DESC:
FIELD OF THE INVENTION:

The present invention relates to a kit for the mass multiplication of bacteria. The present invention also relates to plant nutrients, to the field of microbial fermentation technology and mass multiplication of plant growth promoting bacteria (PGP) in liquid state fermentation methods.

BACKGROUND OF THE INVENTION:

The requirement for agriculture, horticultural growing has increased rapidly since the 1950's as a result there is increase in use of plant growth-promoting bacteria (PGPB) for growing. Plant growth-promoting bacteria (PGPB) satisfies a range of plant growth requirements, seed germination, seedling and plant growth.

Plant growth-promoting bacteria (PGPB) are associated with many, if not all, plant species and are commonly present in diverse environments. The most widely studied group of PGPB is plant growth-promoting rhizobacteria (PGPR), which colonize the root surfaces and the interface between soil and plant roots, commonly referred to as the rhizosphere. The rhizosphere is an environment where bacteria, fungi and other organisms compete for niches and nutrients and for binding to the root structures of the plant. Both deleterious and beneficial bacteria can colonize plant roots. The presence of PGPB within or near plant roots or seeds can lead to a healthier rhizosphere environment (less colonization of deleterious bacteria) and healthier plants. The free living bacteria are deemed to promote plant growth and health of agricultural crops and increased yield. PGPB that colonize the roots and maintain their benefits throughout the growth cycle of the plant are especially desired for application during germination and early growth or as a seed inoculants of agricultural crops.

The mechanisms that PGPB use in promoting seed germination, seedling and plant growth, are diverse and often plant- or cultivar-specific. Several growth-promoting mechanisms are known. These mechanisms involve supplying the plant with nutrients through e.g. phosphate solubilisation and transport towards the roots and atmospheric nitrogen fixation, or synthesizing phytohormones. As an additional advantage, PGPB can also lead to extensive re-modelling of the plant root systems.

Currently extensive of chemical fertilizer like Urea, DAP, Ammonium Sulphate and many other reduces soil fertility and so crop productivity. This chemicals also enters the food systems. Currently extensive uses of chemical pesticide are being increasing in agriculture, This extensive use of chemical pesticide contaminate food system by deposition of pesticide residue in agriculture products and also increases resistance in pests and decreases soil fertility by affecting soil microorganism reproduction and function. To reduce environmental protection and enhance agriculture product safety and quality, reducing the usage of chemicals in agriculture is necessary.

New safe chemical fertilizers and pesticide development is also difficult task. Bio fertilizer and Biological control of various disease is the main direction of development.

Global demand of bio fertilizer bacteria increases but having low number of live bacteria reach to farms. From the production in laboratory and reaching to farmers it takes time about 3 months to 8 months. During this period microbial count decreases to 10 to 99 percentage. This leads to ineffective or less effective product.

Although freeze-drying bacteria prior to mixing with a carrier usually gives a high initial recovery, the bacteria does not always remain stable for long storage periods. Therefore, it would be advantageous to provide a method and kit for dormant bacteria inoculant which can be easily prepared during the time of use and which will be stable over fairly long storage times with high yields of viable bacteria upon re-exposure to moisture.

According to the present invention, there is provided a method and kit for mass multiplication of bacteria such as Bio fertilizer bacteria/ plant growth promoting bacteria.

SUMMARY OF THE INVENTION

One of the aspect of the present invention is to provide a method and kit for mass multiplication of bacteria.

Another aspect of the present invention provides a method and kit for mass multiplication of bacteria wherein the system comprises;
a. Sterile water and Antifungal substance and pH controller in container 1
b. Active strain of Bacteria in container 2
c. Sterile Salt solution for Media in container 3
d. Sterile Carbon Source of Media in container 4

Another aspect of the present invention is to provide a nutrient feeding system and method of uses thereof The present invention provides a complete, cost-effective, simple- to-use, and reproducible, chronologically sequential, and pH balanced nutrient feeding system for growing plants indoors, outdoors and in green houses, wherein the nutrient feeding system comprises a master container which in turn comprises formulation containers, each of which contains packaged nutrient components, namely macro-nutrients, micro-nutrients, carbohydrates, amino acids, enzymes, vitamins, hormones, yeast extracts, beneficial bacteria, beneficial organic ingredients and organic extracts.

Another aspect of the invention is to provide compositions, methods, kits, and systems for efficient and rapid propagation of plants via bioculture.

Another aspect of the present invention to provide a self-contained plant kit that includes a pack of nutrient-rich food to be dispersed in the field easily.

Another aspect of the invention is to provide a mass multiplication of Plant growth promoting bacteria in liquid state fermentation method using antifungal treated media and system to offsite mass multiplication in specialized packaging for plant growth promoting bacteria as well as fungus spore production.

Another aspect of the invention is to provide an advance media composition with method and system to produce these Plant growth promoting bacteria and fungus without any microbiology biotechnology laboratory setup.

Another aspect of the present invention is to provide a complete, cost-effective, simple-to-use, and reproducible, and pH balanced plant nutrient feeding method system plant.

Another aspect of the present invention describes an automated, or semi-automated, low-cost system, which significantly increases the quantity and quality, the number and size of the resulting plants, reduces the cost and shortens the cultivation time. This invention provides novel compositions and an efficient and rapid system for mass propagation of plants.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention is susceptible of embodiment in various forms, there is shown in the drawings and will hereinafter be described a presently preferred, albeit not limiting, embodiment with the understanding that the present disclosure is to be considered an exemplification of the present invention and is not intended to limit the invention to the specific embodiments illustrated.

Bactericidal: the term "bactericidal", as used herein, refers to the ability of a composition or substance to increase mortality or inhibit the growth rate of bacteria.

Diverse plant-associated microorganisms can positively impact plant health and physiology in a variety of ways. These beneficial microbes are generally referred to as plant growth-promoting microorganisms (PGPMs). The term “plant growth-promoting activity”, as used herein, encompasses a wide range of improved plant properties, including, for example without limitation, improved nitrogen fixation, improved root development, increased leaf area, increased plant yield, increased seed germination, increased photosynthesis, or an increased in accumulated biomass of the plant. In various embodiments the improvement is an at least 10% increase or at least 25% increase or at least 50% increase or at least 75% increase or at least a 100% increase in the property being measured. Thus, as non-limiting examples, the microbes may produce an above stated percentage increase in nitrogen fixation, or an above stated increase in total root weight, or in leaf area or in plant product yield (e.g., an above stated percentage increase in plant product weight), or an increased percentage of seeds that germinate within 10 days or 14 days or 30 days, or rate of photosynthesis (e.g., determined by CO2 consumption) or accumulated biomass of the plant (e.g., determined by weight of the plant). The plant product is the item—usually but not necessarily—a food item produced by the plant. The yield can be determined using any convenient method, for example, bushels or pounds of plant product produced per acre of planting. To date, isolated strains of over two dozen genera of microorganisms have been reported to have plant growth-promoting activity and/or biocontrol activity, and new genera and species with similar activities are still being discovered. Additionally, within some bacterial genera, multiple species and subspecies of biocontrol agents have been identified and can be found across multiple spatial scales, from the global level to farm level, and even on single plants. Furthermore, it has been reported that some individual microbial isolates may display biocontrol and/or plant growth-promoting activity not only on the plants or crops from which they were obtained but also on other crops. This indicates the generalist nature of some genotypes, especially those with a wide geographic distribution. As discussed above, if introduced in sufficient numbers and active for a sufficient duration, a single microbial population can have a significant impact on plant health.

The term “plant growth-promoting microorganism”, for the purpose of this disclosure, refers to a microorganism that can enhance plant growth in the absence of pathogens. As discussed in more detail below, examples of direct plant growth promotion include (a) biofertilization, (b) stimulation of root growth, (c) rhizoremediation, and (d) plant stress control. In addition, several PGPMs have been reported to promote plant growth indirectly via mechanisms of biological control, i.e., by reducing the level of disease, for example antibiosis, induction of systemic resistance, and competition with pathogens for nutrients and niches.
Biofertilizers: Microbial fertilizers supply the plant with nutrients and thereby can promote plant growth in the absence of pathogen pressure. Non-limiting examples of microbial isolates that can directly promote plant growth and/yield include N2-fixing bacteria Rhizobium and Bradyrhizobium species that, through symbiotic nitrogen fixation, can form nodules on roots of leguminous plants, in which they convert atmospheric N2 into ammonia which, in contrast to atmospheric N2, can be used by the plant as a nitrogen source. Other examples include Azospirillum species, which are free-living N2-fixers that can fertilize and increase yield of cereal crops such as wheat, sorghum, and maize. Despite Azospirillum's N2-fixing capacity, the yield increase caused by inoculation by Azospirillum is often attributed to increased root development and thus to increased rates of water and mineral uptake. In this respect, several rhizobacteria like Azotobacter spp. have been reported to be capable of producing a wide array of phytohormones (e.g., auxins, cytokinins) and enzymes (e.g., pectinase). Many of these phytohormones and enzymes have been shown to be intimately involved in the infection process of symbiotic bacteria-plant associations which have a regulatory influence on nodulation by Rhizobium.

One of the embodiments of the present invention is to provide a nutrient feeding system and method of uses thereof. The present invention provides a complete, cost-effective, simple- to-use, and reproducible, chronologically sequential, and pH balanced nutrient feeding system for growing plants indoors, outdoors and in green houses, wherein the nutrient feeding system comprises a master container which in turn comprises formulation containers, each of which contains packaged nutrient components, namely macro-nutrients, micro-nutrients, carbohydrates, amino acids, enzymes, vitamins, hormones, yeast extracts, beneficial bacteria, beneficial organic ingredients and organic extracts.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

One of the embodiments of the present invention is to provide compositions, methods, kits, and systems for efficient and rapid propagation of plants via bioculture.

One of the embodiments of the present invention provides a nutrient feeding system, wherein the system comprises;
a. Sterile water and Antifungal substance and pH controller in container 1
b. Active strain of Bacteria in container 2
c. Sterile Salt solution for Media in container 3
d. Sterile Carbon Source of Media in container 4

The method fixes atmospheric nitrogen in soil about 15 to 30 kg per hectare. It also produces antifungal substances which inhibits the growth of harmful fungi and result in lowering the disease occurrences.

One of the embodiments of the present invention is to provide a mass multiplication of Plant growth promoting bacteria in liquid state fermentation method using antifungal treated media and system to offsite mass multiplication in specialized packaging for plant growth promoting bacteria as well as fungus spore production.

One of the embodiments of the present invention is to provide an advance media composition with method and system to produce these Plant growths promoting bacteria and fungus without any microbiology biotechnology laboratory setup.

One of the embodiments of the present invention is to provide a complete, cost-effective, simple-to-use, and reproducible, and pH balanced plant nutrient feeding method system plant.

One of the embodiments of the present invention describes an automated, or semi-automated, low-cost system, which significantly increases the quantity and quality, the number and size of the resulting plants, reduces the cost and shortens the cultivation time. This invention provides novel compositions and an efficient and rapid system for mass propagation of plants.

One of the embodiments of the present invention is to provide a Component of bacteria Growth Media comprising;
a. Nutrients: proteins/peptides/amino-acids.
b. Energy: carbohydrates.
c. Essential metals and minerals: calcium, magnesium, iron, trace metals: phosphates, sulphates etc.
d. Buffering agents: phosphates, acetates etc.
e. Indicators for pH change: phenol red, bromo-cresol purple etc.
f. Selective agents: chemicals, antimicrobial agents.
g. Gelling agent (optional)

One of the embodiments described the method of the Packing material:

1. Should be Autoclavable (Ex. Glass bottle/ Autoclavable PP bottle/ Autoclavable HDMI etc.) bottle/container/pouch with neck of containers contains rubber closure or neck of containers having lock mechanism for sterile transfer of liquid from one container to other.
2. Container 1 is from size of 100 ml to 100 liter in size.
3. Volume of Material filled in Container 1 in the range of 10 percent to 90 percent of container 1 volume.
4. Other container contains concentrated growth media and cultures. One container for Culture. Other containers contain media component. Media container will be in the quantity of 1 to 5 in number.
5. Culture contains at least CFU 10000 of Bacteria to be produced.
6. Strain of Bacteria either single strain or multiple strain combination.
7. Bacteria Culture Container in size from 2 ml to 10-liter size.
8. Media container is in size range from 2 ml to 25-liter volume size.
9. Sterile infusion set/IV set/sterile syringe used for transfer of component from another container to fermenter tank.
10. Component of fermenter tank also be some component of other container according to adjustment of media with container bottle.

One of the embodiments describes the strain of bacteria Azotobacter, Rhizobium, Phosphate solubilizing bacteria, Potash Mobilizing Bacteria and other Plant Growth Promoting bacteria are widely distributed in nature with many different species, which provide nutrition and plant growth hormone to crops.

One of the embodiments describes the strain of bacteria Azotobacter Chroococcum is a free living nitrogen fixing aerobic bacterium. Azotobacter releases ammonia into the soil and improves soil fertility.

One of the embodiments describes the strain of bacteria Rhizobium spp. infects the legume root and form root nodules within which they reduce molecular nitrogen in to ammonia which is readily converted into nitrogen containing compounds. The site of symbiosis is within the root nodules. It produces nodules and multiplies in it. By remaining inside the nodules, it fixes atmospheric nitrogen.

One of the embodiments describes the Acetobacter forms colonies inside crop tissue and makes available nitrogen for plant consumption. Acetobacter also produce growth hormone like IAA for root and shoot growth.

One of the embodiments describes the Phosphate solubilizing bacteria produces organic acids (viz, citric acid, lactic acid, etc.), and enzymes (viz, phytase, nuclease, etc.) that helps, in solubilization of insoluble phosphates and makes it available to plant in utilizable form.

The microbe, Frateuria spp. is a beneficial bacterium capable of mobilizing available Potash into near the roots of the plants. It works well in all types of soil especially, low K content soil.

The present invention pertains to the field, of plant nutrients, in particular to a nutrients kit and method of use. The present invention mainly belongs to the field of microbial fermentation technology and mass multiplication of plant growth promoting bacteria (PGP) in liquid state fermentation methods.

One of the embodiments preparing the plant growth promoting bacteria (PGP) in Kit Component comprising of:
Container 1 = bioreactor with Sterile water + Antifungal substance + pH controller
Container 2 = Cultures strains of Active strain of plant growth promoting bacteria (PGP) / microorganisms
Container 3 = Sterile Salt solution for Media
Container 4 = Sterile Carbon Source of Media

The bioreactor Container 1:

According to the present invention, bioreactor Container 1, which comprises Sterile water, Antifungal substance, pH controller for a complete growth cycle according to a schedule of plant growth promoting bacteria (PGP) / microorganisms. The bioreactor Container 1 is a bottle, cylinder or other suitable container. The bioreactor Container 1 is made of plastic, or any other suitable material. In one embodiment of the invention as illustrated in Figures 1, 2, and 3, the bioreactor Container 1 is a plastic bottle of sufficient dimension to contain the growth of active strain of plant growth promoting bacteria (PGP) / microorganisms. The bioreactor Container 1 shapes and compositions can be used without departing from the scope of the invention.

A bioreactor Container 1 refers to any device or system that supports a biologically active environment. As described herein a bioreactor is a vessel in which plant growth promoting bacteria (PGP) / microorganisms can be grown. A bioreactor Container 1 may be any appropriate shape or size for growing the microorganisms. A bioreactor may range in size and scale from 100 ml to 10 liter and may be made of plastic or stainless steel or any other appropriate material as known and used in the art. The bioreactor Container 1 a batch type bioreactor. For example, a bioreactor may be a chemostat as known and used in the art of microbiology for growing and harvesting microorganisms.

A bioreactor Container 1 is a small container, a batch bioreactor used for processes of growth of plant growth promoting bacteria (PGP) / microorganisms that cannot be used for continuous operations.

Sterile Salt solution for Media Container 2:
The media container 2 comprises Sterile Salt solution. Sterile Salt solution is of sodium chloride. Sodium chloride consists of a single sodium ion that is bonded to a single chlorine ion. The presence of sodium chloride in media maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms. If the salt concentration is not similar, osmosis takes place transporting excess water into or out from the cell. Both of these scenarios can be lead to the death of the cell. The Media Container 2 is a bottle, cylinder or other suitable container. The Media Container 2 is made of plastic, or any other suitable material. In one embodiment of the invention as illustrated in Figures 1, 2, and 3, the Media Container 3 is a plastic bottle of sufficient dimension to store Sterile Salt solution for Media for active strain of plant growth promoting bacteria (PGP) / microorganisms in a controlled condition. The Media Container 2 shapes and compositions can be used without departing from the scope of the invention.

The Cultures strains Container 3:

Cultures strains of the plant growth promoting bacteria (PGP) / microorganisms are stored in air tight container in a controlled conditions in Container 3 of the kit. The Cultures strains Container 3 is a bottle, cylinder or other suitable container. The Cultures strains Container 3 is made of plastic, or any other suitable material. In one embodiment of the invention as illustrated in Figures 1, 2, and 3, the Cultures strains Container 3 is a plastic bottle of sufficient dimension to store active strain of plant growth promoting bacteria (PGP) / microorganisms in a controlled conditions. The Cultures strains Container 3 shapes and compositions can be used without departing from the scope of the invention.

Sterile Carbon Source of Media Container 4:
The Sterile Carbon Source of Media Container 4 provide everything to the plant growth promoting bacteria (PGP) / microorganisms for the cultivation. Sterile Carbon Source of Media Container 4 contains Glucose as ideal carbon source for many bacteria. The plant growth promoting bacteria (PGP) / microorganisms can make everything they need from glucose source. Some bacteria can't use sugars and need carbon dioxide or carbonate or bicarbonate. The Media Container 4 is a bottle, cylinder or other suitable container. The Media Container 3 is made of plastic, or any other suitable material. In one embodiment of the invention as illustrated in Figures 1, 2, and 3, the Media Container 4 is a plastic bottle of sufficient dimension to store Sterile Carbon Source of Media for active strain of plant growth promoting bacteria (PGP) / microorganisms in a controlled conditions. The Media Container 4 shapes and compositions can be used without departing from the scope of the invention.

Process for culture and growth of plant growth promoting bacteria (PGP) / microorganisms:

The Growth in the bioreactor is under aerobic conditions at suitable temperatures and pH for growth. For the plant growth promoting bacteria (PGP) / microorganisms, cell growth is preferable at temperatures between 5 and 37° C., with the preferred temperature being in the range of 15 to 30° C., 15 to 28° C., 20 to 30° C., or 15 to 25° C. The pH of the nutrient medium can vary between 4.0 and 9.0, but the preferred operating range is usually slightly acidic to neutral at pH 4.0 to 7.0, or 4.5 to 6.5, or pH 5.0 to 6.0. Typically, maximal cell yield is obtained in 20-72 hours after inoculation.

Optimal conditions for the cultivation of the microorganisms of this invention will, of course, depend upon the particular strain. However, by virtue of the conditions applied in the selection process and general requirements of most microorganisms, a person of ordinary skill in the art would be able to determine essential nutrients and conditions. The microorganisms would typically be grown in aerobic liquid cultures on media which contain sources of carbon, nitrogen, and inorganic salts that can be assimilated by the microorganism and supportive of efficient cell growth. Preferred carbon sources are hexoses such as glucose, but other sources that are readily assimilated such as amino acids, may be substituted. Many inorganic and proteinaceous materials may be used as nitrogen sources in the growth process. Preferred nitrogen sources are amino acids and urea but others include gaseous ammonia, inorganic salts of nitrate and ammonium, vitamins, purines, pyrimidines, yeast extract, beef extract, proteose peptone, soybean meal, hydrolysates of casein, distiller's solubles, and the like. Among the inorganic minerals that can be incorporated into the nutrient medium are the customary salts capable of yielding calcium, zinc, iron, manganese, magnesium, copper, cobalt, potassium, sodium, molybdate, phosphate, sulfate, chloride, borate, and like ions. Without being limited thereto, use of potato dextrose liquid medium for fungal strains and R2A broth premix for bacterial strains is preferred.

Process Steps for culture and growth of plant growth promoting bacteria (PGP) / microorganisms:

The step (i) of culturing the plant growth promoting bacteria (PGP) / microorganisms is carried out in bioreactor Container 1.

The step (ii) the growth the plant growth promoting bacteria (PGP) / microorganisms suitable culture mediums are required. The stored Sterile Salt solutions for Media in Container 2 are transferred to bioreactor Container 1. The Sterile Salt solution in Container 2 are transferred to through sterile plastic tube and needle set or by sterile syringe or any other sterile condition, for a predetermined time, at a suitable temperature.

The step (iii) The Cultures strains of the plant growth promoting bacteria (PGP) / microorganisms are stored in air tight container in controlled conditions in Container 3.

The Cultures strains of the plant growth promoting bacteria (PGP) / microorganisms in Container 3 are transferred to bioreactor Container 1 through a sterile plastic tube and needle set or by sterile syringe or any other sterile condition in controlled and closed conditions.

The step (iv) the growth the plant growth promoting bacteria (PGP) / microorganisms suitable culture mediums are required. The stored culture medium, Sterile Carbon Source of Media in Container 4 is transferred to bioreactor Container 1. The Sterile Carbon Source of Media in Container 4 are transferred to through sterile plastic tube and needle set or by sterile syringe or any other sterile condition, for a predetermined time, at a suitable temperature.

The step (v) culturing of the plant growth promoting bacteria (PGP) / microorganisms in bioreactor Container 1 is growth is carried in controlled temperature 15-35°C and centrifugation at 50 to 500 RPM on Rotary shaker for 5-15 Days, so as to obtain a bacterial phase and a culture broth phase bacteria.

One of the embodiment describes the strain of plant growth promoting bacteria (PGP) / microorganisms that can be used are: Azotobacter Spp., Rhizobium Species, Acetobacter Species . Other example of bacteria species Pseudomonas, Bacillus megaterium, Bacillus subtilis, Azospirillium, Bacillus spp., Clostridium, Escherichia, Proteus, Haliscomenobacter, Zoogloea, Beggiatoa, Acetobacter, Bacteriodes, Methanobacterium, Nitrosomonas, Nocardia, Camplyobacter, Acinobacter, Sphaerotilus, Spirochaeta, Thiobacillus, Desulfovibrio.

All the media components are either packed in single container of mixture or mixed separate container or different combination with optional to use or not to be used in process. Some of the examples are listed below:

Example 1
For Azotobacter Spp.
Container 1 = Sterile Water (Non Pyrogenic) = 1.5 Liter
+ Calcium Carbonate= 5 gm
Mancogeb 0.35 mg
Container 2 = Media 1
(K2HPO4 300 mg, MgSO4 300 mg, NaCl 300 mg)
Container 3 = Bacteria Culture + Glycerol
Container 4 = Mannitol = 24 gm

Example 2
For Rhizobium Species
Container 1 = Sterile Water (Non Pyrogenic) = 1.5 Liter
+ Calcium Carbonate= 3 gm
Mancogeb 0.35 mg
Container 2 = Media 1
(K2HPO4 750 mg, MgSO4 300 mg, NaCl 150 mg)
Container 3 = Bacteria Culture + Glycerol
Container 4 = Mannitol = 15 gm

Example 3
For Acetobacter Species
Container 1 = Sterile Water (Non Pyrogenic) = 1.5 Liter
+ Calcium Carbonate= 15 gm
Mancogeb 0.35 mg
Container 2 = Media 1
(K2HPO4 750 mg, MgSO4 300 mg, NaCl 150 mg)
Container 3 = Bacteria Culture + Glycerol
Container 4 = Glucose = 5 gm

All the container contains sterile except culture container and the pH is in the range of 6.8 to 7.2.The temperature for operation is 26 to 34 Degree centigrade
One of the embodiment describes the change in the composition according to bacterial growth pattern and species
Group of Growth Material Used in Media

Group Code Component Example
WT Sterile Distilled Water
GM 1 Nutrients Protein/Peptide/Amino Acid Source: Yeast Extract, Peptone, Tryptone,
GM 2 Energy Source Carbohydrate:
Glucose, Dextrose, Mannitol, malic acid, Glycerol, jaggery, Sucrose, Fructose, Molasses, Etc
GM 3 Essential Metal &
Minerals Magnesium Sulphate,
Sodium Chloride,
Calcium Chloride,
Ferrous Sulphate,
Manganese sulphate,
Ferrous EDTA,
Sodium Molybdate,
Boric Acid,
Copper Sulphate,
Zinc Sulphate,
Ferric Chloride,
Ammonium Sulphate,
Ammonium Phosphate,
Potassium Chloride,
Ammonium Chloride
Etc.
GM 4 Acidity Controller Calcium Carbonate
Calcium Phosphate
Rock Phosphate
Etc.
GM 5 Buffers Dipotassium Hydrogen, orthophosphate,
Potassium Dihydrogen phosphate,
Sodium Phosphate,
Sodium dihydrogen phosphate,
Disodium Hydrogen Phosphate,
etc.
GM 6 pH Indicator Congo red,
Phenol Rel,
Bromocresol Purple
GM 7 Anti Microbial Substance Mancozeb,
Metalaxyl,
Hexaconazole,
Fluconazole,
Potassium Sorbate
GM8 Acid Base Chemicals Sodium Hydroxide
Potassium Hydroxide
Hydrochloric acid etc.
GM9 Culture Bacteria Culture

Combination Code Container 1 Container 2 Container 3 Container 4 Container 5 Container 6 Container 7 Container 8 Container 9
M1 KIT
Default Fermenter Tank
WT GM1 GM2 GM3 GM4 GM5 GM6 GM7 GM8

M2 Kit Fermenter Tank
WT +/ GM4 +/ GM7 +/ GM6 +/GM8 GM1 +/ GM2 +/GM3 +/GM4 +/GM5+/GM6 +/GM7 +/GM8 GM2 GM3 GM4 GM5 GM6 GM7 GM8
M3 Kit Fermenter Tank
WT+/GM1 +/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8 GM1
+/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8
GM1
+/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8 GM1
+/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8 GM1
+/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8 GM1
+/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8 GM1
+/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8 GM1
+/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8 GM1
+/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8
M4 Kit Fermenter Tank
WT+/GM1 +/ GM2 +/GM3 +/GM4 +/GM5 +/GM6 +/GM7 +/GM8 GM1 +/ GM2 GM2 GM3 +/ GM5 GM4 GM5 GM6 GM7 GM8

Table 1 describes the list of different ingredients for Container 1

List of ingredient/substance for Container 1
Code Name Ingredient/chemical/media Range
1A Sterile Distilled water 5% to 98 % of Container Volume
1B Calcium Carbonate 0.01 % to 25% w/v
1C Calcium Phosphate 0.01 % to 25% w/v
1D Rock Phosphate 0.01 % to 25 % w/v
1E Mancozeb
(or other antifungal/anti microbial) 0.00001% to0.5 %w/v
(1 mg/liter to 5 gm/liter)
1F Sodium Carbonate 0.01 % to 25% w/v

Table 2 describes the list of different ingredients for Container 2
List of ingredient/substance for Salt Solution Container 2
Code Name Ingredient/chemical/media Range Weight/1 liter of Fermentation volume (Range)
2A Sterile Distilled water 5% to 100 % of Container Volume
2B Magnesium Sulphate 0.01 mg to 15 gm
2C Sodium Chloride 0.01 mg to 15 gm
2D Calcium Chloride 0.01 mg to 15 gm
2E Ferrous Sulphate 0.01 mg to 15 gm
2F Manganese sulphate 0.01 mg to 5 gm
2G Ferrous EDTA 0.01 mg to 15 gm
2H Sodium Molybdate 0.01 mg to 15 gm
2I Boric Acid 0.01 mg to 15 gm
2J Copper Sulphate 0.01 mg to 15 gm
2K Zinc Sulphate 0.01 mg to 15 gm
2L Dipotassium Hydrogen orthophosphate 0.01 mg to 15 gm
2M Potassium Dihydrogen phosphate 0.01 mg to 15 gm
2N Ferric Chloride 0.01 mg to 15 gm
2O Ammonium Sulphate 0.01 mg to 15 gm
2P Ammonium Phosphate 0.01 mg to 15 gm
2Q Potassium Chloride 0.01 mg to 15 gm
2R Ammonium Chloride 0.01 mg to 15 gm
2S Potassium Sulphate 0.01 mg to 45 gm
2T Magnesium Chloride 0.01 mg to 15 gm
2V Disodium hydrogen phosphate 0.01 mg to 15 gm
2W Disodium phosphate 0.01 mg to 15 gm
2X Sodium Acetate
2Y Potassium Nitrate
2z
2AA

Table 3 describes List of ingredient/substance Container 3
List of ingredient/substance Container 3
Code Name Ingredient/chemical/media Range
3A Sterile Distilled water 5% to 98 % of Container Volume
3B Culture (minimum 1 x 102 CFU Bacteria) 1% to 98% of container 3
3C Glycerol +/Polyvinylpyrrolidone +/gum arabic 0.01 % to 60% w/v

Table 4 describes List of ingredient/substance Container 4
List of ingredient/substance Container 4
Code Name Ingredient/chemical/media Range Weight/1 liter of Fermentation volume (Range)
4A Sterile Distilled water 5% to 98 % of Container Volume
4B Mannitol 0.01 % to 40% w/v 1 mg to 100 gm
4C Glucose (Dextrose) 0.01 % to 40% w/v 1 mg to 100 gm
4D Malic Acid 0.01 % to 40% w/v 1 mg to 100 gm
4E Glycerol 0.01 % to 40% w/v 1 mg to 100 gm
4F Yeast Extract 0.01 % to 40% w/v 1 mg to 100 gm
4G Jaggery 0.01 % to 40% w/v 1 mg to 100 gm
4H Molasses 0.01 % to 40% w/v 1 mg to 100 gm
4I peptone 0.01 % to 40% w/v 1 mg to 100 gm
4J Pancreatic Digest of Gelatin 0.01 % to 40% w/v 1 mg to 100 gm
4K Tryptone 0.01 % to 40% w/v 1 mg to 100 gm

Table 5 describes List of ingredient/substance Container 5
List of ingredient/substance Container 5
Code Name Ingredient/chemical/media Range
5A Sterile Distilled water 5% to 98 % of Container Volume
5B NaOH +/ KOH 0.01 % to 40% w/v
5C HCl 0.01 % to 30% w/v

Few Example of Kit Composition

Sr No. Bacteria Species Container 1 Container 2 Container 3 Container 4 Container 5
1 Azotobacter Spp. 1A +1B+1E 2A +2B +2C +2E +2F +2L + 2N 3A + 3B +3C 4A +4B -
2 Acetobacter Spp.
Gluconobacter Spp. “ 4F + 4A 3A + 3B +3C 4A + 4C -
3 Rhizobium Spp. “ 2A +2B +2C 3A + 3B +3C 4A + 4B + 4C +4F -
4 Azospirillum “ 2A + 2B +2C +2D + 2G +2F +2H + 2I +2J +2K +2L 3A + 3B +3C 4A + 4D 5A +5B
5 Bacillus Megaterium 1A + 1C +1E 2A +2B +2Q +2O +2F +2E 3A + 3B +3C 4A + 4C +4F -
6 Frateuria Aurantia 1A +1B+1E 2A + 2B 3A + 3B +3C 4A + 4C +4F +4I -
7 Pseudomonas
“ 2A+2S +2T 3A + 3B +3C 4A + 4J -
8 Bacillus Subtilis “ 2A + 2C + 4F “ 4A + 4K -
9 Nitrosomonas 1A +1B+1F 2A + 2B +2D +2O +2J +2E +2M “ 4A +2M +2V +-5B -
10 Proteus “ 2A + 2V +2L “ 4A + 4F -
11 Norcardia

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.
,CLAIMS:We Claim,

[Claim 1]. A kit for mass multiplication of bacteria comprises;
a. A bioreactor Container 1 comprises sterile water, Antifungal substance and pH buffer.
b. Media container 2 comprises of Sterile Salt solution for Media;
c. Bacteria container 3 comprises active strains of microorganisms;
d. Media container 4 comprises Sterile Carbon Source of Media.
Wherein bioreactor Container 1 is bottle with neck and contains rubber closure for sterile transfer of liquid from container 2, container 3 and container 4;
Wherein Bacteria container 3 is airtight sealed container;
Wherein Media container 2 and Media container 4 are plastic bottle;
Sterile Salt solution for Media are transferred form Container 3, active strain of microorganisms are transferred form Container 3, Carbon Source of Media are transferred form Container 4 to bioreactor Container 1 through a sterile tube and needle set in a step wise process in an controlled and closed conditions for culturing the microorganisms.

[Claim 2]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, method for mass multiplication of bacteria comprises as
step (i) A bioreactor Container 1 comprises sterile water, Antifungal substance and pH buffer and Sterile Salt solution for culturing and growth of the microorganisms pH in the range of 6.8 to 7.2;
step (ii) the stored Sterile Salt solution for Media in Container 2 is transferred to bioreactor Container 1; the Sterile Salt solution in Container 2 are transferred to through sterile plastic tube and needle set or by sterile syringe or any other sterile condition;
step (iii) active strain of microorganisms in Container 3 are transferred to bioreactor Container 1 through a sterile plastic tube and needle set or by sterile syringe or any other sterile condition, in a controlled and closed conditions;
step (iv) Sterile Carbon Source of Media in Container 4 is transferred to bioreactor Container 1 through sterile tube and needle set in a controlled and closed conditions;
Step (iv) culturing of the microorganisms is at temperature 15°C - 35°C, pH 6.8 to 7.2 and centrifugation at 50 to 500 RPM for 5-15 Days.

[Claim 3]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, active strains of plant microorganisms is growth promoting bacteria (PGP)

[Claim 4]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, bioreactor Container 1 is bottle with neck wherein neck of containers is having lock mechanism.

[Claim 5]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, bioreactor Container 1 is from size of 100 ml to 100 liter in size.

[Claim 6]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, Container 3, Bacteria Culture Container 3 in size from 2 ml to 10 liter size.

[Claim 7]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, Container 3, Media container is in size range from 2 ml to 25 liter volume size

[Claim 8]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, Volume of Material filled in Container 1 in the range of 10 percent to 90 percent of container 1 volume.

[Claim 9]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, growth Material Used in Media are Sterile Distilled Water, Nutrients, Energy Source, Essential Metal & Minerals, Acidity Controller, Buffers, pH Indicator, Anti Microbial Substance, Acid Base Chemicals, active Culture.

[Claim 10]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, Nutrients are selected from Protein, Peptide, Amino Acid Source, Yeast Extract, Peptone, Tryptone.

[Claim 11]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, Energy Source are selected from Carbohydrate, Glucose, Dextrose, Mannitol, malic acid, Glycerol, jaggery, Sucrose, Fructose, Molasses.

[Claim 12]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, Essential Metal & Minerals are selected from Magnesium Sulphate, Sodium Chloride, Calcium Chloride, Ferrous Sulphate, Manganese sulphate, Ferrous EDTA, Sodium Molybdate, Boric Acid, Copper Sulphate, Zinc Sulphate, Ferric Chloride, Ammonium Sulphate, Ammonium Phosphate, Potassium Chloride, Ammonium Chloride.

[Claim 13]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, Acidity Controller are selected from Calcium Carbonate, Calcium Phosphate, Rock Phosphate.

[Claim 14]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, Buffers are selected from Dipotassium Hydrogen, orthophosphate, Potassium Dihydrogen phosphate, Sodium Phosphate, Sodium dihydrogen phosphate, Disodium Hydrogen Phosphate.

[Claim 15]. A kit for mass multiplication of bacteria claimed in claim 1 wherein, pH Indicator is selected from Congo red, Phenol Rel, Bromocresol Purple.

[Claim 16]. A kit for mass multiplication of bacteria claimed in claim 1 wherein Anti Microbial Substance are selected from Mancozeb, Metalaxyl, Hexaconazole, Fluconazole, Potassium Sorbate.

[Claim 17]. A kit for mass multiplication of bacteria claimed in claim 1 wherein Base Chemicals are selected from Sodium Hydroxide, Potassium Hydroxide

[Claim 18]. A kit for mass multiplication of bacteria claimed in claim 1 wherein Acid are selected from Hydrochloric acid.

[Claim 19]. A kit for mass multiplication of bacteria claimed in claim 1 wherein ingredients for Container 1 comprises:
i. Sterile Distilled water
ii. Calcium Carbonate
iii. Calcium Phosphate
iv. Rock Phosphate
v. Antifungal and anti microbial
vi. Sodium Carbonate

[Claim 20]. A kit for mass multiplication of bacteria claimed in claim 1 wherein ingredients for Container 2 comprises:
i. Sterile Distilled water
ii. Magnesium Sulphate
iii. Sodium Chloride
iv. Calcium Chloride
v. Ferrous Sulphate
vi. Manganese sulphate
vii. Ferrous EDTA
viii. Sodium Molybdate
ix. Boric Acid
x. Copper Sulphate
xi. Zinc Sulphate
xii. Dipotassium Hydrogen orthophosphate
xiii. Potassium Dihydrogen phosphate
xiv. Ferric Chloride
xv. Ammonium Sulphate
xvi. Ammonium Phosphate
xvii. Potassium Chloride
xviii. Ammonium Chloride
xix. Potassium Sulphate
xx. Magnesium Chloride
xxi. Disodium hydrogen phosphate
xxii. Disodium phosphate
xxiii. Sodium Acetate
xxiv. Potassium Nitrate

[Claim 21]. A kit for mass multiplication of bacteria claimed in claim 1 wherein ingredients for Container 3 comprises:
i. Sterile Distilled water
ii. Active Culture
iii. Glycerol
iv. Polyvinylpyrrolidone
v. gum Arabic

[Claim 22]. A kit for mass multiplication of bacteria claimed in claim 1 wherein ingredients for Container 4 comprises:
i. Sterile Distilled water
ii. Mannitol
iii. Glucose (Dextrose)
iv. Malic Acid
v. Glycerol
vi. Yeast Extract
vii. Jaggery
viii. Molasses
ix. peptone
x. Pancreatic Digest of Gelatin
xi. Tryptone

[Claim 23]. A kit for mass multiplication of bacteria claimed in claim 1 wherein ingredients for Container 5 comprises:
i. Sterile Distilled water;
ii. Base;
iii. acid.

[Claim 24]. A kit for mass multiplication of bacteria claimed in claim 1 wherein active culture contains at least 1 x 108 CFU of Bacteria to be produced.

[Claim 25]. A kit for mass multiplication of bacteria claimed in claim 1 wherein Sterile infusion set comprises IV set, sterile syringe, sterile tubes, used for transfer of component from other container to fermenter tank.