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WO2011154960A1 - Matière nouvelle et procédé de préparation de formulation bactérienne utilisant des bactéries fixatrices d'azote qui fixent l'azote atmosphérique et le rendent disponible pour les plantes cultivées - Google Patents

Matière nouvelle et procédé de préparation de formulation bactérienne utilisant des bactéries fixatrices d'azote qui fixent l'azote atmosphérique et le rendent disponible pour les plantes cultivées Download PDF

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Publication number
WO2011154960A1
WO2011154960A1 PCT/IN2010/000388 IN2010000388W WO2011154960A1 WO 2011154960 A1 WO2011154960 A1 WO 2011154960A1 IN 2010000388 W IN2010000388 W IN 2010000388W WO 2011154960 A1 WO2011154960 A1 WO 2011154960A1
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WIPO (PCT)
Prior art keywords
nitrogen
pac
culture
nitrogen fixing
fixing bacteria
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Ceased
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PCT/IN2010/000388
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English (en)
Inventor
Chetan S. Patel
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PATEL BABUBHAI C
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PATEL BABUBHAI C
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Application filed by PATEL BABUBHAI C filed Critical PATEL BABUBHAI C
Priority to PCT/IN2010/000388 priority Critical patent/WO2011154960A1/fr
Publication of WO2011154960A1 publication Critical patent/WO2011154960A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F11/00—Other organic fertilisers
    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like

Definitions

  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Hup hydrogen uptake ability
  • the present invention provides recombinant plasmids pIJ1008 and pIJ1007 which enable it to be transferred.
  • 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.
  • the invention relates to a method 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.
  • 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.
  • 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.
  • 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.
  • the invention relates to a process and apparatus used in inoculating leguminous plants with nitrogen fixing bacteria.
  • 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.
  • 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.
  • 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.
  • the mixture is dried to form dried product and the latter is shipped to users.
  • 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.
  • 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.
  • 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 (N 2 ) 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 4 + ) and nitrate (N0 3 + ) 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.
  • NH 4 + ammonium
  • N0 3 + nitrate
  • 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.
  • N 2 atmospheric nitrogen
  • NH 3 ammonia
  • 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.
  • 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.
  • 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.
  • 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 MgS0 4 7H 2 0, 5 gm Yeast extract, 5 gm Peptone, 20 gm Agar agar and 1000 ml distilled water)
  • Step-2 Starter culture (In 100 ml flask)
  • Step-3 Growth culture (In 1000 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 MgS0 4 7H 2 0, 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 MgS0 4 7H 2 0, 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 MgS0 4 7H 2 0, 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
  • 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.
  • 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.
  • 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:

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Abstract

L'invention concerne des matières et des procédés pour obtenir ou améliorer la fixation d'azote chez des plantes à partir de bactéries de culture pures telles que Azotobacter chrococcum ou Azotobacter vinelandii ou Acetobacter xylinum ou Gluconacetobacter diazotrophicus ou Azospirillum lipoferum qui fixent l'azote atmosphérique de l'air et le rendent disponible pour les plantes. L'efficacité d'un engrais chimique est également augmentée.
PCT/IN2010/000388 2010-06-09 2010-06-09 Matière nouvelle et procédé de préparation de formulation bactérienne utilisant des bactéries fixatrices d'azote qui fixent l'azote atmosphérique et le rendent disponible pour les plantes cultivées Ceased WO2011154960A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014163471A1 (fr) * 2013-04-05 2014-10-09 Valorhyze Procede de formulation stable d'un produit biofertilisant a base d'une souche fixatrice d'azote atmospherique, azospirillum brasillense
CN107548280A (zh) * 2014-07-28 2018-01-05 氮技术有限公司 农业方法
US9957509B2 (en) 2011-06-16 2018-05-01 The Regents Of The University Of California Synthetic gene clusters
US9975817B2 (en) 2015-07-13 2018-05-22 Pivot Bio, Inc. Methods and compositions for improving plant traits
US11479516B2 (en) 2015-10-05 2022-10-25 Massachusetts Institute Of Technology Nitrogen fixation using refactored NIF clusters
US11565979B2 (en) 2017-01-12 2023-01-31 Pivot Bio, Inc. Methods and compositions for improving plant traits
US11678668B2 (en) 2018-06-27 2023-06-20 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US11946162B2 (en) 2012-11-01 2024-04-02 Massachusetts Institute Of Technology Directed evolution of synthetic gene cluster
US11993778B2 (en) 2017-10-25 2024-05-28 Pivot Bio, Inc. Methods and compositions for improving engineered microbes that fix nitrogen
US12151988B2 (en) 2017-10-25 2024-11-26 Pivot Bio, Inc. Gene targets for nitrogen fixation targeting for improving plant traits
US12281980B2 (en) 2020-05-01 2025-04-22 Pivot Bio, Inc. Measurement of nitrogen fixation and incorporation
US12281299B2 (en) 2019-03-19 2025-04-22 Massachusetts Institute Of Technology Control of nitrogen fixation in rhizobia that associate with cereals
US12391624B2 (en) 2018-07-11 2025-08-19 Pivot Bio, Inc. Temporally and spatially targeted dynamic nitrogen delivery by remodeled microbes
US12421519B2 (en) 2019-01-07 2025-09-23 Pivot Bio, Inc. Plant colonization assays using natural microbial barcodes
US12478068B2 (en) 2020-05-01 2025-11-25 Pivot Bio, Inc. Stable liquid formulations for nitrogen-fixing microorganisms

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Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10662432B2 (en) 2011-06-16 2020-05-26 The Regents Of The University Of California Synthetic gene clusters
US9957509B2 (en) 2011-06-16 2018-05-01 The Regents Of The University Of California Synthetic gene clusters
US12209245B2 (en) 2011-06-16 2025-01-28 The Regents Of The University Of California Synthetic gene clusters
US11946162B2 (en) 2012-11-01 2024-04-02 Massachusetts Institute Of Technology Directed evolution of synthetic gene cluster
WO2014163471A1 (fr) * 2013-04-05 2014-10-09 Valorhyze Procede de formulation stable d'un produit biofertilisant a base d'une souche fixatrice d'azote atmospherique, azospirillum brasillense
CN107548280A (zh) * 2014-07-28 2018-01-05 氮技术有限公司 农业方法
US10919814B2 (en) 2015-07-13 2021-02-16 Pivot Bio, Inc. Methods and compositions for improving plant traits
US10934226B2 (en) 2015-07-13 2021-03-02 Pivot Bio, Inc. Methods and compositions for improving plant traits
US12497342B2 (en) 2015-07-13 2025-12-16 Pivot Bio, Inc. Methods and compositions for improving plant traits
US10556839B2 (en) 2015-07-13 2020-02-11 Pivot Bio, Inc. Methods and compositions for improving plant traits
US11739032B2 (en) 2015-07-13 2023-08-29 Pivot Bio, Inc. Methods and compositions for improving plant traits
US10384983B2 (en) 2015-07-13 2019-08-20 Pivot Bio, Inc. Methods and compositions for improving plant traits
US9975817B2 (en) 2015-07-13 2018-05-22 Pivot Bio, Inc. Methods and compositions for improving plant traits
US11479516B2 (en) 2015-10-05 2022-10-25 Massachusetts Institute Of Technology Nitrogen fixation using refactored NIF clusters
US11565979B2 (en) 2017-01-12 2023-01-31 Pivot Bio, Inc. Methods and compositions for improving plant traits
US12151988B2 (en) 2017-10-25 2024-11-26 Pivot Bio, Inc. Gene targets for nitrogen fixation targeting for improving plant traits
US11993778B2 (en) 2017-10-25 2024-05-28 Pivot Bio, Inc. Methods and compositions for improving engineered microbes that fix nitrogen
US11678667B2 (en) 2018-06-27 2023-06-20 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US11963530B2 (en) 2018-06-27 2024-04-23 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US12268212B2 (en) 2018-06-27 2025-04-08 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US12290074B2 (en) 2018-06-27 2025-05-06 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US12471599B2 (en) 2018-06-27 2025-11-18 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US11678668B2 (en) 2018-06-27 2023-06-20 Pivot Bio, Inc. Agricultural compositions comprising remodeled nitrogen fixing microbes
US12391624B2 (en) 2018-07-11 2025-08-19 Pivot Bio, Inc. Temporally and spatially targeted dynamic nitrogen delivery by remodeled microbes
US12421519B2 (en) 2019-01-07 2025-09-23 Pivot Bio, Inc. Plant colonization assays using natural microbial barcodes
US12281299B2 (en) 2019-03-19 2025-04-22 Massachusetts Institute Of Technology Control of nitrogen fixation in rhizobia that associate with cereals
US12281980B2 (en) 2020-05-01 2025-04-22 Pivot Bio, Inc. Measurement of nitrogen fixation and incorporation
US12478068B2 (en) 2020-05-01 2025-11-25 Pivot Bio, Inc. Stable liquid formulations for nitrogen-fixing microorganisms

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