DESC:microbial formulation along with chemical fertilizer. The invention also relates to a method to obtain composition of microbial enriched macronutrients for use in agriculture.

Background of the invention:
Chemical fertilizers have been used from decades in agriculture to provide essential macronutrients such as phosphorus and potassium. However, continuous use or overuse of chemical fertilizers have negative impact on soil and environment. Microbial based fertilizers are observed to be among prime agricultural components that improve crop productivity, and soil health which contribute to sustainable ecosystem. Application of fertilizers in minimum rational quantity combined with microbial enrichment will reduce need of chemical fertilizers, thereby preventing chemical leaching into soil.

Further exploration in terms of application of combined use of microbes and chemical fertilizer can improve the sustainability of soil in terms of improving soil microflora which in turn effects crop growth, defense against pest and crop yield. Microbes play important role in degrading the macronutrients, specifically P and K to a form which is easy available to the plants. Use of microbes affects the soil properties, reduces abiotic stress, and make it feasible for others beneficial plant associated microbes to flourish.

CN101163653B relates to an enhanced fertilizer comprising fertilizer particles, lactic acid bacteria and bacteria of the Baciliaceae family. The invention also relates to an enhancer for a fertilizer and a soil additive to enhance plant growth. The invention further describes the methods for increasing growth, development or yield of a plant and methods of enhancing a soil for increasing growth, development or yield of a plant.

WO2013148278A1 provides Microbial composition comprising liquid fertilizer and processes for agricultural use. The compositions comprises a microbial composition and liquid fertilizer, preferably a liquid fertilizer that contains at least soluble nitrogen. The composition can be applied immediately to soil, foliage, seeds or seedlings. Alternatively, the composition may be activated by incubation for up to about seven days to form an activated composition. The incubation conditions influence the properties of the consortium after incubation. In one embodiment, the compositions comprise one or more lactic acid producing bacteria, one or more nitrogen fixing bacteria, and liquid fertilizer comprising soluble nitrogen. In preferred embodiments, there are multiple genera and/or species of lactic acid bacteria and nitrogen fixing bacteria.

There is huge use of chemical fertilizers depleting soil with essential nutrients and posing serious environmental threats. The present invention addresses the above issues by providing a composition of microbial enriched essential macronutrients for use in agriculture. The composition enhances crop characteristics in various ways.

Object of the invention:
Accordingly, the main object of the present invention is to provide a composition of microbial enriched macronutrients formulation to enhance the availability of essential plant nutrients, improving soil properties, enhance crop yield and simultaneously reducing chemical need.

Another object of the present invention is to provide a composition of water-soluble formulation of augmented essential macronutrients that contain at least microbes, phosphorus and potassium together.
Yet another object of the present invention is to provide a composition comprising a novel microbial formulation along with chemical fertilizer.

Yet another object of the present invention is to provide a novel microbial formulation with potash mobilizing, phosphate and zinc solubilizing efficiency.

Yet another object of the present invention is to provide a composition of potash mobilizing bacteria and phosphorus solubilizing bacteria along with chemical fertilizer.

Yet another object of the present invention is to provide a composition in which microbes are compatible with chemical fertilizer and shows a longer shelf life of at least 24 months in the composition.

Yet another object of the present invention is to provide a method to obtain composition of microbial enriched macronutrients for use in agriculture.

Summary of the present invention:
It will nevertheless be understood that no limitation of the scope of the invention is thereby intended by way of embodiments and examples. Such alterations and further modifications in the present invention, and such further applications of the principles of the invention as would normally occur to those skilled in the art are to be construed as being within the scope of the present invention.

It will be understood by those skilled in the art that the summary of the invention provided herein is exemplary and explanatory of the invention and are not intended to be restrictive thereof. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The composition, methods, and examples provided herein are only illustrative and not intended to be limiting.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more steps of method or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other, steps or components. Appearances of the phrase "in a preferred embodiment”, “in an embodiment", “in another embodiment” and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

Accordingly, the present invention provides a composition of microbial enriched essential macronutrients formulation comprising at least one microbe mixed with essential macronutrients or chemical fertilizer. The composition of microbial enriched essential macronutrients of the present invention enhances crop production, increase plant defense and improve soil characteristics in various ways.

The composition comprises a novel microbial formulation capable to mobilize potash, solubilize phosphate and zinc effectively. The microbial formulation of the present invention is novel with high cell density using optimized media.

Present invention also provides a process of preparation of microbial formulation that is water soluble, stable for at least 24 months and compatible with chemical fertilizer. The chemical fertilizer mainly includes essential macronutrients such as phosphorus and potassium. The chemical fertilizer may include essential macronutrients such as phosphorus, potassium and zinc or any other essential nutrient(s).

In one non-limiting embodiment, the composition comprises microbial formulation of bacteria selected from one or more different species of Bacillus or one or more different species of potash mobilizing bacteria or one or more different species of phosphate solubilizing bacteria or one or more different species of zinc solubilizing bacteria or any combination thereof, and chemical fertilizer comprising soluble phosphorus and potassium.

In another non-limiting embodiment, the composition comprises microbial formulation of bacteria selected from one or more different species of Bacillus or one or more different species of potash mobilizing bacteria or one or more different species of phosphate solubilizing bacteria or one or more different species of zinc solubilizing bacteria or any combination thereof, and chemical fertilizer comprising soluble phosphorus, potassium and zinc.

The composition is provided in water soluble form. The shelf life of the composition at least 24 months. The composition is effective in terms of pot and field trials on different crops.

In another non-limiting embodiment, the composition enhances the growth of wheat crop.

DETAILED DESCRIPTION OF THE INVENTION WITH NON-LIMITING EMBODIMENTS AND EXAMPLES
The present invention provides a composition of microbial enriched essential macronutrients formulation comprising microbial formulation of at least one microbe mixed with essential macronutrients. The composition of microbial enriched essential macronutrients of the present invention enhances crop production, increase plant defense and improve soil characteristics in various ways.

The composition comprises a novel microbial formulation and essential macronutrients in a ratio ranging from 1:10 to 1:2. The formulation is capable to mobilize and/or solubilize essential macronutrients such as, but not limiting to potash, phosphate, zinc, iron, sulphur, magnesium, calcium and the like effectively.

In a preferred embodiment, the composition comprises a novel microbial formulation and essential macronutrients in a ratio of 1:5.

In another preferred embodiment, the composition comprises a novel microbial formulation and essential macronutrients in a ratio of 1:2.

The microbial formulation of the present invention is obtained by culture using optimized media under specific conditions to achieve high cell density of micro-organism ranging from 1 × 108 to 1 × 1010 colony forming units per ml (cfu/ml). The microbial formulation thus obtained is water soluble and compatible with chemical fertilizers and/or synthetic macronutrients.

Pure culture of desired microorganism has been grown in an aqueous medium at the temperature ranging from 25°C to 40°C and at pH ranging from 5.5 to 8.0 for a period of time to achieve above 90% sporulation to obtain fermented broth. The fermented broth contains microbial count ranging from 1 × 108 to 1 × 1010 cfu/ml.
The aqueous medium used for growth of micro-organism comprises a carbon source, one or more nitrogen source and salts in water.

The carbon source is selected from dextrose, jaggery, sucrose, glycerol, soluble starches. The one or more nitrogen source is selected from yeast extract, rice bran extract, barley extract, meat extract, soy peptone, fish peptone, casamino acids, casein (tryptone), gelatin hydrolysate, proteose peptone, bacteriological peptone, barley extract. The salts are selected from magnesium sulfate, manganese sulfate, calcium chloride, sodium chloride, calcium carbonate, ammonium ferrous sulfate.

In one embodiment, the aqueous medium comprises the ingredients mentioned in Table 1:
Table 1. Media composition for growth of micro-organism
S.no. Description Concentration (%, w/v)
1 Carbon source (dextrose, jaggery, sucrose, glycerol, soluble starches) 1 to 5%
2 Nitrogen source (yeast extract, rice bran extract, barley extract, meat extract) 0.1 to 3%
3 Nitrogen source (soy peptone, fish peptone, casamino acids, casein (tryptone), gelatin hydrolysate, proteose peptone, bacteriological peptone) 0.1 to 3%
4 Nitrogen source (yeast extract, rice bran extract, barley extract, meat extract) 0.1 to 3%
5 Salts (magnesium sulfate, manganese sulfate, calcium chloride, sodium chloride, calcium carbonate, ammonium ferrous sulfate) 0.001 to 5 %
6 Water q.s.

In a preferred embodiment, the micro-organism to obtain microbial formulation of the present invention is Bacillus aerius. The aqueous medium for growth of Bacillus aerius comprises starch 1%, cornsteep liquor 0.20%, peptone 0.50%, beef extract 0.30%.

The microbe Bacillus aerius was isolated from rock phosphate mine of Jhamarkotra, Udaipur Rajasthan. Samples were collected in sterile containers. The organism is deposited at IMTECH (Institute of Microbial Technology), Chandigarh, India bearing accession no. MTCC 25483 dated December 03, 2021.

The process of preparation of microbial formulation that is water soluble and compatible with synthetic macronutrients and/or chemical fertilizer comprises
(a) taking suitable amount of water soluble carrier in a mixing blender,
(b) transferring suitable amount of broth containing microbial formulation into a sprayer tank,
(c) adding broth into the water soluble carrier in the mixing blender via specialised sprayer in mist form, wherein
- first half quantity of the broth is added to the carrier in the mixing blender and kept still for 5-10 mins to avoid over-wetting of the carrier,
- remaining half quantity of the broth is added after 10-20 mins. into the water soluble carrier in the mixing blender,
(d) running the mixing blender until the mixture dries to be in free flow condition to obtain powder form, said powder is the microbial formulation with high cfu count,
(g) mixing the essential macronutrients with the microbial formulation into the blender in specific ratio to obtain composition of microbial enriched essential macronutrient formulation.

The essential micronutrients are selected from synthetic macronutrients or chemical fertilizers or a combination thereof.

The suitable amount of the water soluble carrier and the broth containing microbial formulation ranging in the ratio from 1:20 to 1:30. The water soluble carrier is selected from dextrose, maltodextrin, soluble starch, corn starch.

The specific ratio of microbial formulation and synthetic macronutrients and/or chemical fertilizer ranges from 1:10 to 1:2.

The composition is capable of mobilizing macronutrients such as, but not limiting to potash, phosphate, zinc, iron, sulphur, magnesium, calcium and the like effectively.

The high cfu count of the micro-organism in the microbial formulation is 1x108 to 5x109 cfu/g.
The macronutrients of the composition are selected from phosphorus, potassium, zinc , iron, sulphur, magnesium, calcium or a combination thereof.

The essential macronutrients can be readily available chemical fertilizers or synthetic macronutrients or a combination thereof.

In one non-limiting embodiment, the composition comprises microbial formulation of bacteria selected from one or more different species of Bacillus or one or more different species of potash mobilizing bacteria or one or more different species of phosphate solubilizing bacteria or one or more different species of zinc solubilizing bacteria or any combination thereof, and chemical fertilizer and/or synthetic macronutrients comprising soluble phosphorus, potassium, zinc, iron, sulphur, magnesium, calcium or a combination thereof.

In another non-limiting embodiment, the composition comprises microbial formulation of bacteria selected from one or more different species of Bacillus or one or more different species of potash mobilizing bacteria or one or more different species of phosphate solubilizing bacteria or one or more different species of zinc solubilizing bacteria or any combination thereof, and chemical fertilizer comprising soluble phosphorus, potassium and zinc.

The phosphate solubilizing micro-organism is selected from Pseudomonas fluorescens, Burkholderia cepacian, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundi, Proteus mirabalis, Serratia marcenscens, Bacillus subtilis, Pseudomonas putida, Pseudomonas mendocina, Bacillus licheniformis, Klebsiella aerogenes, Rhizobium leguminosarum, Pseudomonas striata, Bacillus polymyxa, Azotobacter, Azospirillum, Enterobacter asburiae, Pseudomonas fragi, Serratia liquefaciens, Serratia marcescens, Bacillus thuringiensis, Pantoea agglomerans, Sinorhizobium meliloti, Streptomyces sp. strain PGPA39, Penicillium bilaiae Chalabuda, Rhizobium RASH6, Bacillus megaterium, Azotobacter coroocoocum, lentus Azospirillum brasilens, Bacillus circulans, Planococcus rifietoensis, Agrobacterium spp.

The potash mobilizing micro-organism is selected from Frateuria aurantia, Bacillus mucilaginous, Bacillus subtilis, Azotobacter, Agrobacterium tumefaciens, Bacillus mucilaginosus, Bacillus globisporus, Bacillus pasteurii, Bacillus megaterium, Bacillus coagulans, Bacillus metallica, Bacillus firmus, Bacillus cereus, Bacillus mycoides, Bacillus amyloliquefaciens, Bacillus licheniformis, Burkholderia, Microbacterium, Paenibacillus glucanolyticus, Brevibacillus, Enterobacter hormaechei, Enterobacter cloacae, Pseudomonas putida, Pseudomonas azotoformans, Klebsiella variicola, Alcaligenes piechaudii, Rhizobium Pusense, Pantoea ananatis, Rahnella aquatilis

Zinc mobilizing bacteria is selected from Pseudomonas fluorescens, Bacillus subtilis, Bacillus megaterium, Bacillus cerius, Bacillus thuringiensis, Bacillus pumilis, Gluconacetobacter diazotrophicus, Klebsiella spp., Acinetobacter calcoaceticus, Bacillus aryabhattai, Pseudomonas taiwenensis, Serratia spp., Rhizobium spp. RL9, Serratia liquefaciens FA-2, Serratia marcescens, Pseudomonas fragi, Pantoea dispersa, Pantoea agglomerans.

The essential micronutrients are selected from synthetic macronutrients or chemical fertilizers or a combination thereof.

In a non-limiting embodiment, the composition enhances the growth of wheat crop.

The composition of microbial enriched essential macronutrient formulation of the present invention is packed in LDPE poly bags. The stability of the composition is at least 24 months.

Stability studies
The stability studies of the composition comprising microbial formulation of Bacillus aerius, and chemical fertilizer/ synthetic macronutrients were conducted at two different conditions i.e accelerated (37°C) and real time (25°C). These stability sample were tested for the colony-forming units (CFU) per gram, using the serial dilution method, The Water-Soluble Microbial Enriched Essential Macronutrient powder was suspended in sterile saline and mixed well by vortexing. After 5 minutes of revitalization, the cell suspension was serially diluted in sterile saline. For the cfu of Bacillus aerius, the dilutions are plated in duplicates. The agar plates were incubated aerobically for three days at 30°C. Plates with 30 - 300 colonies are chosen for counting of colony forming units. The result is reported as average CFU/g of Bacillus aerius sample, calculated from the duplicates.
The results are shown in Tables 2 and 3. Table 2 shows storage stability results (CFU/g) of composition at the accelerated conditions. Table 3 shows storage stability results (CFU/g) of Bacillus aerius at the real time conditions.

Table 2. Storage stability results (CFU/g) of Water-Soluble Microbial Enriched Essential Macronutrient powder at 37 °C.
Time (day) Bacillus spp. formulated in water soluble powder (CFU/g) at 37°C
0 day 8 × 107
7 days 8 × 107
14 days 7 × 107
21 days 5 × 107
28 days 3 × 107

Table 3. Storage stability results (CFU/g) of Water-Soluble Microbial Enriched Essential Macronutrient powder at 25°C.
Time (months) Bacillus spp. formulated in water soluble powder (CFU/g) at 25°C
0 day 8 × 107
1st month 8 × 107
2nd month 8 × 107
3rd month 8 × 107
4th month 8 × 107
5th month 7 × 107
6th month 7 × 107
7th month 6 × 107
8th month 6 × 107
9th month 5 × 107
10th month 4 × 107
11th month 3 × 107
12th month 3 × 107

Thus, the composition of the present invention is compatible with chemical fertilizer or synthetic macronutrients. The macronutrients mainly include essential macronutrients such as phosphorus, potassium, zinc or any other essential nutrient(s).

The composition is provided in water soluble form. The shelf life of the composition at least 24 months. The composition is effective in terms of pot and field trials on different crops.

CLAIMS:

We claim:
1. A composition of microbial enriched essential macronutrients formulation that enhances crop characteristics, said composition comprises
- microbial formulation of at least one microbe,
- essential macronutrients,
wherein said formulation is water soluble and compatible with chemical fertilizers and/or synthetic macronutrients, capable of mobilizing and/or solubilizing macronutrients such as, but not limiting to, potash, phosphate, zinc, iron, sulphur, magnesium, calcium and the like effectively.

2. The composition as claimed in claim 1, wherein said at least one microbe of the microbial formulation is selected from one or more different species of Bacillus or one or more different species of potash mobilizing bacteria or one or more different species of phosphate solubilizing bacteria or one or more different species of zinc solubilizing bacteria or any combination thereof.

3. The composition as claimed in claim 2, wherein said phosphate solubilizing bacteria is selected from Pseudomonas fluorescens, Burkholderia cepacian, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundi, Proteus mirabalis, Serratia marcenscens, Bacillus subtilis, Pseudomonas putida, Pseudomonas mendocina, Bacillus licheniformis, Klebsiella aerogenes, Rhizobium leguminosarum, Pseudomonas striata, Bacillus polymyxa, Azotobacter, Azospirillum, Enterobacter asburiae, Pseudomonas fragi, Serratia liquefaciens, Serratia marcescens, Bacillus thuringiensis, Pantoea agglomerans, Sinorhizobium meliloti, Streptomyces sp. strain PGPA39, Penicillium bilaiae Chalabuda, Rhizobium RASH6, Bacillus megaterium, Azotobacter coroocoocum, lentus Azospirillum brasilens, Bacillus circulans, Planococcus rifietoensis, Agrobacterium spp.
4. The composition as claimed in claim 2, wherein said potash mobilizing bacteria is selected from Frateuria aurantia, Bacillus mucilaginous, Bacillus subtilis, Azotobacter, Agrobacterium tumefaciens, Bacillus mucilaginosus, Bacillus globisporus, Bacillus pasteurii, Bacillus megaterium, Bacillus coagulans, Bacillus metallica, Bacillus firmus, Bacillus cereus, Bacillus mycoides, Bacillus amyloliquefaciens, Bacillus licheniformis, Burkholderia, Microbacterium, Paenibacillus glucanolyticus, Brevibacillus, Enterobacter hormaechei, Enterobacter cloacae, Pseudomonas putida, Pseudomonas azotoformans, Klebsiella variicola, Alcaligenes piechaudii, Rhizobium Pusense, Pantoea ananatis, Rahnella aquatilis.

5. The composition as claimed in claim 2, wherein said zinc solubilizing bacteria is selected from Pseudomonas fluorescens, Bacillus subtilis, Bacillus megaterium, Bacillus cerius, Bacillus thuringiensis, Bacillus pumilis, Gluconacetobacter diazotrophicus, Klebsiella spp., Acinetobacter calcoaceticus, Bacillus aryabhattai, Pseudomonas taiwenensis, Serratia spp., Rhizobium spp. RL9, Serratia liquefaciens FA-2, Serratia marcescens, Pseudomonas fragi, Pantoea dispersa, Pantoea agglomerans.

6. The composition as claimed in claim 1, wherein said essential macronutrients are selected from phosphorus, potassium, zinc, iron, sulphur, magnesium, calcium or a combination thereof.

7. The composition as claimed in claim 1, wherein said essential micronutrients are selected from synthetic macronutrients or chemical fertilizers or a combination thereof.

8. The composition as claimed in claim 1, wherein said microbial formulation and said essential macronutrients are in a ratio ranging from 1:10 to 1:2.
9. The composition as claimed in claim 1, wherein said microbial formulation and said essential macronutrients are in a ratio ranging from 1:5.

10. The composition as claimed in claim 1, wherein said microbial formulation and said essential macronutrients are in a ratio ranging from 1:2.

11. The composition as claimed in claim 1, wherein viable cell count of the microbe in the microbial formulation is 1x108 to 5x109 cfu/g.

12. A composition as claimed in claim 1, wherein said at least one microbe is Bacillus aerius.

13. The composition as claimed in claim 1, wherein said composition is stable at room temperature for at least 24 months.

14. A process of preparation of composition of claim 1, said process comprising
(a) growing pure culture of desired microbe in an aqueous medium at the temperature ranging from 25°C to 40°C and at pH ranging from 5.5 to 8.0 for a period of time to achieve above 90% sporulation to obtain fermented broth with desired cfu count,
(b) taking suitable amount of water soluble carrier in a mixing blender,
(c) transferring suitable amount of broth containing microbial formulation into a sprayer tank,
(d) adding broth into the water soluble carrier in the mixing blender via specialised sprayer in mist form, wherein
- first half quantity of the broth is added to the carrier in the mixing blender and kept still for 5-10 mins to avoid over-wetting of the carrier,
- remaining half quantity of the broth is added after 10-20 mins. into the water soluble carrier in the mixing blender,
(e) running the mixing blender until the mixture dries to be in free flow condition to obtain powder form, said powder is the microbial formulation with high cfu count,
(f) mixing the essential macronutrients with the microbial formulation into the blender in specific ratio to obtain composition of microbial enriched essential macronutrient formulation.

15. The process as claimed in claim 14, wherein said suitable amount of the water soluble carrier and the broth containing microbial formulation ranging in the ratio from 1:20 to 1:30.

16. The process as claimed in claim 14, wherein said water soluble carrier is selected from dextrose, maltodextrin, soluble starch, corn starch.

17. The process as claimed in claim 14 wherein said fermented broth contains microbial count ranging from 1 × 108 to 1 × 1010 cfu/ml.

18. The process as claimed in claim 14, wherein said aqueous medium used for growth of micro-organism comprises a carbon source, one or more nitrogen source and salts in water.

19. The process as claimed in claim 18, wherein said carbon source is selected from dextrose, jaggery, sucrose, glycerol, soluble starches.

20. The process as claimed in claim 18, wherein said one or more nitrogen source is selected from yeast extract, rice bran extract, barley extract, meat extract, soy peptone, fish peptone, casamino acids, casein (tryptone), gelatin hydrolysate, proteose peptone, bacteriological peptone, barley extract.

21. The process as claimed in claim 18, wherein said salts are selected from magnesium sulfate, manganese sulfate, calcium chloride, sodium chloride, calcium carbonate, ammonium ferrous sulfate.

22. The process as claimed in claim 14, wherein said essential micronutrients are selected from synthetic macronutrients or chemical fertilizers or a combination thereof.