprayed dry at definite 160 °C in let and 70 to 72 °C out let temp in spray dryer unit

9. A composition and method of preparing bacterial based product according to claim 1, wherein, material dried in spray dryer unit is then collected in clean stainless steel vessels and stored at 5 °C up to further use

10. A composition and method of preparing bacterial based product according to claim 1 , wherein, at the time of formulation, material previously dried is mixed with dextrose @ 10 % ( 10 gm bacterial powder with 90 gm dextrose powder) and the final output thus obtained is packed in 100 gm, 250 gm and 500 gm by using pouch packing machine as nutrient supplier or plant growth promoter bio fertilizer.