WO2011154960A1

WO2011154960A1 - Advance material and method of preparation of bacterial formulation using nitrogen fixing bacteria that fix atmoshpheric nitrogen and make available to crop plant

Info

Publication number: WO2011154960A1
Application number: PCT/IN2010/000388
Authority: WO
Inventors: Chetan S. Patel
Original assignee: Patel, Babubhai C.
Priority date: 2010-06-09
Filing date: 2010-06-09
Publication date: 2011-12-15

Abstract

The subject invention concerns materials and methods for providing or enhancing nitrogen fixation in plants from pure culture bacteria like Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter xylinum or Gluconacetobacter diazotrophicus or Azospirillum lipoferum that fixes the atmospheric nitrogen from air and makes it available to plants. It also increases efficiency of chemical fertilizer.

Description

TITLE- Advance Material And Method Of Preparation Of Bacterial Formulation Using Nitrogen Fixing Bacteria That Fix Atmoshpheric Nitrogen And Make Available To Crop Plant.

PREAMBLE OF INVENTION- This invention in particular describes 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 advanced 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. 6548289, wherein, the invention relates to the method for increasing the rate of conversion of atmospheric nitrogen into ammonia in a microorganism of the genus Rhizobium, by increasing the intracellular level of an activator protein which is capable of activating the transcription of DNA of the microorganism encoding one or more proteins capable of effecting such conversion in the microorganism.

In the existing system as given in United States Patent Application No. 20090137390, wherein, the invention concerns materials and methods for providing or enhancing nitrogen fixation in plants. The invention provides for the use of nitrogen fixing bacteria that are

[i] PRIOR ART-

In the existing system as given in United States Patent Application No. 20090137390, wherein, the invention concerns materials and methods for providing or enhancing nitrogen fixation in plants. The invention provides for the use of nitrogen fixing bacteria that are isolated from nitrogen efficient plants. Plants for which enhanced nitrogen fixation is desired are inoculated with an effective amount of nitrogen firing bacteria of the invention. In an exemplified embodiment, the bacteria is Klebsiella Kp342. The subject invention also concerns means to increase the number of free-living nitrogen-fixing bacteria in plants. Mutants of beneficial endophytic bacteria that are resistant to plant defense responses can be used to colonize a plant in numbers higher than a wild type or a non-mutated bacteria can colonize a plant. The higher number of bacteria colonizing the plant provide for more nitrogen fixation for the plant. The subject invention concerns methods for producing non- leguminous plants that are capable e of utilizing atmospheric nitrogen by colonization with a nitrogen fixing endophyic bacteria that is resistant to plant defense responses. The subject invention also concerns the plants produced by the subject method. The subject invention also concerns methods for producing the mutant endophytic bacteria. The subject invention also concerns the mutant endophytic bacteria produced using the subject methods. In the existing system as given in United States Patent Application No. 4711656, wherein, the invention relates to legumes inoculated with tryptophan catabolic variants (tan variants) of wild-type bradyrhizobia that are characterized by an enhanced capacity to fix atmospheric nitrogen as compared to parent strains. Responses of the symbiotic system to the variants include an auxinic stimulation of root growth, an enhancement of nodulation, and an increase in the nitrogen-fixation activities of the nodulated root systems.

In the existing system as given in United States Patent Application No. 5183759, wherein, the invention relates to the sequences of a Rhizobium bacteria responsible for competitiveness with respect to plant nodulation that have been isolated and permanently transferred to superior nodulating Rhizobium genome. This has resulted in a stable construct that can form a plant inoculant that yields effective nodulation, while reducing the risk of suppression by other bacteria in the environment.

In the existing system as given in United States Patent Application No. 3704546, wherein, the invention relates to inoculated cells from plants are caused to grow aseptically in a synthetic liquid culture medium to produce a mass of undifferentiated plant cells. These are aseptically incoulated with a symbiotic microorganism to produce a synthetic symbiotic system that is capable of fixing atmospheric nitrogen. The end product contains available protein material.

In the existing system as given in United States Patent Application No. 5113619, wherein, the invention relates to a 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 United States Patent Application No. 4567146, wherein, the invention relates to a Bacteria of the genus Rhizobium nodulate legumes and are responsible for nitrogen fixation. The energetics of this process is improved if hydrogen uptake ability (Hup) is imparted to the bacteria to "recycle" some of the hydrogen lost during the nitrogen fixation. Such Hup is observed in a naturally occurring strain of Rhizobium leguminosarum but it was not known how to transfer it to other strains. The present invention provides recombinant plasmids pIJ1008 and pIJ1007 which enable it to be transferred. These plasmids are formed from a transmissible plasmid, synthesized to include a drug-resistance marker, and a plasmid of the naturally occurring strain. Cultures containing the plasmids have been deposited in a culture collection and can be used, after appropriate further crosses as described, to impart Hup to other strains of Rhizobium. In addition, cultures containing the starting transmissible plasmids (pVW5JI and pVW3JI) have been deposited and can be used to prepare strains of Rhizobium containing the same or similar recombinant plasmids. Plant growth analysis in laboratory tests demonstrate that rhizobial strains containing these recombinant nodulation plasmids which confer Hup⁺ ability are superior to the corresponding Rhizobium field isolates in terms of overall plant growth dependent on symbiotic nitrogen fixation.

In the existing system as given in United States Patent Application No. 4136486, wherein, the invention relates to a method is described which uses a composition including a small non-toxic amount (less than about 0.001 part per part of the composition) of a water soluble adhesive agent, particularly an alkali metal salt of carboxymethyl cellulose, with water and with nitrogen fixing bacteria for applying the bacteria on the surfaces of seeds of the plant family leguminosae or on particles surrounding the seeds including soil particles or combinations thereof. The nitrogen fixing bacteria are of a symbiotic species and are selectively isolated from effectively nodulated plants of the variety to be inoculated so that they are efficient in inducing effective root nodulation in the plants. Novel compositions are described which include the bacteria mixed with water and the adhesive agent and which are prepared by a manufacturer, frozen for shipment, and then thawed and substantially diluted with water by the farmer or other user for application.

In the existing system as given in United States Patent Application No. 5041383, wherein, the invention discloses a method for producing novel strains of Rhizobium bacteria for legume inoculants which are both competitive and have high nitrogen fixation characteristics. The method involves first isolating and identifying the naturally predominant strains of the Rhizobium species of interest in a particular locale and then mutagenizing that strain to produce mutant strains which retain the competitiveness and add enhanced nitrogen fixation capabilities.

In the existing system as given in United States Patent Application No. 20080190158, wherein, the invention relates to biofertilizers have been identified as an alternative to chemical fertilization to increase soil fertility and crop production using sustainable farming. Treatment is with at least a proprietary formulation of a nitrogen-fixing con=X sortium and Azospirillum brasilense. The application of biofertilizers resulted in higher biomass compared to chemical fertilizer treatment. Chlorophyll and nitrogen levels in these grass plants are also likely to be improved.

In the existing system as given in United States Patent Application No. 5586411, wherein, the invention relates to methods and compositions for increasing the availability of soluble phosphate and fixed nitrogen for legume:Rhizobium symbioses involving co-inoculating legume seeds with a phosphate-solubilizing soil fungus, Penicillium bilaii, and Rhizobium spp. prior to planting. The two types of microorganisms do not appear to compete for nutrients in the rhizosphere of legumes and the P. bilaii provides a source of available phosphorus for use by the plant without adversely affecting the nitrogen fixation ability of the Rhizobium spp.; and indeed such ability is enhanced. The invention is used to increase the efficiencies of nodulation, nitrogen fixation and legume crop production.

In the existing system as given in United States Patent Application No. 3976017, wherein the invention relates to a process and apparatus used in inoculating leguminous plants with nitrogen fixing bacteria is disclosed. The process involves the preparation of an aqueous dispersion of the bacteria from a suitable source material and its dispensation to the soil in the fluid state and through the use of a separate dispenser from that handling the seeds. The apparatus includes a soil opening implement that forms a furrow during use and separate means for delivering the aqueous dispersion and seed to the furrow. A positive displacement pump provides quantitative dispensation of the bacteria.

In the existing system as given in United States Patent Application No. 20040229365, wherein the invention relates to a promintron sequence derived from an intervening sequence of the a gene of Agrobacterium rhizogenes strain A4 is described. The sequence is able to drive gene expression within bacteroids in all stages of nodule development in order to obtain, over the developmental time of the nodule, a constitutive expression of the gene(s) of interest. Uses of said sequence, derived vectors and recombinant bacteria are also described.

In the existing system as given in European Patent Application No. EP1186581, wherein the invention relates to nitrogen-free composition comprises: (a) 50-90 wt.% powdered stone, preferably a mixture of different minerals; (b) 5-50 wt.% triple layered clay minerals; (c) 1- 25 wt.% zeolite; (d) 1 -5 wt.% binder, especially dextrin; (e) nitrogen-fixing bacteria; and (f) 0.1-10 wt.% dry mixture comprising magnesium, sodium, copper, manganese, zinc and/or iron sulfate. An Independent claim is also included for the preparation of the composition.

In the existing system as given in United States Patent Application No. 5093262, wherein the invention relates to produce organic fertilizer which is effective for sterile agricultural land, a mixture comprising coral reef powder, domestic animal excreta, sewage disposal waste and ground cellulose derived from plant fiber is prepared and it is well mixed in the presence of organic compounds and inorganic salts. Thereafter, it is subjected to cultivating in a multi-bath type cultivating bath for a predetermined period of time while temperature and pH are adjusted properly. Cultivation is achieved with the addition of bacilli including nitrogen fixing bacillus such as Azotobactor vinelandii, Bacillus megaterum, Rhizobium leguminosarm or the like, Trichoderma virde acting as raw fiber decomposing bacillus, Candida utilus acting as yeast fungus and green algae, each of which is cultivated under the operating condition of symbiosis cultivation. The pH in the cultivating bath is maintained in the range of 5.5 to 7.5, preferably in the range of 6 to 7. After completion of cultivation, the mixture is dried to form dried product and the latter is shipped to users. In the existing system as given in United States Patent Application No. 4782022, wherein the invention discloses isolation and characterization of a gene which activates nitrogen fixation genes of Rhizobium meliloti when that bacterium is in a symbiotic relationship with a plant is disclosed. This newly discovered gene, designated fix D, can activate the nifHD promoter. A method of making this inducible gene constitutive is presented. This is useful for making nifHD constitutive. The combination of the fixD promoter with heterologous structural genes is taught. Such combinations are useful for limiting expression of an encoded protein to rhizobia involved in a symbiotic relationship with a plant. Plasmids and methods useful in performance of this invention are also disclosed.

PARENT CASE DATE:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application number 336/MUM/2010, filed February, 02, 2010, now pending.

BACKGROUND OF THE 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 (NH₄ ⁺) 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. 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. 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).

As is well known by any expert in the art, there have been hitherto made a number of proposals as to a method of producing organic fertilizer. To practice the conventional methods various kinds of raw materials and processes were employed but each of them has drawbacks in terms of procurement of raw material and cost.

OBJECT OF THE INVENTION

Hence, the present invention has been made with the foregoing background in mind and its object resides in providing a material and method of rationally producing a high quality of organic fertilizer at the highest economical level using raw material which can be easily procured at the most inexpensive cost.

STATEMENT OF INVENTION-

The inventor has invented a composition and method for improving crop yields, involving increasing nitrogen fixation in nitrogen fixing bacteria from pure culture of bacteria like Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter xylinum or Gluconacetobacter diazotrophicus or Azospirillum lipoferum.

DETAILED DESCRIPTION OF INVENTION-

The subject invention concerns materials and methods for providing for or enhancing nitrogen fixation in plants. The invention provides for the material and method in preparing nitrogen fixing bacteria from pure culture of bacteria like Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter xylinum or Gluconacetobacter diazotrophicus or Azospirillum lipoferum.

Material and Method used in preparing the above said innovative combination is as under

Step-1 Nucleus culture (Maintain culture)

Pure culture of bacteria {Azotobacter chrococcum or Azotobacter vinelandii or Acetobacter xylinum or Gluconacetobacter diazotrophicus or Azospirillum lipoferum) is inoculated aseptically on plate having 20 ml PAC-010 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-010 agar media is 5 gm Dextrose, 1 gm MgS0₄7H₂0, 5 gm Yeast extract, 5 gm Peptone, 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-010 broth media with the help of inoculating loop, and allowing it 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-010 agar media is 5 gm Dextrose, 1 gm MgS0₄7H₂0, 5 gm Yeast extract, 5 gm Peptone 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-010 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-010 agar media is 5 gm Dextrose, 1 gm MgS0₄7H₂0, 5 gm Yeast extract, 5 gm Peptone 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-010 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-010 agar media is 5 gm Dextrose, 1 gm MgS0₄7H₂0, 5 gm Yeast extract, 5 gm Peptone 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-010 media (Composition for one liter of PAC-010 agar media is 5 gm Dextrose, 1 gm MgS0₄7H₂0, 5 gm Yeast extract, 5 gm Peptone 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

Step-6 Bacterial Separation

Final output from big fermentor is centrifuge by using advance centrifugation technology. By this separated bacteria are use for formulation of product whereas remaining liquid is throughout.

Step-7 Formulation

Recovered bacteria form centrifuge is mix in a set of ration with suitable carrier composed of zero TDS water etc and then homogenized it for equivalently of formulation.

Step-8 Packaging

Material prepared as above is then packed in 100 ml, 250 ml and 500 ml packing by using bottle or pouch packing machine.

Step-9 - 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 ml per seed required per hector

For soil application add 100 ml to 250 ml per hector

Claims

CLAIMS I claim,

1. A material 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 Gluconacetobacter diazotrophicus or Azospirillum lipoferum; the method comprising steps of nucleus culture, starter culture, growth culture, seed fermentor, production fermentor, bacterial separation, formulation and packaging.

2. A material 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 Gluconacetobacter diazotrophicus or Azospirillum lipoferum aseptically on two to three plates having 20 ml PAC-010 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-010 broth media contains 5 gm Dextrose, 1 gm MgSO₄7H₂O, 5 gm Yeast extract, 5 gm Peptone, 20 gm Agar agar and 1000 ml distilled water.

3. A material 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-010 broth media with the help of inoculating loop, and allowing it 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-010 broth media contains 5 gm Dextrose, 1 gm MgS0₄7H₂0, 5 gm Yeast extract, 5 gm Peptone and 1000 ml distilled water.

4. A material and method of preparing nitrogen fixing bacteria according to claim 1, further comprising of inoculating aseptically 50 ml culture from grown culture as per method recited in claim 3 in 500 ml PAC-010 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-10 broth media contains agar media is 5 gm Dextrose, 1 gm MgS0₄7H₂0, 5 gm Yeast extract, 5 gm Peptone and 1000 ml distilled water.

5. A material 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-010 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-10 broth media contains 5 gm Dextrose, 1 gm MgS0₄7H₂0, 5 gm Yeast extract, 5 gm Peptone and 1000 ml distilled water

6. A material 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-010 agar media, wherein, composition for one liter of PAC - 010 agar media is 5 gm Dextrose, 1 gm MgS0₄7H₂0, 5 gm Yeast extract, 5 gm Peptone 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 material and method of preparing nitrogen fixing bacteria according to claim 1, wherein final output from big fermenter is centrifuge by using advance centrifugation technology; this separated bacteria are use for formulation of product whereas remaining liquid is thrown out.

8. A material and method of preparing nitrogen fixing bacteria according to claim 1, wherein, recovered bacteria form centrifuge is mix in a set of ration with suitable carrier composed of zero TDS water etc and then homogenized it for equivalently of formulation.

9. A material and method of preparing nitrogen fixing bacteria according to claim 1 , further comprising of packing the final output as nitrogen fixing bacterial product in 100 ml, 250 ml, 500 ml by using bottle or pouch packing machine; distribution to farmer, retailer, distributor etc

10. A material and method of preparing nitrogen fixing bacteria according to claim 1, the final material prepared has to be used in following form dose in one Acre: In case of Seed treatment add 100 ml per seed required per hector; for soil application add 100 ml to 250 ml per hector.