WO2011099019A1

WO2011099019A1 - Composition and method of preparation of bacterial based product that fix atmospheric nitrogen from air and makes available to plant

Info

Publication number: WO2011099019A1
Application number: PCT/IN2010/000177
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 improved nitrogen fixing bacterial based product from pure culture bacteria like Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter xylinum or Azospirillum lipoferum that fixes the atmospheric nitrogen from air and makes it available to plants. It also increases efficiency of chemical fertilizer.

Description

Composition and Method of Preparation of Bacterial Based Product that fix atmospheric Nitrogen from air and makes available to plant.

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 a bacterial based product that fix atmospheric nitrogen from air and makes available to plant.

PRIOR ART-

In the existing system as given in United States Patent Application No. 5113619 Granted Patent wherein the composition for application to seed is disclosed, the composition including bacteria and adherent. The adherent is a biopolymer secreted naturally from bacteria and also acts as matrix for protecting bacteria applied to the seed. The invention is concerned particularly with a method and composition for adhering nitrogen-fixing bacteria to legume seed using a biopolymer secreted by the bacteria to be adhered.

In the existing system as given in Indian Patent Application No. 1096 MUM/2002 - Patent Application wherein the invention relates to a Bio-Power micronized compound from active principles of inactivated soil beneficial bacterial fortified with Blue green algae for improving soil condition to mobilize nutrients for absorption of plant.

[i] In the existing system as given in United States Patent Application No. 6939688 - Granted Patent wherein the invention relates to the biological addition to organic- mineral fertilizers contains two bacteria associations taken in the ratio 1-2:0.5-1, with one of the associations containing Azotobacter chroococcum and Beijerinckia fluminensis nitrogen binding bacteria in the ratio 1-0.5:1-0.5 and the other association containing Bacillus megaterium and Bacillus mucilaginosis bacteria, which decompose phosphorus- and potassium containing compounds, in the ratio 1-5:0.5-2, as well as a preservative and micro- and macroelements

In the existing system as given in United States Patent Application No. 20090266125- Patent Application where in invention provides, in some embodiments, a plant fertilizing composition and a method of producing the plant fertilizer. Other embodiments of the invention provide natural, organic, non-pathogenic enzymes for soil and water immobilization and microencapsulated bacterial enzymatic complexes. Various embodiments of the invention are particularly suited for use in agriculture to increase the health and yield of crop plants. In other embodiments, the composition may be utilized to reduce waterborne contamination.

In the existing system as given in Indian Patent Application No. 2108/DEL/2004 - Patent application wherein the invention relates to A plant growth stimulator useful for

i'

promoting and maintaining plant growth, wherein said stimulator is post-microbial culture inoculation nutrient medium consisting of black tea and a carbohydrate source, wherein the culture is consisting of Acetobacter xylinum, Candida sp. and Zygosaccharomyces rouxii, and a process for the preparation of the plant growth stimulator, and further, a method of promoting and maintaining the growth of plants using the plant stimulator of claim 1 having multiple disease resistance activity. In the existing system as given in United States Patent Application No. 7053025 - Granted Patent wherein the invention relates to plant growth stimulator useful for promoting and maintaining plant growth, wherein said stimulator is post-microbial culture inoculation nutrient medium consisting of black tea and a carbohydrate source, wherein the culture is consisting of Acetobacter xylinum, Candida sp. and Zygosaccharomyces rouxii, and a process for the preparation of the plant growth stimulator, and further, a method of promoting and maintaining the growth of plants using the plant stimulator having multiple disease resistance activity.

In the existing system as given in WIPO Patent Application No. PCT/US 1996/012480 - Patent Application wherein said A new microbial cellulose with high water content and process for making the new microbial cellulose utilizes a trough containing a biological medium and a cellulose producing microorganism. A plurality of disks are partly submerged in the medium and moved through the medium. Cellulose is formed on the surface of the disks which is later harvested.

In the existing system as given in United States Patent Application No. 2009031 1752- Patent Application where in the invention relates to methods for improving the yield of microbial processes that use lignocellulose biomass as a nutrient source. The methods comprise conditioning a composition comprising lignocellulose biomass with an enzyme composition that comprises a phenol oxidizing enzyme. The conditioned composition can support a higher rate of growth of microorganisms in a process. In one embodiment, a laccase composition is used to condition lignocellulose biomass derived from non- woody plants, such as corn and sugar cane. The invention also encompasses methods for culturing microorganisms that are sensitive to inhibitory compounds in lignocellulose biomass. The invention further provides methods of making a product by culturing the production microorganisms in conditioned lignocellulose biomass. In the existing system as given in United States Patent Application No. 5664368 - Patent Application where in the invention relates to a process for developing plants of novel type being capable of nitrogen-fixation also in their leaves. The process of the invention comprises inoculating plant protoplasts, cells, tissues, embryos or organs grown and/or treated under in vitro conditions with bacteria belonging to the family of Azotobacteraceae, then cocultivating the thus-obtained culture at a temperature of 15° to 35° C. and, if desired, propagating and/or regenerating the whole plant under in vitro conditions on or in a culture medium containing nitrogen and main carbon source(s) utilizable only by the plant cells as well as optionally other additives. The process of the invention ensures a well-balanced growth of the plant together with the bacteria.

OBJECT OF THE INVENTION Traditionally, the known bacterial product for nitrogen fixation has adopted different process. Whereas the present invention is based upon Nitrogen fixing bacteria from pure culture of bacteria like Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter x linum or Azospirillum lipoferum that fix the atmospheric nitrogen from air and makes it available to plants. It also increases efficiency of chemical fertilizer.

STATEMENT OF INVENTION- The inventor has invented a composition and method of making a bacterial based product that fix atmospheric nitrogen from air and makes available to plant. It also increases efficiency of chemical fertilizer

DETAILED DESCRIPTION OF INVENTION-

Plants require more than just water and sunlight to grow. They also require many nutrients found in the soil. One of the most important nutrients required for plant growth is nitrogen. Nitrogen is used to build plant proteins and nucleic acids, including DNA.

Nitrogen is found naturally in the atmosphere and in the soil. Even though there is an abundance of nitrogen available, the most common form of nitrogen (N₂) cannot be used by plants. Nitrogen can be combined chemically with oxygen or hydrogen to form types of nitrogen compounds that plants can use. These nitrogen compounds can be added to the soil in the form of ammonium (NH ) and nitrate (N0₃ ⁺) fertilizer. Plants grow well when fertilizer containing nitrogen is added to the soil, but this method can be expensive and has to be repeated each time the nitrogen in the soil is used up.

Bacteria to the rescue! Bacteria are small, single-celled organisms that live in nearly every environment on Earth. Some of these organisms are able to live in extreme environments and have many amazing capabilities. Some species of bacteria are able to turn milk into cheese while others can reproduce in less than twenty-four hours. Nitrogen fixing bacteria can turn the nitrogen in the soil into usable nitrogen compounds like ammonium and nitrate ions. These bacteria can attach themselves to the roots of some plants, forming little growths called nodules or some species of bacteria may leave separately as non-symbioant. In case of symbiotic association the bacteria receive nutrients and protection from the plant roots and the plants get their fill of nitrogen. Both organisms benefit from this symbiotic relationship. Legumes and clover in particular, readily form this symbiotic relationship with bacteria where as in case of non symbiotic bacteria they are beneficial to wide range of plants Nitrogen Fixing Bacteria captures the free nitrogen from the atmosphere and convert into nitrates and nitrites which can be absorbed by the plants. Nitrogen fixing bacterium 'fixes' or sequester nitrogen from the atmosphere and utilize the nitrogen to generate cell constituents. Nitrogen Fixation may be defined as a natural process, by which soil bacterium, algae and plants manufacture useable nitrogen for plants from the atmosphere. Nitrogen fixing bacteria include a diverse group of prokaryotes, reaching into phylogenetically distinct groups of archaea and bacteria. In general nitrogen fixing organisms are unified only on the basis of their metabolic ability to "fix" nitrogen or otherwise convert N ₂ (atmospheric nitrogen) to a water soluble or other such form that is available for plant use such as, for example NH ₃ (ammonia).

Accordingly, in one aspect, this invention provides the composition and method of preparing nitrogen fixing bacteria from pure culture of bacteria like Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter xylinum or Azospirillum lipoferum that fix the atmospheric nitrogen from air and makes it available to plants. It also increases efficiency of chemical fertilizer.

Material and Method used in preparation of nitrogen fixing bacteria are as under

Step-1 Nucleus culture (Maintain culture)

Pure culture of bacteria {Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter xylinum or Azospirillum lipoferum) is inoculated aseptically on plate having 20 ml PAC-08 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-08 agar media is 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0.1 gm FeS0₄, 2 gm CaC0₃, 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-08 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_f (Composition for one liter of PAC-08 broth media is 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0.1 gm FeS0₄, 2 gm CaC0₃ 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-08 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-08 broth media is 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0.1 gm FeS0₄, 2 gm CaC0₃ and 1000 ml 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-08 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-08 broth media is 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0.1 gm FeS0₄, 2 gm CaC0₃ 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-08 media (Composition for one liter of PAC-08 broth media is 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0.1 gm FeS0₄, 2 gm CaC0₃ 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 and melto dextrin @ 1 to 2 % which act as a drying agent or with 5 % skim milk power and agitate continuously by using agitator.

Step-7

Mix culture prepared as per step-6, is then Spray dried at definite 160 °C in let and 70 to 72 °C out let temp in spray dryer unit

Step-8 Collecting dry bacterial powder

Material dried in spray dryer unit is then collected in clean stainless steel vessels and stored at 5 °C up to further use

Step-9 Mix with suitable carrier

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

Step-10 Packaging

Material prepared as above is then packed in 100 gm, 250 gm and 500 gm packing by using pouch packing machine. 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 an improved nitrogen fixing bacterial based product for plant that fixes atmospheric nitrogen from air and makes it available to plant from pure culture of bacteria like Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter xylinum or Azospirillum lipoferum, the method comprising steps of nucleus culture, starter culture, growth culture, seed fermentor, production fermentor, mixing of final output, drying, collecting, mixing with dextrose and collecting.

2. A composition and method of preparing nitrogen fixing bacteria according to claim 1 , comprising of inoculating pure culture of bacteria such as Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter xylinum or Azospirillum lipoferum aseptically on two to three plates having 20 ml PAC-08 Agar media, whereas such two to three plates are generally inoculated and maintained in BOD incubator at 27 ± 1 °C for 6 to 7 days with 12/12 hour lighting cycle, wherein, composition for one liter of PAC-08 agar media contains 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0.1 gm FeS0₄, 2 gm CaC0₃, 20 gm Agar agar and 1000 ml distilled water.

3. A composition and method of preparing nitrogen fixing bacteria according to claim 1 , further comprising of inoculating aseptically the culture grown on plate as per method recited in claim 2 in a flask of 250 ml capacity having 100 ml PAC- 08 broth media with the help of inoculating loop, and allowing to grow at 27 ± 1 °C for at least 3 to 4 days and for superior growth, the flask is put on shaker with agitation of media at 150 RPM, wherein composition for one liter of PAC-08 broth media contains 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0.1 gm FeS0₄, 2 gm CaC0₃ and 1000 ml distilled water.

4. A composition and method of preparing nitrogen fixing bacteria according to claim 1 , further comprising of inoculating aseptically 50 ml culture from this grown culture as per method recited in claim 3 in 500 ml PAC-08 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-08 broth media contains 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0.1 gm FeS0₄, 2 gm CaC0₃ and 1000 ml distilled water.

5. A composition and method of preparing nitrogen fixing bacteria according to claim 1 , further comprising of one (1) lit grown culture as per method recited in claim 4 being inoculated aseptically in small fermenter having 10 litter PAC-08 broth media, and allowing 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- 08 broth media contains 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0, 1 gm FeS0₄, 2 gm CaC0₃ and 1000 ml distilled water.

6. A composition and method of preparing nitrogen fixing bacteria according to claim 1, further comprising of Ten (10) lit culture grown as per method recited in claim 5 being inoculated aseptically in big fermenter having 200 litter PAC-08 media, wherein, composition for one liter of PAC-08 broth media contains 20 gm Sucrose, 1 gm K₂HP0₄, 0.5 gm MgS0₄7H₂0, 0.5 gm NaCl, 0.1 gm FeS0₄, 2 gm CaC0₃ 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

7. A composition and method of preparing nitrogen fixing bacteria according to claim 1, further comprising of final out-put of claim 6 from big fermentor being mixed with starch @ 4 to 5 %, which acts as a stabilizer, and melto dextrin @ 1 to 2 %, which acts as a drying agent or with 5 % skim milk powder and agitate, continuously by using agitator.

8. A composition and method of preparing nitrogen fixing bacteria according to claim 1, further comprising of process of mix culture prepared as per claim 7, which is then sprayed dry at definite 160°C in let and 70 to 72 °C out let temperature in spray dryer unit.

9. A composition and method of preparing nitrogen fixing bacteria according to claim 1 , further comprising of collecting the material dried in spray dryer unit as recited in claim 8 in clean stainless steel vessels and storing at 5 °C up to further use.

10. A composition and method of preparing nitrogen fixing bacteria according to claim 1, further comprising of mixing the each 10 gm bacterial power previously dried as recited in claim 9 with 90 gm of dextrose powder and packing the final output as nitrogen fixing bacterial product.