FIELD OF THE INVENTION

The present invention provides a novel microbial consortium capable of solubilizing Phosphorus and Potassium in soil as well as fixing atmospheric Nitrogen. The present invention also relates to liquid formulation of microbial consortium of Nitrogen fixing and Phosphorus and Potassium (NPK) solubilizing bacteria. The present invention also provides a method of preparing the novel microbial consortium and the Liquid Formulation of microbial consortium having extended shelf life. Further the present invention also provides a novel liquid medium that is economical and supports the growth of microbial consortium formulation.

BACKGROUND OF THE INVENTION

Microorganisms play a very important role in improving the soil fertility and providing nutrients to the plant (Tilak et al. 2005). Biofertilizers play a very significant role in improving soil fertility by fixing atmospheric nitrogen, both, in association with plant roots and without it, solubilise insoluble soil phosphates and potash minerals and produces plant growth substances in the soil. Biofertilizer contains microorganisms which promote the adequate supply of nutrients to the host plants and ensure their proper development of growth and regulation in their physiology. Effective living microorganisms are used in the preparation of biofertilizers.

The plant rhizosphere is a unique environment characterized by the continuous supply of low molecular weight compounds exuded from the roots. The rhizosphere supports a large and metabolically active microbial population that can be several orders of magnitude higher than in non-rhizosphere soil (Schloter et al. 1997). Interactions between the plant and microorganisms and among rhizosphere microorganisms are largely unknown and recent works show that these interactions are complex and are dependent on multiple traits (Lugtenberg and Dekkers 1999). Although the rhizosphere appears to be too complex to allow its manipulation, specific bacteria can be applied to seed or roots, which cause an alteration in the composition of the rhizosphere. In addition to the manipulation of the microorganisms to discourage disease causing organisms, it should be possible to promote the activity of beneficial ones, such as arbuscular mycorrhizal (AM) fungi, plant growth promoting rhizobacteria (PGPR) and Rhizobium sp. Thus the focus of attention has now shifted from plant-microbe interactions to plant-microbe-microbe interactions. Attempts have been made not only to highlight increase in biological activities in two or three member associations of organisms (Barea et al. 2004) but also to decipher the mechanisms involved in such interactions (Ma et al. 2003). Such syntropic associations are of ecological importance with implied agricultural significance. It is important, therefore, to identify the best strains of beneficial microbes for the planting situation, verify their compatibility and combined efficacy, both in vitro and in vivo, and employ this combination inoculum to real agricultural situation as part of the management and production practices. Compatible combinations have been tested in field, yield increases have been attained but with limited success and consistency. The application of biofertilizer containing mycorrhizal fungus and three species of bacteria namely N-fixer (Azotobacter chroococcum), P solubilizer (Bacillus megaterium) and K solubilizer (Bacillus mucilaginous) significantly increased the growth of Z. mays. Microbial inoculum not only increased the nutritional assimilation of plant (total N, P and K), but also improved soil properties, such as organic matter content and total N in soil (Wu et al. 2005). Coinoculation of phosphate solubilizing bacteria (PSB) Bacillus megaterium var. phosphaticum and potassium solubilizing bacteria (KSB) Bacillus mucilaginosus along with rock P and rock K in nutrient limited soil planted with pepper and cucumber significantly enhanced soil availability of P, the uptake of N, P and K by shoots and roots, and the growth of both pepper and cucumber (Han et al. 2006). Co-inoculation of waste mica with B. mucilaginosus and A. chroococcum A-41 resulted in highest biomass production and nutrient acquisition in sudan grass. Co-inoculation of bacterial strains maintained consistently highest amounts of available K and N in soils even at 150 days of crop growth than other treatments (Basak and Biswas, 2010).

The carrier based microbial inoculants are generally lignite, coal (or) charcoal based. The major disadvantages associated with these carriers are shorter shelf life, poor quality, high contamination and unpredictable field performance. The cost of solid carrier based inoculant production is high as it is labour and energy intensive process, involving milling, sieving and correcting pH (Somasegaran and Hoben, 1994). Liquid inoculant formulation is one solution to the problems associated with processing of solid carriers. The use of various broth cultures amended with substances that promote cells survival in the package and after application for seed (or) soil. Additives to liquid inoculant formulations should have a role in protecting Azospirillum cells on seed at high temperature and during desiccation. Many kinds of polymers have been used for inoculant production because of their ability to limit heat transfer, their good rheological properties and high water activities (Mugnier and Jung 1985). Liquid formulation of Azospirillum amended with different chemical additives was found to be more effective as compared to carrier based inoculants (Kumaresan and Reetha 2011). Formulations with different combinations of microorganisms have been developed and patent granted. However a single liquid formulation of compatible microbes providing all the three major nutrients- nitrogen, phosphorus and potassium and with extended shelf life of one year has not been reported so far.

Biofertilizers are being essential component of organic farming are the preparations containing live or latent cells of efficient strains of nitrogen fixing, phosphate solubilizing or cellulolytic microorganisms used for application to seed and, soil bio-priming or composting areas with the to enhance the effective microorganisms and accelerate those microbial processes which augment enhance the availability of nutrients that can be easily assimilated by plants.

U.S. Pat. No. 5,697,186 discloses the use of microorganisms to enhance crop productivity and, more specifically, to the use of flocculated forms of bacteria, particularly Azospirillum and Rhizobium, or a combination thereof, as crop inoculants and delivery systems for other agriculturally beneficial microorganisms.

U.S. Pat. No. 4,551,164 discloses a composition of bacteria, specially Bacillus, and algae and methods for plant growth promotion. More particularly the invention concerns microbial plant growth promoting compositions and methods for their use.

U.S.Pat. No. 7,097,830 discloses synergistic bioinoculant composition comprising Bacillus strains isolated from cows, either individually or in all possible combinations, and optionally a carrier, with each of the strains showing plant growth-promoting activity.

U.S. Pat. No. 4,155,737 discloses to a process of inoculating microorganisms in plants in a polymer gel in which are embedded microorganisms. The invention is intended for controlling the productivity of cultivated plants.

WO2007110686 application discloses a synergistic composition of at least one strain of Trichodermaharzianum or a combination thereof which is useful as bioinoculant.

U.S. Pat. No.20120015806 discloses a novel formulation of microbial consortium based bioinoculant for wide spread use in agriculture practices. A microbial formulation for plant growth with customized solution comprises at least seven beneficial bacteria; at least two beneficial fungi; at least one yeast; and at least one compound which extends the effective life time of said formulation.
EP 2150610 Biofertilizer formulation discloses strains of psychrophilic bacteria isolated from the rizosphere of Deschampsiaantarctica are characterized and biofertilizer compositions comprising one or more of these psychrophilic bacteria strains are disclosed.

The Nitrogen (N), phosphorus (P) and potassium (K) or the NPK are the major macronutrients required by the plants for healthy and balanced plant growth. These nutrients are added as chemical fertilizers or can be made available to the plants through the activity of microorganisms. Phosphorus is second only to nitrogen in mineral nutrients most commonly limiting the growth of crops. However most of the P added to the soil gets fixed and is not available to the plant. Likewise there are considerable amounts of insoluble K reserves in many soils, most of which exist in alumino-silicate minerals from which K cannot be absorbed directly by plants. Although consortia of microbes have been used for metabolizing soil nutrients as discussed in the above sections, yet none of the presently available methods or consortia of microbes have been very effective in solving the problem of metabolizing NPK nutrients. Therefore there is a dire need to develop such microbial consortias which not only provide an efficient combination of the NPK metabolizing microbes but also have economically beneficial extended shelf life.

SUMMARY OF THE INVENTION

Accordingly, the main embodiment of the present invention provides a microbial consortium comprising ofAzotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillusdecolorationis (MTCC 25044).

Another embodiment of the present invention provides a microbial consortium as herein described wherein the consortium fixes atmospheric Nitrogen and solubilizes, Phosphorus and Potassium in soil to be available as nutrients for plants.

Another embodiment of the present invention provides a microbial consortium as herein described, wherein the said consortium is a liquid formulation.

Another embodiment of the present invention provides a microbial consortium as herein described wherein the consortium enables to augment 25-30 Kg of Nitrogen per hectares, 20-25 Kg of Phosphorus per hectares and 10-15 Kg of Potassium per hectares.

Another embodiment of the present invention provides a medium for culturing that is economical and supports the growth of all three bacteria and said medium comprising of 2.5g of Sodium glutamate, 10g of Glucose, 0.5g Yeast Extract, 2g Sodium chloride, 0.5g Magnesium sulphate, 1.0g Dipotassium hydrogen phosphate, 0.1g Calcium carbonate.

Yet another embodiment of the present invention provides a method of preparing a microbial consortium, said method comprising steps of:
(a) preparing an inoculum of Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044) by culturing them individually for 48 h at 30°C in new designed medium;
(b) transferring 10% inoculum to fermenter vessel (5 liter capacity) and growing the said inoculum for 48-72 h at 30°C;
(c) mixing the cultures of Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044) in 1:1:1 ratio in a sterilized container;
(d) adding additional ingredients and mixing them with NDM containing the culture mixture; and
(e) obtaining a liquid formulation of microbial consortium

Another embodiment of the present invention provides a Liquid Formulation of microbial consortium, said composition comprising of a microbial consortium as herein described, a medium as herein described and additives selected from 2% PVP or 1% PEG, 0.6% of Glycerol and 0.5% of Sucrose.

Another embodiment of the present invention provides a microbial consortium as herein described wherein the said consortium has a shelf-life of at least one year.

Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the the said Formulation can be used for seed treatment, seedling dip in case of transplanted crops and as soil treatment for trees.
Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the use of the said Formulation results in increase in yield of agricultural crops in the range of 5-30%.
Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the said Formulation is cost effective and reduce inputs of artificial fertilizers.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Testing of bacterial cultures for compatibility based on cross streak method
Figure 2: Colony morphology of cultures used in the formulation.
Figure 3: Comparison of solubilization of P and K by single culture and consortium in qualitative assay.
Figure 4: Qualitative comparison of potassium solubilizing efficiency of liquid formulation of consortium before and after incubation for 12 months
Figure 5: Qualitative comparison of phosphorus solubilizing efficiency of liquid formulation of consortium before and after incubation for 12 months.

DETAILED DESCRIPTION

While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown by way of example in the figures and tables and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.

The tables and protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

Definitions:
For the purposes of this invention, the following terms will have the meaning as specified therein:

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that one or more processes or composition/s or systems or methods proceeded by "comprises... a" does not, without more constraints, preclude the existence of other processes, sub-processes, composition, sub-compositions, minor or major compositions or other elements or other structures or additional processes or compositions or additional elements or additional features or additional characteristics or additional attributes.

As used herein, the term “Stickler” when used in the context of the present invention refers to polymers soluble in water and used for adhesion of microbes to seed.

As used herein, the term “Shelf-Life” when used in the context of the present invention refers to the length of time that a bacteria/bacterial consortium may be stored without becoming unfit for use or sale.
As used herein, the term “Seed Bacterization” when used in the context of the present invention refers tocoating or treatment of seeds with Liquid formulation of microbial consortium as herein described.
As used herein, the term “Biofertilizer” when used in the context of the present invention refers tosubstance which contains living microorganisms which, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant.
As used herein, the term “Solubilization” when used in the context of the present invention refers to a process by which unavailable form (insoluble) of soil minerals are converted into available form (soluble) in soil through microorganisms/biological process.
As used herein, the term “P-Solubilization or Phosphorus Solubilization” when used in the context of the present invention refers to a process in which microorganisms release inorganic phosphorus (PO4--) from insoluble phosphorus deposits in soil mainly through production of organic acids. The P-Solubilization or Phosphorus Solubilization microorganism as used in context of the present invention is Burkholderia cepacia (having MTCC 25043).
As used herein, the term “K-Solubilization or Potassium Solubilization” when used in the context of the present invention refers to a process in which microorganisms release potassium (K+ ions) from insoluble potassium bearing silicate minerals in soilmainly through production of organic acids.The K-Solubilization or Potassium Solubilization microorganism as used in context of the present invention is Bacillus decolorationis (having MTCC 25044).
As used herein, the terms “Consortium or Consortia or Microbial Consortium or Microbial Consortia” when used in the context of the present invention refers to mixture of multiple beneficial microorganisms having capacity to increase plant growth as described in the present invention.Consortium or Consortia or Microbial Consortium or Microbial Consortia as described in the context of present invention comprises of Azotobacter chroococcum, (MTCC 25044), Burkholderia cepacia (having MTCC 25043) andBacillus decolorationis (having MTCC 25044).

As used herein, the terms “Nitrogen Fixing Bacteria/N-fixing organism/ Nitrogen fixing/N2-fixing” when used in the context of the present invention refers to microorganisms that reduce atmospheric nitrogen into ammonia by the action of enzyme nitrogenase.The Nitrogen Fixing Bacteria/N-fixing organism/ Nitrogen fixing/N2-fixing microorganism as used in context of the present invention is Azotobacter chroococcum, (MTCC 25044).

As used herein, the terms “Phosphorus fixing bacterial or P-fixing bacteria or Phosphorus Solubilizing or P-Solubilizing bacteria” when used in the context of the present invention refers to a group of beneficial bacteria capable of hydrolysinginsoluble soil phosphate into soluble forms by secreting several organic acids. The KPhosphorus fixing bacterial or P-fixing bacteria or Phosphorus Solubilizing or P-Solubilizing bacteriaas used in context of the present invention is Bacillus decolorationis (having MTCC 25044).
As used herein, the terms “Potassium fixing bacterial or K-fixing bacteria or Potassium Solubilizing or K-Solubilizing bacteria” when used in the context of the present invention refers to a group of beneficial bacteria capable of releasing potassium from insoluble potassium bearing minerals in soil.The Potassium fixing bacterial or K-fixing bacteria or Potassium Solubilizing or K-Solubilizing bacteria as used in context of the present invention is Bacillus decolorationis (having MTCC 25044)
As used herein, the terms “Newly Designed Medium or NDM or Present Medium or Liquid Medium or New Liquid Medium” when used in the context of the present invention refers to development of a novel medium which favors the optimum growth and survival of individual bacterial cultures used in microbial consortium of liquid formulation. The Newly Designed Medium or NDM or Present Medium or Liquid Medium or New Liquid Medium as described in the context of the present invention comprises of necessary ingredients as herein described which is enables the survival and growth of the microbes as herein described in context of the present invention.More specifically NDM as herein described comprises of .5g of Sodium glutamate, 10g of Glucose, 0.5g Yeast Extract, 2g Sodium chloride, 0.5g Magnesium sulphate, 1.0g Dipotassium hydrogen phosphate, 0.1g Calcium carbonate along with 2% PVP, 1% PEG, 0.6% of Glycerol and 0.5% of Sucrose.

As used herein, the terms “Liquid Formulation of Microbial Consortium or Liquid Microbial Formulation or Formulationor Newly Designed Formulation or NDF or Present Formulation or New Formulation or New Liquid Formulation or Consortium Formulation or Consortia Formulation or Liquid Formulation” when used in the context of the present invention refers to a Liquid consortium comprising mixture of functionally different bacterial cultures (e.g. Nitrogen fixing bacteria, phosphorus solubilizing bacteria and potassium solubilizing bacteria) in a new liquid medium as herein described.
As used herein, the terms “Relative % of Functional Activity” when used in the context of the
present invention refers to comparison of functional traits or activity (K and P solubilization) in consortium formulation at initial time and after 12 months incubation with respect to the control.

The present invention provides a novel microbial consortium comprising of Nitrogen fixing, Phosphorus and Potassium (NPK) solubilizing bacteria. The microbial consortium comprises of nitrogen fixing bacteria Azotobacter chroococcum(MTCC Accession No. 25045), phosphorus solubilizing bacteria is Burkholderia cepacia(MTCC Accession No. 25043) and potassium solubilizing bacterial Bacillus decolorationis (MTCC Accession No. 25044).

Further in another aspect the present invention provides a novel and unique Liquid formulation of NPK providing bacteria was developed that consists of a nitrogen fixing Azotobacter chroococcum(MTCC Accession No. 25045), phosphorus solubilizing bacteria is Burkholderia cepacia(MTCC Accession No. 25043) and potassium solubilizing bacterial Bacillusdecolorationis (MTCC Accession 25044). In another aspect the present invention provides that the microbial strains of the consortium are compatible and coexist with each other in the liquid formulation.

In another aspect the present invention provides that ainoculation with the Liquid Formulation of the microbial consortium augments 25-30 kg of Nitrogen per hectare, 20-25 kg Phosphorus per hectareand 10-15 kg potassium per hectare.

In another aspect the present invention provides the use and application of the Microbial consortium and its Liquid Formulation for use in agricultural crops and non-agricultural plants.

One aspect of the present invention provides that the Liquid formulation of NPK is useful for cereals, millets, pulses, vegetables, fiber crops and oil producing commercial crops.

In another aspect the present invention provides that the Microbial consortium and its Liquid Formulation most suited for different type of soils. Particularly the said Microbial consortium and its Liquid Formulation as herein described in useful for soils with pH in the range of 6.5 to 8.5.

In another aspect the present invention provides that the Microbial consortium and its Liquid Formulation is convenient and easy to use and handle

In another aspect the present invention provides that the Microbial consortium and its Liquid Formulation there is No loss in plant growth promoting activities, even on long storage.

In another aspect the present invention provides a microbial consortium and its Liquid formulation in which the microbes are compatible, coexist and also maintain a high cfu count throughout its storage.

In another aspect the present invention provides a microbial culture and it’s Liquid Formulation which has a shelf life of at least one year.

In another aspect the present invention provides a microbial culture and its Liquid Formulation that maintains soil health and keeps soils biologically active.

Accordingly, the main embodiment of the present invention provides a microbial consortium comprising Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillusdecolorationis (MTCC 25044).

Another embodiment of the present invention provides a microbial consortium as herein described wherein the consortium fixes Nitrogen andsolubilizes Phosphorus and Potassium in soil to be available as nutrients for crop plants.

Another embodiment of the present invention provides a microbial consortium as herein described, wherein the said consortium is a liquid formulation.

Another embodiment of the present invention provides a microbial consortium as herein described the microbes are mixed in the ratio of 1:1:1 wherein the cfu count of Azotobacter chroococcum (MTCC 25045) is 2 ? 1011, Burkholderia cepacia (MTCC 25043) is 3 ? 1011 and of Bacillus decolorationis (MTCC 25044) is 4 ? 1011.

Another embodiment of the present invention provides a microbial consortium as herein described wherein the consortium enables to augment 25-30 kg of Nitrogen per hectare, 20-25 kg of Phosphorus per hectare and 10-15 kg of Potassium per hectare.

Another embodiment of the present invention provides a microbial consortium as herein described wherein the said consortium has a shelf-life of at least one year.

Yet another embodiment of the present invention provides a method of preparing a microbial consortium, said method comprising steps of:
(a) preparing an inoculum of Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044) by culturing them individually for 48 h at 30°C in new designed medium;
(b) transferring 10% inoculum to fermenter vessel (5 liter capacity) and growing the said inoculum for 48-72 h at 30°C;
(c) mixing the cultures of Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis(MTCC 25044) in 1:1:1 ratio in a sterilized container;
(d) adding additional ingredients and mixing them with NDM containing the culture mixture; and
(e) obtaining a liquid formulation of microbial consortium.

Yet another embodiment of the present invention provides a method of preparing a microbial consortium as herein described wherein the microbes are mixed in the ratio of 1:1:1 wherein the cfu count of Azotobacter chroococcum (MTCC 25045) is 2 ? 1011, Burkholderia cepacia (MTCC 25043) is 3 ? 1011 and of Bacillus decolorationis (MTCC 25044) is 4 ? 1011.

Yet another embodiment of the present invention provides a method of preparing a microbial consortium as herein described wherein the additional ingredientsselected from 2% PVP, 1% PEG, 0.6% of Glycerol and 0.5% of Sucrose.

Another embodiment of the present invention provides a medium for culturing microbial consortium said medium comprising of 2.5g of Sodium glutamate, 10g of Glucose, 0.5g Yeast Extract, 2g Sodium chloride, 0.5g Magnesium sulphate, 1.0g Dipotassium hydrogen phosphate, 0.1g Calcium carbonate.

Another embodiment of the present invention provides a medium as herein described wherein said medium is highly economical and effective for formulating the microbial consortium.

Another embodiment of the present invention provides a medium as herein described for the formulation of microbial consortium as herein described.

Another embodiment of the present invention provides a Liquid Formulation of microbial consortium, said composition comprising of a microbial consortium as herein described, a medium as herein described and additives selected from 2% PVP or 1% PEG, 0.6% of Glycerol and 0.5% of Sucrose.

Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the said Liquid Formulation microbes are mixed in ratio of 1:1:1 wherein the cfu count of Azotobacter chroococcum (MTCC 25045) is 2?1011, Burkholderia cepacia (MTCC 25043) is 3?1011 and of Bacillus decolorationis (MTCC 25044) is 4?1011.

Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the shelf life of the formulation is at least one year.

Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the microbial consortium retains its functional attributes even on long storage.

Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the microbial consortium of the formulation augments 25-30 kg of Nitrogen per hectare, 20-25 kg of Phosphorus per hectare and 10-15 kg of Potassium per hectare.

Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the seed bacterization with the formulation can improve the seed germination, growth and development of both agricultural crops and non-agricultural seeds and plants.

Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the use of the said Formulation results in increase in yield of agricultural crops in the range of 5-30%.

Yet another embodiment of the present invention provides a Liquid Formulation as herein described wherein the said Formulation is cost effective and reduce inputs of artificial fertilizers.

Another embodiment of the present invention provides for use of the microbial consortium comprising Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillusdecolorationis (MTCC 25044) for as formulation for use in enhancing nitrogen fixation and solubilizing Phosphorus and Potassium.

In another embodiment the present invention provides for use of microbial consortium as herein described for improving seed germination, growth and development of both agricultural crops and non-agricultural seeds and plants.

Yet another embodiment of the present invention provides the use of Liquid Formulation as herein described for increasing the yield of agricultural crops in the range of 5-30%.

Yet another embodiment of the present invention provides a microbial consortium as herein described for increasing the yield of agricultural crops in the range of 5-30%.

Yet another embodiment of the present invention provides for use of microbial consortium comprising Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044) for fixing atmospheric Nitrogen and solubilizing Phosphorus and Potassium in soil to be available as nutrients for crop plants.

Yet another embodiment of the present invention provides for use of New Liquid Medium for culturing microbial consortium comprising Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044).

Yet another embodiment of the present invention provides for use of Liquid Formulation comprising a microbial consortium in new liquid medium for augmenting atmospheric Nitrogen and solubilizing Phosphorus and Potassium in soil to be available as nutrients for crop plants.

The invention will now be explained with the help of following examples. However, the scope of the invention should not be limited to these examples as the person skilled in the art can easily vary the proportion of the ingredients and combinations.

EXAMPLES

EXAMPE 1
Selection of Compatible Microbial Strains
A. K-solubilizing Microbial Strains

Among the K solubilizing isolates, Pseudomonas aeruginosa (A) inhibited the growth of all the cultures tested (Burkholderia cepacia, Azotobacterchroococcum). Bacillus firmus (B) inhibited the growth of Azotobacterchroococcum W5 (Figure 1).

Among K solubilizers, both Bacillus decolorationis and Bacillus halodurans were compatible with other strains. Since Bacillus decolorationis was efficient for K solubilization as compared to B. halodurans it was selected for developing consortium (Table 1).

Table 1: K solubilizing bacterial isolates tested for compatibility
Isolates Code on plate Belongs to K released in broth (ppm)
TSS A Pseudomonas aeruginosa 30.2
IARI-SL-8 B Bacillus firmus 32.8
IARI-SL-13 C Bacillus decolorationis 32.8
IARI-SL-21 D Bacillus halodurans 29

For the P solubilization, strain PS-27 (MTCC 2503) was selected for development of consortium as this strain was found to be best in P-solubilization.

Table 2: P-solubilizing bacterial isolate tested for compatibility
Isolate Code on plate Belongs to
PS-27 (MTCC 2503) 2 Burkholderia cepacia(earlierPseudomonasstriata)

Among the various strains of N-fixing bacteria which were tested, the Azotobacter chroococcumstrain W5 (MTCC 25045) was selected because it was not only highly efficient in nitrogen fixation but also influence the growth and yield of diverse and variety of vegetable, fruit and crop plants.

Table 3: Nitrogen fixing bacteria tested for compatibility
Isolate Code on Plate Belongs to
W5 (MTCC 25045) 4 Azotobacter chroococcum

EXAMPLE 2
Growth media for different organisms
At present different growth media are used for growth of different microbes. More specifically it is known that Jensen’s N free Medium is used for culturing Azotobacter species (Jensen, 1954), Pikovskaya's medium is used for culturing Burkholderia cepacia, and Aleksandrov medium is used for culturing Bacillus decolorationis. The use of different media for different microbial cultures is an expensive proposition for farmers and breeders because it requires them to prepare separate inoculums of different microbes in different media before using them in the seeds or soil.

One of the most unexpected and surprising advantage of the present invention is that the bacterial strains of the present invention, namely Azotobacter chroococcum, Burkholderia cepacia and Bacillus decolorationis can be cultured in single medium i.e. in the new designed medium or NDM. The NDM of the present invention (Table 4) is unique as it not only enables growth and survival of the bacterial strains as herein described but also enables the microbial consortia to be stored for an extended period and thus has a long shelf-life. In other words the NDM of the present invention is common to all the three microbes and economically cheaper. The Table 5 provides the cost effective comparison between the three known media versus the single NDM. It has been found that the average cost of three known media taken together (namely Jensen’s N free Medium, Pikovskaya's medium and Aleksandrov medium) is higher than the cost of the single NDM medium of the present invention. Accordingly, the cost of 1 liter NDM is INR 10.59/- while the combined cost of 1 liter media for three different media is INR12.01 (INR 8.95+INR 9.81+INR 17.29/3).

Table 4: The composition of Newly Designed Medium or Novel Medium is:

Ingredients Concentration
Sodium glutamate 2.5 g
Glucose 10 g
Yeast Extract 0.5 g
Sodium chloride 2.0 g
Magnesium sulphate 0.5 g
Dipotassium hydrogen phosphate 1.0 g
Calcium carbonate 0.1g
Distilled Water 1000 mL
pH 7.2

Table 5: Comparison of cost of newly designed medium with conventionally used medium
Newly Designed Medium (NDM) Aleksandrov medium Pikovaskaya medium Jensen’s medium
Constituents Amount (g/l) Cost (Rs.) Constituents Amount (g/l) Cost (Rs.) Constituents Amount (g/l) Cost (Rs.) Constituents Amount (g/l) Cost
(Rs.)
Sodium glutamate 2.5 2.43 Glucose 5 2.15 TCP 5 3.15 Sucrose 20 14.6
Glucose 10 4.3 MgSO4. 7H2O 0.5 0.20 Glucose 10 4.3 Dipotassium phosphate 1 1.04
Sodium chloride 2 0.58 FeCl3 0.005 0.003 (NH4)2SO4 0.5 0.17 Magnesium sulphate 0.5 0.20
Magnesium sulphate 0.5 0.20 CaCO3 0.1 0.06 NaCl 0.2 0.06 Sodium chloride 0.5 0.15
Yeast extract 0.5 1.98 Calcium phosphate 2 1.26 KCl 0.2 0.10 Ferrous sulphate 0.1 0.05
Potassium phosphate (dibasic) 1 1.04 Potassium minerals 2 5.28 MgSO4.7H2O 0.1 0.04 Sodium molybdate 0.005 0.03
Calcium carbonate 0.1 0.06 Yeast Extract powder 0.5 1.98 Calcium carbonate 2 1.22
Ferrous sulphate 0.005 0.003
MnSO4 0.005 0.005
Total amount/litre of medium (in INR) 10.59 8.95 9.81 17.29

EXAMPLE 3

Increasing the Microbial Survival and Shelf-life of Microbial Formulations

Increasing the Microbial Survival

NDM broth was tried in combination with different additional ingredients to increase the survival of Burkholderia cepacia, Azotobacterchroococcum and Bacillus decolorationis cells in a liquid formulation. The various additional ingredients like PVP 2%, PVA 2%, polyethylene glycol (PEG) 1%, Ethylene glycol (EG) 1%, Glycerol 0.6%, Trehalose 0.5% and Sucrose (0.5%)were added to 100 mL of NDM broth individually and in combinations. The details of the combinations are given in Table 6. One ml of log phase culture of Burkholderia cepacia, Azotobacterchroococcum or Bacillus decolorationis was inoculated individually in each broth. Control (without any chemical ingredient) was also maintained and the flasks were incubated on rotary shaker at 280C. The broth cultures were analysed for viable cell population at 7 days interval uptoone month. The results are presented in Table 6. Among the various formulations prepared, based on the cfu count, the formulations 6, 11, 16 and 21were found to be best to support the growth of all the three bacterial for 1 month and thus were further selected for shelf life study.

Increased Shelf-Life Microbial Formulations
Formulations 6, 11, 16 and 21 were tested for shelf life by recording cfu counts at monthly intervals for a period of 12 months. The results are presented in Table 7. Based on the results, both formulations 6 and 11 were stable and maintained a population count of more than 109 for each bacteria even after one year of incubation. Amongst the two, NDM + 1%PEG + 0.6%Glycerol + 0.5% Sucrose was finally selected. This was because PVP has to be heated prior to mixing and requires an additional step during preparation.

Table 6: Effect of additives on population count of Burkholderia cepacia (P), Azotobacterchroococcum(N) and Bacillus decolorationis (K) after 30 days.

Formulations 1 week 2 weeks 3 weeks 4 weeks
K P N K P N K P N K P N
Formulation 1 (Control)
Newly Designed Medium (NDM) 6.7 x 108 4.4 x 107 7.3 x 107 8.3 x 1010 7.6 x 109 9.6 x 109 3.3 x 108 8.3 x 107 7.6 x 107 6.7 x 107 4.5 x 106 5.7 x 106
Formulation 6
NDM + PVP + Glycerol + Sucrose 9.8 x 1010 8.9 x 1010 9.1 x 1010 2.5 x 1011 2.4 x 1011 2.8 x 1011 2.1 x 1011 2.2 x 1011 2.6 x 1011 1.9 x 1011 2.1 x 1011 2.3 x 1011
Formulation 11
NDM +PEG + Glycerol + Sucrose 9.3 x 1010 8.9 x 1010 7.9 x 1010 2.9 x 1011 2.7 x 1011 3.1 x 1011 2.3 x 1011 2.1 x 1011 2.8 x 1011 1.9 x 1011 1.9 x 1011 2.2 x 1011
Formulation 16
NDM + EG + Glycerol + Sucrose 5.9 x 108 4.7 x 108 8.4 x 107 1.7 x 1010 1.1 x 1010 8.6 x 109 9.7 x 109 5.1 x 109 6.9 x 109 4.9 x 108 3.3 x 109 1.3x 109
Formulation 21
NDM + PVA + Glycerol + Sucrose 8.7 x 109 9.1 x 109 1.3 x 1010 2.3 x 1011 2.1 x 1011 2.7 x 1011 6.9 x 1010 8.7 x 1010 8.1 x 1010 4.1 x 1010 6.8 x 1010 4.3 x 1010
Note: The following concentration of additives were used in medium: PVP (2%), PVA (2%), EG (1%), PEG (1%), Glycerol (0.6%), Sucrose (0.5%0 and Trehalose (0.5%)

Table 7: Population count (cfu) of Burkholderia cepacia (P), Azotobacterchroococcum (N) and Bacillus decolorationis (K) after 12 months of incubation at room temperature.

Time in months Formulation No. 6 Formulation No. 11
N P K N P K
0 day 2.6 x 1011 9.7 x 1010 3.68 x 1011 4.0 x 1010 6.56 x 1011 5.56 x 1011
1 2.8 x 1011 2.1 x 1011 2.92 x 1011 1.9 x 1011 1.64 x 1011 2.68 x 1011
2 2.6 x 1011 1.28 x 1011 2.72 x 1011 1.64 x 1011 1.44 x 1011 2.48 x 1011
3 2.88 x 1011 8.7 x 1010 2.6 x 1011 1.72 x 1011 1.28 x 1011 1.98 x 1011
4 9.2 x 1010 7.6 x 1010 8.3 x 1010 3.3 x 1011 1.3 x 1011 1.28 x 1011
5 5.6 x 1010 6.3 x 1010 2.1 x 1010 1.2 x 1011 8.5 x 1010 1.72 x 1011
6 3.8 x 1010 5.2 x 1010 3.3 x 1010 8.12 x 1010 1.22 x 1011 1.72 x 1011
7 8.5 x 1010 3.8 x 1010 5.2 x 1010 1.8 x 1011 8.7 x 1010 1.09 x 1011
8 7.9 x 1010 4.4 x 1010 4.7 x 1010 1.3 x 1011 1.18 x 1011 9.8 x 1010
9 4.1 x 1010 3.6 x 1010 2.9 x 1010 1.06 x 1011 6.8 x 1010 1.32 x 1011
10 2.8 x 1010 2.2 x 1010 2.3 x 1010 6.4 x 1010 5.4 x 1010 9.3 x 1010
11 9.4 x 109 7.3 x 109 4.8 x 1010 4.3 x 1010 4.3 x 1010 8.7 x 1010
12 7.2 x 109 4.5 x 109 8.4 x 109 3.2 x 109 2.8 x 109 1.6 x 109

EXAMPLE 4
Functional attributes exhibited by individual bacterium and Microbial/Bacterial consortium
In order to look for any synergism in functional attributes in the consortium, the values of K and P solubilization was compared with single cultures individually and in consortium. The Qualitative and Quantitative analysis revealed based on measurement of zone of solubilization, that there was a significant increase in both K and P solubilization in consortium as compared to single culture.(Table 8, Figure3 and Table 9).
Table 8.Comparison of solubilization of P and K by single culture and consortium in qualitative assay.

Single culture Consortium
Colony Size Total Size Halo size Relative % of efficiency Colony Size Total Size Halo size Relative % of efficiency
K-solubilization 8 13.25 5.25 65.63 8 15.25 7.25 90.63
P-solubilization 9 20.5 11.5 127.78 9 20.5 11.5 127.78

Table 9.Comparison of solubilization of P and K by single culture and consortium in quantitative assay.

Single Consortium
Available K (in µg/mL) 35.6 36.9
Relative % of functional activity 100 103.65
Available P (in µg/mL) 63.8 78.8
Relative % of functional activity 100 123.51

Functional attributes of consortium before and after incubation for 12 months
The two attributes, that is, P and K solubilization by the consortium formulation was studied both at the initial and final time of incubation. The results revealed that after 12 months of incubation, there was a slight decrease in both P and K solubilization based on qualitative assay on plates by measuring the size of the colony and the halo zone size(Table 10, Figures 4 and 5). Quantitative estimation also revealed a slight decrease in available K after 12 month of incubation. However P solubilization activity was slightly enhanced after the incubation period of 12 months (Table 11).

Table 10: Functional activity in liquid formulations at the initial and final time of incubation of consortium.
Formulations Functional attribute Initial After 12 months
Colony Size (mm) Total Size (mm) Halo size (mm) *Relative % of efficiency Colony Size (mm) Total Size (mm) Halo size (mm) Relative % of efficiency
6 K-solubilization 7 12.25 5.25 75 7 11.66 4.66 66.57
P-solubilization 7 11.5 4.5 64.3 6 9.25 3.25 54.17
11 K-solubilization 8 12.66 4.66 58.25 9 13.25 4.25 47.22
P-solubilization 4 9.25 5.25 131.25 6 10.75 4.75 79.17
*Relative % of efficiency = Total size- Colony size / Colony size x 100

Table11: Quantitative comparison ofP and K solubilizing efficiency of liquid formulation of consortium before and after
incubation for 12 months.
Formulations K-solubilization P-solubilization
Initial After 12 months Initial After 12 months
Formulation No. 6 Amount released (in µg/mL) 32.8 29.8 62.2 66.7
Relative % of functional activity 100 90.85 100 107.23
Formulation No. 11 Amount released (in µg/mL) 33.2 28.7 61.7 63.4
Relative % of functional activity 100 86.45 100 102.76

EXAMPLE 5

Development of liquid formulation of NPK for enhanced shelf life

1. Medium composition used for formulation of consortium
Low cost medium was designed for liquid Biofertilizer production in bulk amount. The compositions of medium (g/L) are: 2.5 Sodium glutamate, 10 Glucose, 0.5 Yeast Extract, 2 Sodium chloride, 0.5 Magnesium sulphate, 1 Dipotassium hydrogen phosphate, 0.1 Calcium carbonate. The pH of medium was adjusted to 7.2 by adding 1 M NaOH.

2. Growth condition of cultures in new designed medium
Initially individual culture was grown for 48 hours at 30 °C in new designed medium for inoculum preparation. After inoculum preparation, 10% inoculum was inoculated in fermenter vessel (5 lt capacity)and grown for 48-72 h at 30 °C. The cfu count of each culture was recorded following dilution plate count method and was found to be 2 x 1011, 3x 1011 and 4x 1011 for Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillusdecolorationis(MTCC 25044) respectively.

3. Mixing of three cultures: The strains of three cultures were mixed with each other in 1:1:1 ratio in an sterilized container, wherein the cfu count of strain Azotobacter chroococcum (MTCC 25045) was 2 x 1011; of strain Burkholderia cepacia (MTCC 25043) was 3x 1011 and of strain Bacillusdecolorationis (MTCC 25044) was and 4x 1011.

4. Composition of Additives used in liquid formulation of NPK –

The formulation was prepared by using different additives. The compositions of additives used in formulation are as follow –

Table 12: Additional Additives in the Liquid Formulation.
Additives name Final concentration in formulation (%)
Polyethylene glycol 1
Glycerol 0.6
Sucrose 0.5

EXAMPLE 6
Method of Application in the Fields
It can be applied as seed treatment, seedling dip in case of transplanted crops and as soil treatment for trees. It is diluted in required amount of water. Stickler is not needed for its application. Seed bacterization with this biofertilizer can improve germination of seeds leading to better seedling health and vigour. The Table 13 exemplifies that that microbial consortium of the present invention can be applied to various crop plants.

Dosage for Application:

1.For seed application: For treating seeds for one acre of land area, a bottle of 100 ml to be diluted to 1000 mL with water and poured over seeds. The bacterial suspension is sprinkled on the seeds and the seeds are thoroughly mixed so as to have a uniform coating. Application ofinoculant to the seed surface prior to sowing is the traditional, most commonly used and most user-friendly means of inoculation. The seeds are spread uniformly for drying on a gunny bag or cement floor in shade avoiding direct sunlight.
2.For seedling application: In case of transplanted crops, the seedlings can be treated. Prepare a suspension of inoculant by mixing 200 mL of inoculant with1.5 lt of water. Dip the roots of the seedlings in the suspension for 5-10 minutes and then the seedlings can be transplanted.
3. For orchard trees: In case of drip irrigation, adjust the volume of the inoculant based on the number of trees to be inoculated. The inoculant used per tree is 10 mL.
4.Flood irrigation: In case flood irrigation has to be done, 500 mL of inoculant is required for 1 acre of land.

Table 13:Influence of inoculation of NPK consortium on yield of crop plants and saving of chemical fertilizer

Crop Yield increase (%) or other benefits Saving of chemical fertilizer
Rice 5-30% N- 25-30 kg/ha
P- 10-15 kg/ha
K- 2-5 kg/ha
Wheat
10- 12% N- 25-30 kg/ha
P- 10-15 kg/ha
K- 2-5 kg/ha
Maize
5-7% N- 25-30 kg/ha
P- 10-15 kg/ha
K- 2-5 kg/ha
Citrus
NPK and Zn solubilizer was used in 8 acres of citrus orchard. Two acres were used as control.
In 2 acres of control area, the incidence of diseases like leaf minor, lemon caterpillar & canker was more. There were deficiency symptoms of micronutrients. However in 8 acres where biofertilizers were used incidence of diseases was markedly less and
deficiency of micronutrients was not seen. The size and quality of fruit was excellent.

References:
1. Bardiya MC and Gaur AC 1974: Isolation and screening of microorganisms dissolving low-grade rock phosphate. Folia Microbial., 19,386-389
2. Barea JM, Azcon R, Azcon-Aguilar C (2004) Mycorrhizal fungi and PGPR. In: Kamp RM, Calvete JJ, Choli-Papadopoulou T (eds). Principles and practice: methods in proteome and protein analysis. Springer, Berlin Heidelberg New York
3. Basak, B.B. and Biswas, D.R. (2010). Co-inoculation of potassium solubilizing and nitrogen fixing bacteria on solubilization of waste mica and their effect on growth promotion and nutrient acquisition by a forage crop. Biol. Fertil. Soils. 46: 641-648.
4. Han, H.S. Supanjani, and Lee K.D.(2006). Effect of co-inoculation with phosphate and potassium solubilizing bacteria on mineral uptake and growth of pepper and cucumber.Plant Soil Environ.,52(3): 130-136.
5. Kumaresan G. and Reetha D. (2011) Survival of Azospirillumbrasilensein liquid formulation amended with different chemical additives. Journal of Phytology 2011, 3: 48-51
6. Lugtenberg BJJ, Dekkers LC (1999) What makes Pseudomonas bacteria rhizosphere competent? Environ Microbiol 1:9--13
7. Ma W, Guinei FC, Glick BR (2003) Rhizobium leguminosarum biovar viciae1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants. Appl Environ Microbiol 69:4396—4402
8. Mugnier J and Jung G (1985) Survival of bacteria and fungi in relation to water activity and the solvent properties of water in biopolymer. Appl. Environ. Microbiol. 50, 108-14.
9. Schloter M, Wiehe W, Assmus B, Steindl H, Bekke H, Hoflick G, Hartman A (1997) Root colonization of different plants by plant -growth promoting Rhizobium leguminosarumbv. trifolii R39 studied with monospecific polyclonal antisera. Appl Environ Microbiol 63:2038--2046
10. Somasegaran P and Hoben JH (1994), Hand book for rhizobia, methods in legume-rhizobium technology. Springer-Verlag, New York, Inc. New York.
11. Tilak, K.V.B.R., Ranganayaki, N., Pal, K.K., De, R., Saxena, A.K., Nautiyal, C.S., Mittal, S., Tripathi, A. K. and Johri, B.N. 2005. Diversity of plant growth and soil health supporting bacteria. Current Science 89: 136-150.
12. Wu, S.C.; Cao, Z.H.; Li, Z.G.; Cheung, K.C. and Wong M.H. (2005). Effect of biofertilizer containing N2-fixer, P and K solubilizers and AM fungi on maize growth: a green house trial. Geoderma.125: 155-166.
13. US7097830.
14. US20030202999.
15. US20070060477.
16. US20080318777.
17. US20090308121.
18. US2010015806.
19. EP0127642

Claims:We Claim:

1. A microbial consortium comprising Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044).

2. The microbial consortium as claimed in claim 1, wherein the consortium fixes atmospheric Nitrogen and solubilizes Phosphorus and Potassium in soil to be available as nutrients for crop plants.

3. The microbial consortium as claimed in claim 1, wherein the said consortium is a liquid formulation.

4. The microbial consortium as claimed in claims 1 and 3, wherein microbes are present in a ratio of 1:1:1 wherein the cfu count of Azotobacter chroococcum (MTCC 25045) is 2?1011, Burkholderia cepacia (MTCC 25043) is 3?1011 and of Bacillus decolorationis (MTCC 25044) is 4?1011.

5. The microbial consortium as claimed in claims 1-4, wherein the consortium enables to augment 25-30 kg of Nitrogen per hectares , 20-25 kg of Phosphorus per hectares and 10-15 kg of Potassium per hectares.

6. The microbial consortium as claimed in claims 1-5, wherein the said consortium has a shelf-life of at least one year.

7. A method of preparing a microbial consortium, said method comprising steps of:
(a) preparing an inoculum of Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044) by culturing them individually for 48 h at 30°C in new designed medium;
(b) transferring 10% inoculum to fermenter vessel (5 liter capacity) and growing the said inoculum for 48-72 h at 30°C;
(c) mixing the cultures of Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044) in 1:1:1 ratio in a sterilized container;
(d) adding additional ingredients and mixing them with NDM containing the culture mixture; and
(e) obtaining a liquid formulation of microbial consortium.

8. The method of preparing a microbial consortium, as claimed in claim 7, wherein each culture of the bacteria is mixed in the ratio of 1: 1: 1 in the microbial consortium, wherein the cfu count of Azotobacter chroococcum (MTCC 25045) is 2?1011, Burkholderia cepacia (MTCC 25043)is 3?1011 and of Bacillusdecolorationis (MTCC 25044)is 4?1011.

9. The method of preparing a microbial consortium as claimed in claim 7, wherein the additional ingredients selected from 2% PVP, 1% PEG, 0.6% of Glycerol and 0.5% of Sucrose.

10. A Liquid Medium for culturing microbial consortium said medium comprising of 2.5g of Sodium glutamate, 10g of Glucose, 0.5g Yeast Extract, 2g Sodium chloride, 0.5g Magnesium sulphate, 1.0gDipotassium hydrogen phosphate, 0.1g Calcium carbonate.

11. The liquid medium as claimed in claim 10, wherein said medium is economical and effective for formulating the microbial consortium.

12. The liquid medium as claimed in claim 10, for the formulation of microbial consortium as claimed in claim 1.

13. A Liquid Formulation of microbial consortium, said formulation comprising of a microbial consortium as claimed in claim 1 in a liquid medium as claimed in claim 10 and additives selected from 2% PVP, 1% PEG, 0.6% of Glycerol and 0.5% of Sucrose.

14. The Liquid Formulation as claimed in claim 13, wherein the said Liquid Formulation the microbes are mixed in the ratio of 1:1:1 wherein the cfu count of Azotobacter chroococcum (MTCC 25045) is 2?1011, Burkholderia cepacia (MTCC 25043) is 3?1011 and of Bacillus decolorationis (MTCC 25044) is 4?1011.

15. The Liquid Formulation as claimed in claims 13-14, wherein the shelf life of the formulation is at least one year.

16. The Liquid Formulation as claimed in claims 12-15, wherein the microbial consortium of the formulation augments 25-30 kg of Nitrogen per hectare , 20-25 kg of Phosphorus per hectare and 10-15 kg of Potassium per hectare.

17. The Liquid Formulation as claimed in claims 12-16, wherein the seed bacterization with the formulation can improve the seed germination, growth and development of both agricultural crops and non-agricultural seeds and plants.

18. The Liquid Formulation as claimed in claims 12-17, wherein the use of the said Formulation results in increase in yield of agricultural crops in the range of 5-30%.

19. The Liquid Formulation as claimed in claims 12-18, wherein the said Formulation is cost effective and reduces inputs of chemical fertilizers.

20. Use of microbial consortium comprising Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044) for fixing atmospheric Nitrogen and solubilizing Phosphorus and Potassium in soil to be available as nutrients for crop plants.

21. Use of New Liquid Medium for culturing microbial consortium comprising Azotobacter chroococcum (MTCC 25045), Burkholderia cepacia (MTCC 25043) and Bacillus decolorationis (MTCC 25044).

22. Use of Liquid Formulation comprising a microbial consortium in new liquid medium for augmenting atmospheric Nitrogen and solubilizing Phosphorus and Potassium in soil to be available as nutrients for crop plants.