Description:Description of the invention with respect to drawings:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
In the following description, reference is made to the accompanying drawings that form a part hereof and in which are shown by way of illustration at least one specific embodiment. The following description provides additional specific embodiments. It is to be understood that still other embodiments are contemplated and may be made without departing from the scope or spirit of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense. While the present disclosure is not so limited, an appreciation of various aspects of the disclosure will be gained through a discussion of the examples provided below.
The present invention is a synergistic formulation comprising of; an efficient phosphate solubilizing microbial strain (MTCC 25312) which is compatible with the chemical fertilizer (N:P:K 19:19:19; 8:16:24; 10:54:10; 13:14:13). Synergistic formulation of phosphate solubilizing bacteria (PSB) with the chemical fertilizer improves the nutrient availability, improves nutrient uptake ability, improves soil health and micro biota and reduces the chemical usage efficiency. The present invention helps to improve consistent performance, faster action compared to chemical fertilizers, more efficacy across various climatic and geographic zones, and increased shelf life. Synergistic formulation of phosphate solubilizing bacteria (PSB) with the chemical fertilizer will improve the nutrient availability, improves nutrient uptake ability, improves soil health and microbiota and reduces the chemical usage efficiency.
In another embodiment, process for preparation of synergistic formulation is disclosed. Selection of Bacillus megaterium Strain: First a strain of Bacillus megaterium that has high phosphate-solubilizing efficiency is selected. It is ensured that the strain is stable and can survive under different environmental conditions, particularly in the presence of fertilizers.
Inorganic Water-Soluble Fertilizers: in second step, fertilizers are chosen that are compatible with the biological activity of Bacillus megaterium. Common inorganic water soluble fertilizers like N:P:K 19:19:19; 8:16:24; 10:54:10; 13:14:13 are considered.
Compatibility Testing: The compatibility of the bacterial strain is tested with various water-soluble fertilizers at different concentrations. The fertilizers are not adversely affect the viability and phosphate-solubilizing activity of Bacillus megaterium.
Formulation Components: a carrier is used that is compatible with both Bacillus megaterium and water-soluble fertilizers. Options include: Solid formulations: Peat, talc, lignite, dextrose can be used as carriers for solid formulations.
Encapsulating agents may be added to maintain the shelf life of the bacterial cells. For liquid formulations, substances like glycerol, skim milk powder and maltodextrin may protect the cells from desiccation or osmotic pressure.
Mode of Application: The formulation can be designed for fertigation (fertilizer combined with irrigation water) or as a foliar spray. It is ensured that the formulation remains stable and active when mixed with water and fertilizers, and during the application process.
Shelf Life and Storage: shelf-life studies are conducted to determine how long the formulation can be stored without losing microbial viability or fertilizer solubility. It is Stored in shade and dry conditions to prolong the life of the formulation.
In another embodiment, the ingredients and the steps of the present invention are as follows:
1. Microbial cell biomass (80% moisture) made up of NPK and phosphorus solubilizing bacteria : 100 g
2. Skim milk powder: 10 g
3. Precipitated silica: 100 g
4. Dextrose: 790 g
Procedure:
1. Prepare Microbial Biomass:
o Begin by collecting 100 g of microbial cell biomass containing 80% moisture. This microbial biomass should be freshly prepared and maintained at a suitable temperature to retain its viability.
2. Add Skim Milk Powder:
o Slowly add 10 g of skim milk powder to the microbial biomass.
o Mix thoroughly until the skim milk powder is uniformly distributed throughout the biomass. The skim milk acts as a encapsulating agent, helping to stabilize the cells during the drying process and enhancing their shelf life.
3. Incorporate Precipitated Silica:
o Add 100 g of precipitated silica to the above mixture.
o Mix the components well to ensure that the silica absorbs any excess moisture and aids in maintaining the free-flowing nature of the final product. Precipitated silica serves as a moisture absorbent and helps prevent clumping during storage.
4. Add Dextrose:
o Finally, add 790 g of dextrose to the mixture.
o Homogenize the entire mixture to achieve a uniform blend. Dextrose serves as a carbohydrate source for preserving the microbial cells and helps in maintaining cell viability during storage. It also acts as a stabilizer for the cells during the drying process.
5. Homogenization and Drying:
o After the homogenization, the formulation can be subjected to spray drying or freeze-drying if a powdered form is required. Drying will further reduce the moisture content, prolonging the shelf life of the formulation.
o Ensure the product is dried to a moisture level that is optimal for long-term storage and stability.
6. Packaging:
o Once the formulation is thoroughly mixed and dried (if necessary), it should be packaged in moisture-proof, air-tight containers.
o Store the product in a cool, dry place to ensure prolonged shelf life and maintain the viability of the microbial cells.
Key Considerations:
• Skim milk powder serves as an encapsulating agent that shields the microbial cells during drying and storage, improving overall cell survival.
• Precipitated silica helps to absorb any excess moisture, preventing degradation of the microbial cells and clumping of the formulation.
• Dextrose provides a carbohydrate source that can help preserve cell viability and maintain product stability during long-term storage.
This formulation is designed to optimize the shelf life and efficacy of microbial-based products, ensuring that the active ingredients remain viable and effective over time.
In the formulation process, the addition of skim milk powder may be employed to stabilize the bacterial cells during the drying process. This may be complemented by the inclusion of stabilizers, such as glycerol or sucrose, which can further maintain the shelf life of the bacterial cells. The incorporation of precipitated silica may serve to absorb moisture and prevent clumping, thereby ensuring the formulation remains free-flowing. Dextrose may be added to preserve cell viability, acting as a carbohydrate source that supports the microbial cells during storage. The microbial biomass, which may be prepared with a moisture content of about 80%, can be homogenized with these components to achieve a uniform blend. This blend may then be subjected to drying techniques such as spray drying or freeze-drying to reduce moisture content, thereby prolonging the shelf life of the formulation. The final product may be packaged in moisture-proof, air-tight containers and stored in cool, dry conditions to ensure prolonged viability and stability. This approach may enhance nutrient efficiency and soil health by improving nutrient availability, nutrient uptake ability, and reducing chemical usage. The formulation may also improve soil health and microbiota, offering potential advantages over traditional chemical fertilizers.
In the context of the formulation, the phosphate solubilizing microbial strain, specifically Bacillus Megaterium, may be utilized to enhance nutrient availability and nutrient uptake ability. This microbial strain may also contribute to improving soil health and microbiota, while potentially reducing chemical usage. The formulation may include a combination of chemical fertilizers and phosphate solubilizing bacteria (PSB), which can be designed to work synergistically. The chemical fertilizers may be selected to be compatible with the biological activity of Bacillus Megaterium, ensuring that the microbial activity is not inhibited by high concentrations of nitrogen or salts. The formulation may be prepared by selecting a strain of Bacillus Megaterium with high phosphate-solubilizing efficiency, ensuring its stability and survival under various environmental conditions, particularly in the presence of fertilizers. Compatibility testing may be conducted to ensure that the bacterial strain remains viable and active in the presence of different concentrations of water-soluble fertilizers. The formulation may be designed to be stable and active when mixed with water and fertilizers during the application process, which can be achieved through the use of suitable carriers and stabilizers. The formulation may be stored in cool, dry conditions to prolong its shelf life and maintain the viability of the microbial cells. The potential benefits of this formulation may include improved nutrient availability, enhanced nutrient uptake ability, better soil health, and reduced chemical usage, which may lead to more consistent performance and faster action compared to chemical fertilizers alone. Additionally, the formulation may exhibit more efficacy across various climatic and geographic zones, with an increased shelf life.
In the context of the formulation, the preparation of microbial biomass may involve the collection and maintenance of microbial cell biomass, which can be essential for ensuring the viability of the microbial component. The microbial biomass may be prepared by collecting a specific quantity of microbial cell biomass, which may contain a certain moisture content to retain its viability. This step may be crucial for the subsequent formulation process, as it may provide the foundational microbial component necessary for the synergistic formulation. The microbial biomass may be freshly prepared and maintained at a suitable temperature to ensure its viability throughout the formulation process.
The preparation of microbial biomass may be followed by the addition of skim milk powder, which may act as an encapsulating agent. The skim milk powder may be added slowly to the microbial biomass and mixed thoroughly until it is uniformly distributed throughout the biomass. This may help stabilize the microbial cells during the drying process and enhance their shelf life. The encapsulating nature of skim milk powder may be attributed to its ability to shield the microbial cells from adverse conditions during drying and storage.
Incorporating precipitated silica into the formulation may serve as a moisture absorbent, which may be essential for maintaining the free-flowing nature of the final product. The precipitated silica may be added to the mixture and mixed well to ensure that it absorbs any excess moisture. This may prevent clumping during storage, thereby preserving the integrity of the formulation. The moisture-absorbing properties of precipitated silica may contribute to the overall stability and longevity of the formulation.
The addition of dextrose to the formulation may provide a carbohydrate source that can help preserve cell viability during storage. Dextrose may be added to the mixture and homogenized to achieve a uniform blend. This may also serve as a stabilizer for the microbial cells during the drying process, further enhancing the formulation's stability. The presence of dextrose may ensure that the microbial cells remain viable and effective over time, contributing to the formulation's prolonged shelf life.
The homogenization and drying process may involve subjecting the formulation to spray drying or freeze-drying if a powdered form is required. This step may further reduce the moisture content, prolonging the shelf life of the formulation. The drying process may be optimized to achieve a moisture level that is suitable for long-term storage and stability. The homogenization may ensure that the formulation is uniformly blended, which may be critical for maintaining the consistency and effectiveness of the final product.
Finally, the packaging of the formulation in moisture-proof, air-tight containers may be necessary to ensure prolonged shelf life and maintain the viability of the microbial cells. The product may be stored in a cool, dry place to prevent degradation and preserve its effectiveness. The packaging and storage conditions may be carefully controlled to optimize the formulation's shelf life and ensure that the active ingredients remain viable and effective over time.
In the context of the formulation process, the action of homogenization and drying may be employed to achieve a uniform blend and reduce moisture content. This process can involve techniques such as freeze-drying or spray drying, which may be selected based on the desired characteristics of the final product. The homogenization step may ensure that all components are evenly distributed, which can be crucial for maintaining the stability and efficacy of the formulation. The drying process may further reduce the moisture content, which can be essential for prolonging the shelf life of the formulation. This step may correlate with the actions of achieving a uniform blend and reducing moisture. The processes involved in this, such as freeze-drying and spray drying, may be critical in ensuring that the formulation remains stable and active during storage and application. Additionally, factors such as temperature control and drying time may play a role in optimizing the drying process to achieve the desired moisture level. The potential advantage of this process may lie in its ability to enhance the stability and shelf life of the formulation, ensuring that the active ingredients remain viable and effective over time.
According to an embodiment, the formulation may be stored in moisture-proof, air-tight containers to prolong its life. The storage conditions may be cool and dry to ensure the prolonged shelf life of the formulation. The packaging process may involve placing the formulation in containers that prevent moisture ingress, thereby maintaining the integrity of the formulation. The storage conditions can be optimized to prevent any degradation of the formulation, ensuring that the active components remain viable over time. The packaging and storage steps may be crucial in maintaining the formulation's effectiveness, as they can protect the formulation from environmental factors that may otherwise compromise its stability and efficacy.
The process may involve the addition of an encapsulating agent, such as skim milk powder, to the microbial biomass. This encapsulating agent may be mixed thoroughly with the biomass to ensure uniform distribution. The role of the encapsulating agent may be to stabilize the microbial cells during subsequent drying processes, thereby enhancing their shelf life. The encapsulating agent may also contribute to maintaining the viability of the microbial cells throughout storage.
Following this, a moisture absorbent, such as precipitated silica, may be incorporated into the mixture. The moisture absorbent may be mixed well to ensure it effectively absorbs any excess moisture present in the formulation. This step may be crucial in maintaining the free-flowing nature of the final product and preventing clumping during storage. The moisture absorbent may thus play a vital role in preserving the physical integrity of the formulation.
Subsequently, a carbohydrate source, such as dextrose, may be added to the mixture. The carbohydrate source may serve to preserve the microbial cells and maintain their viability during storage. Additionally, it may act as a stabilizer for the cells during the drying process, further contributing to the overall stability and effectiveness of the formulation.
The process may then proceed to the homogenization of the PSB cell biomass with the encapsulating agent, moisture absorbent, and carbohydrate source to obtain a uniform mixture. This homogenization step may be essential to achieve a consistent blend, ensuring that all components are evenly distributed throughout the formulation.
The mixture may then be subjected to a drying process, which may involve spray drying or freeze-drying, to obtain the synergistic formulation in a powdered form. The drying process may be designed to reduce the moisture content of the formulation to a level that is optimal for long-term storage and stability. This step may be critical in prolonging the shelf life of the formulation and ensuring the microbial cells remain viable over time.
Finally, the synergistic formulation may be packaged in moisture-proof, air-tight containers and stored in a cool, dry place. This packaging step may be vital in protecting the formulation from environmental factors that could compromise its stability and effectiveness. By storing the product under these conditions, the shelf life of the formulation may be significantly extended, ensuring that the active ingredients remain viable and effective for an extended period.
In step 102, the process may involve the addition of an encapsulating agent to the PSB cell biomass, which is then mixed thoroughly to ensure uniform distribution throughout the biomass. The encapsulating agent, identified as skim milk powder, may serve to stabilize the microbial cells during the drying process, potentially enhancing their shelf life. This step may be crucial in maintaining the viability of the PSB, as the encapsulating agent may act as a stabilizer, safeguarding the cells against adverse conditions that could arise during subsequent processing stages. The thorough mixing of the encapsulating agent with the PSB cell biomass may ensure that each cell is adequately coated, thereby maximizing the encapsulating effect. This may be essential for preserving the functional integrity of the PSB, which is critical for the formulation's effectiveness in solubilizing phosphate when applied in agricultural settings. The use of skim milk powder as an encapsulating agent may be particularly advantageous due to its compatibility with the PSB and its ability to enhance the formulation's stability and shelf life.
In step 104, the process may involve the addition of a moisture absorbent to the PSB cell biomass, ensuring that the moisture absorbent effectively absorbs any excess moisture. This step may be crucial in maintaining the free-flowing nature of the formulation and preventing clumping during storage. The moisture absorbent, identified as precipitated silica, may be incorporated into the PSB cell biomass. The action of incorporating precipitated silica may serve the purpose of absorbing moisture and preventing clumping. This step may be essential for ensuring the stability and longevity of the formulation, as excess moisture can lead to degradation and reduced efficacy of the microbial cells. The use of precipitated silica as a moisture absorbent may be a strategic choice to maintain the desired physical properties of the formulation, allowing it to remain effective over time. The process may be designed to ensure that the moisture absorbent is thoroughly mixed with the PSB cell biomass, achieving a uniform distribution that maximizes its moisture-absorbing capabilities. This step may be part of a larger process aimed at creating a stable and effective synergistic formulation of phosphate solubilizing bacteria with water-soluble fertilizers.
In the context of step 106, the process may involve the addition of a carbohydrate source to the PSB cell biomass. This step may be crucial in ensuring the preservation of the microbial cells' viability during storage. The carbohydrate source, identified as dextrose, may serve as a stabilizer for the cells throughout the drying process. The addition of dextrose can potentially enhance the formulation's stability by providing a necessary energy source for the microbial cells, thereby maintaining their viability over extended periods. This may be aligned with the broader objective of the formulation, which is to improve nutrient availability, nutrient uptake ability, soil health, and microbiota, while also reducing chemical usage. The incorporation of dextrose may be seen as a strategic measure to ensure that the microbial cells remain active and effective when the formulation is eventually applied in agricultural settings. This step may be part of a larger sequence of actions that collectively aim to optimize the formulation's performance and shelf life, ensuring that it remains a viable and effective solution for enhancing agricultural productivity.
In step 108, the process may involve the homogenization of the PSB cell biomass with the encapsulating agent, the moisture absorbent, and the carbohydrate source to obtain a mixture. This step may be crucial in ensuring that all components are uniformly distributed, which can potentially enhance the stability and effectiveness of the formulation. The homogenization process may involve thorough mixing to achieve a consistent blend, which may be essential for the subsequent drying process. The encapsulating agent, such as skim milk powder, may serve to stabilize the bacterial cells during this process, while the moisture absorbent, like precipitated silica, may help in maintaining the free-flowing nature of the mixture by absorbing excess moisture. The carbohydrate source, such as dextrose, may play a role in preserving the viability of the microbial cells, ensuring that they remain active and effective during storage and application. This step may be integral to the overall formulation process, as it may set the foundation for achieving a stable and effective synergistic formulation of phosphate solubilizing bacteria with water-soluble fertilizers.
In the context of the synergistic formulation process, the drying of the mixture may be a critical step to obtain the formulation in a powdered form. This step may involve subjecting the mixture to either spray drying or freeze-drying techniques. The choice between these drying methods may depend on the desired characteristics of the final product, such as particle size and moisture content. Spray drying may involve atomizing the mixture into a hot drying medium, which can result in rapid moisture removal and the formation of fine powder particles. Alternatively, freeze-drying may involve freezing the mixture and then reducing the surrounding pressure to allow the frozen water in the mixture to sublimate directly from the solid phase to the gas phase. This method may be particularly useful for preserving the viability of sensitive components within the formulation. The drying process may be essential to achieve a moisture level that is optimal for long-term storage and stability of the formulation. The powdered form of the synergistic formulation may facilitate ease of handling, transportation, and application, while also ensuring that the active components remain viable and effective over time.
In process steps, the process may involve the packaging of the synergistic formulation into moisture-proof, air-tight containers. This can be crucial for maintaining the integrity and effectiveness of the formulation. The packaging may serve to protect the formulation from environmental factors such as moisture and air, which can potentially degrade the product. The containers used may be specifically designed to prevent any ingress of moisture, thereby ensuring that the formulation remains in a stable and active state. Additionally, the storage of the packaged formulation in a cool, dry place may be emphasized to further prolong its shelf life. This storage condition can help in maintaining the viability of the phosphate solubilizing bacteria (PSB) within the formulation, ensuring that they remain effective when applied. The careful packaging and storage of the formulation may thus play a significant role in preserving its quality and efficacy over time.
Examples
Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.
The following is the experimental data which shows that by the reaction of various NPK and phosphate solubilizing cultures.

Example 1:
taking 5 % of the NPK (N:P:K 19:19:19) in the Soyabean casein digest broth. The phosphate solubilizing cultures were inoculated and kept on orbital shaker for 48 h at 120 rpm. Optical density values were recorded at 520 nm using UV Vis Spectrophotometer ELICO S244. Among all, KLBM01 do not show high survivability till 15% NPK concentration.
Example 2:
taking 10 % of the NPK (N:P:K 19:19:19) in the Soyabean casein digest broth. The phosphate solubilizing cultures were inoculated and kept on orbital shaker for 48 h at 120 rpm. Optical density values were recorded at 520 nm using UV Vis Spectrophotometer ELICO S244. Among all, KLBM01 do not show high survivability for 10% NPK concentration.
Example 3:
taking 15 % of the NPK (N:P:K 19:19:19) in the Soyabean casein digest broth. The phosphate solubilizing cultures were inoculated and kept on orbital shaker for 48 h at 120 rpm. Optical density values were recorded at 520 nm using UV Vis Spectrophotometer ELICO S244. Among all, KLBM01 shows high survivability for 15% NPK concentration.
Screening and isolation of potent phosphate solubilizing bacteria:
Soil samples were collected from the agricultural lands of Bowrampet village, Telangana where excessive chemical fertilizers were used from the past 3 decades. Soil samples were collected from a depth of 10-15 cm deep and screened on the Pikovasky’s agar medium containing Tricalcium phosphate.
Total 12 PSB were screened based on the zone of phosphate solubilization. Among all the PSB’s obtained, KLBM 01 showed high zone of phosphate solubilization and which was used for further studies.

Example 1:Screening and isolation of potent phosphate solubilizing bacteria:
Soil samples were collected from the agricultural lands of Bowrampet village, Telangana where excessive chemical fertilizers were used from the past 3 decades. Soil samples were collected from a depth of 10-15 cm deep and screened on the Pikovasky’s agar medium containing Tricalcium phosphate.
Total 12 PSB were screened based on the zone of phosphate solubilization. Among all the PSB’s obtained, KLBM 01 showed high zone of phosphate solubilization and which was used for further studies.

Graph demonstrating phosphate solubilization zones of phosphate solubilizing isolates
The potent phosphate solubilizing strain KLBM01 was characterized by using the 16S rRNA sequencing. The DNA sequencing was performed in Bioserve technologies, Hyderabad. Obtained raw DNA sequence was quality checked using Chromas pro 2.0 and the primer or chimera sequences were trimmed. Good DNA sequences were exported to the NCBI Genbank and based on the similarity score the KLBM01 was identified as Bacillus megaterium which is now renamed as Priestia megaterium.
The Bacillus megaterium KLBM01 was patent deposited in the Microbial culture collection, Chandigarh under the IDA Budapest treaty. The accession number obtained was MTCC 25312.
KLBM01 Bacillus megaterium gene sequencing is as follows:
TCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAACTGATTAGAAGCTTGCTTCTATGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTGTAAGACTGGGATAACTTCGGGAAACCGAAGCTAATACCGGATAGGATCTTCTCCTTCATGGGAGATGATTGAAAGATGGTTTCGGCTATCACTTACAGATGGGCCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACAAGAGTAACTGCTTGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGAAAAGCGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTTTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACTCTAGAGATAGAGCGTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAAAGGGCTGCAAGACCGCGAGGTCAAGCCAATCCCATAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGAGTAACCGTAA
Example 2:Compatibility of the PSB with various NPK (19:19:19) concentrations:
To test the compatibility of PSB with NPK water soluble fertilizers, NPK 19:19:19 was taken as the test formulation. Compatibility of PSB with various concentrations was studied by taking 5, 10, 15 and 20% of the NPK (N:P:K 19:19:19) in the Soyabean casein digest broth. The phosphate solubilizing cultures were inoculated and kept on orbital shaker for 48 h at 120 rpm. Optical density values were recorded at 520 nm using UV Vis Spectrophotometer ELICO S244. Among all, KLBM01 showed high survivability till 15% NPK concentration.
For NPK : 19:19:19:

Survivability of PSB isolates at various concentrations of NPK 19:19:19

Similarly, total viable count of the PSB at various concentrations was assed. Among all the PSB isolates, KLBM01 showed high survivability at 15% NPK concentration with 8.9x108/ml and 4.2x106at 20% concentration of NPK.

Graph showing total viable count of microbes at various concentrations of NPK 19:19:19
Example 3:Testing KLBM01 on other water-soluble formulations:
Survivability of the KLBM01 was studied on other water-soluble formulations viz. NPK 8:16:24, 10:54:10, 13:14:13. Results obtained were on par with the results of NPK 19:19:19.

Graph showing total viable count of microbes at various concentrations of NPK water soluble formulations

The enumeration of the microbial count at 15% concentration of all the three grades was done which showed promising results in combination with 8:16:24 (1.4x109/ml), 10:54:10 (1.2x109/ml), 13:14:13 (1.5x109/ml).
Example 4:Optimized formulation to improve the shelf life of the synergistic product:

Ingredients Composition
Cell biomass (80% moisture) 100 g
Skim milk powder 10 g
Precipitated silica 100 g
Dextrose 790 g
Procedure:
• Take 100 g of microbial cell biomass
• Add 10 g of skim milk powder
• Mix it thoroughly until uniformity is noticed
• Add 100 g of precipitated silica to the above mixture
• Add 790 g of dextrose to the above mixture and homogenize it Usage:
Mix 10 Kg of the phosphate solubilizing bacteria of 1.0x1010/g to 40 kg of water-soluble formulations.
Example 5: Testing the shelf life of the formulated product with the different grades of water-soluble formulations:
Shelf-life studies were carried out and the formulated product showed high stability even at 12 month and CFU/ml is 1.2x108.
Shelf-life studies
Month Compatibility with 19:19:19 CFU/g Compatibility with 8:16:24 CFU/g Compatibility with 10:54:10 CFU/g Compatibility with 13:14:13 CFU/g
0 2.5x109 3.4x109 1.9x109 2.8x109
1 2.2x109 3.2x109 1.7x109 2.6x109
2 1.4x109 2.9x109 1.5x109 2.3x109
3 1.0x109 2.8x109 9.2x108 1.7x109
4 9.2x108 2.2x109 8.8x108 1.5x109
5 8.4x108 1.4x109 8.3x108 9.7x108
6 8.1x108 9.0x108 6.4x108 7.8x108
7 7.4x108 8.0x108 5.5x108 6.9x108
8 6.5x108 7.7x108 4.3x108 6.4x108
9 5.7x108 7.2x108 3.8x108 5.6x108
10 4.8x108 6.5x108 2.2x108 5.2x108
11 3.3x108 4.3x108 1.3x108 4.8x108
12 1.2x108 3.8x108 1.1x108 2.3x109
Testing the product efficiency
Pot trials were conducted on maize, and the results demonstrated enhanced performance with the application of PSB (Phosphate Solubilizing Bacteria) combined with water-soluble fertilizer (19:19:19). Maize plants treated with this combination exhibited an average shoot length of 205.45 ± 3.96 cm, shoot dry weight of 86.5 ± 1.23 g, root length of 58.4 ± 0.95 cm, and root dry weight of 16.7 ± 0.7 g

Graph showing the shoot lengths of the maize plants in all the treatments

Graph showing the shoot dry weight of the maize plants in all the treatments

Graph showing the root lengths of the maize plants in all the treatments

Graph showing the root dry weights of the maize plants in all the treatments
The following examples shows that the phosphorus content in the leaves are tested and it is as follows:
1)110 gms of RP Green leaves of Maize sample is collected. The sample is then digested using a strong acid (HNO3) and sometimes perchloric acid (hclo4) to break down the organic matter releasing the phosphorus in soluble form .The phosphorus content is determined by colorimetric analysis.The digested sample is reacted with ammonium molybdate and reduced to blue colour by ascorbic acid. The intensity of the colour proportional to the phosphorus concentration is measured using a spectrophotometer at wavelength 880nm and the result is it contains phosphate 53mg/100 mg.
2) 140 gm of 19:19:19 leaves of Maize sample is collected. The sample is then digested using a strong acid (HNO3) and sometimes perchloric acid (hclo4) to break down the organic matter releasing the phosphorus in soluble form .The phosphorus content is determined by colorimetric analysis.The digested sample is reacted with ammonium molybdate and reduced to blue colour by ascorbic acid. The intensity of the colour proportional to the phosphorus concentration is measured using a spectrophotometer at wavelength 880nm and the result is it contains phosphate 75mg/100gm.
3) 140 gm of PSB (phosphate solubilizing bacteria) green leaves of Maize sample is collected. The sample is then digested using a strong acid (HNO3) and sometimes perchloric acid (hclo4) to break down the organic matter releasing the phosphorus in soluble form .The phosphorus content is determined by colorimetric analysis.The digested sample is reacted with ammonium molybdate and reduced to blue colour by ascorbic acid. The intensity of the colour proportional to the phosphorus concentration is measured using a spectrophotometer at wavelength 880nm and the result is it contains phosphate 82mg/100gm.
4) 19:19:19 leaves of Maize sample+ PSB (phosphate solubilizing bacteria) green leaves of Maize sample is collected. The sample is then digested using a strong acid (HNO3) and sometimes perchloric acid (hclo4) to break down the organic matter releasing the phosphorus in soluble form .The phosphorus content is determined by colorimetric analysis.The digested sample is reacted with ammonium molybdate and reduced to blue colour by ascorbic acid. The intensity of the colour proportional to the phosphorus concentration is measured using a spectrophotometer at wavelength 880nm and is given and the result is it contains phosphate 99mg/100 gm.

Advantages of formulation containing phosphate solubilizing Bacillus Megaterium compatible with inorganic water-soluble fertilizers
1) The present invention (formulation) improves the nutrient availability, improves nutrient uptake ability, improves soil health and micro biota and reduces the chemical usage efficiency.
2) The present invention helps to improve consistent performance, faster action compared to chemical fertilizers.
3) Advanced stabilization techniques prevent degradation over time, such as encapsulation or use of preservatives, resulting into increased shelf life of the formulation.
4) The present invention improves the bioavailability of the synergistic formulation thereby improving more efficacy of the phosphate solubilizing strain across various climatic and geographic zones, ensuring uniform effectiveness regardless of soil conditions.
5) Synergistic formulation of phosphate solubilizing bacteria (PSB) with the chemical fertilizer improves the nutrient availability, improves nutrient uptake ability, improves soil health and microbiota and reduces the chemical usage efficiency.
6) Complex material is converted into soluble material, thereby increasing Faster nutrient dissolution ultimately enhancing solubility.
7) Minimize exposure to moisture and air, preserving product integrity, thereby retaining Moisture Resistance.

, Claims:Claims:
1. A synergistic formulation of phosphate solubilizing bacteria (PSB) with water-soluble fertilizers, comprising:
a PSB having high phosphate-solubilizing efficiency and compatibility with various water-soluble fertilizers at different concentrations without adversely affecting its viability and phosphate-solubilizing activity, wherein the PSB is stable and can survive under different environmental conditions particularly in the presence of the water-soluble fertilizers;
an encapsulating agent that stabilizes the PSB during a drying process and enhances its shelf life;
a moisture absorbent that maintains a free-flowing nature of the formulation and prevents clumping during storage; and
a carbohydrate source that preserves the PSB and maintains its viability during storage;
wherein the formulation has a prolonged shelf life with the PSB remaining viable, and the formulation is stable and active when mixed with water and the water-soluble fertilizers during an application process.
2. A method of preparing a synergistic formulation of phosphate solubilizing bacteria (PSB) with water-soluble fertilizers, the method comprising:
collecting a PSB cell biomass;
adding an encapsulating agent to the PSB cell biomass and mixing thoroughly until the encapsulating agent is uniformly distributed throughout the PSB cell biomass;
adding a moisture absorbent to the PSB cell biomass and mixing well to ensure the moisture absorbent absorbs excess moisture;
adding a carbohydrate source to the PSB cell biomass;
homogenizing the PSB cell biomass with the encapsulating agent, the moisture absorbent, and the carbohydrate source to obtain a mixture;
drying the mixture to obtain the synergistic formulation, wherein the synergistic formulation is in a powdered form; and
packaging the synergistic formulation in moisture-proof, air-tight containers and storing the synergistic formulation in a cool, dry place;
wherein the PSB has high phosphate-solubilizing efficiency and compatibility with various water-soluble fertilizers at different concentrations without adversely affecting its viability and phosphate-solubilizing activity, and the PSB is stable and can survive under different environmental conditions particularly in the presence of the water-soluble fertilizers.
3. The synergistic formulation of claim 1, wherein the PSB is Bacillus megaterium.
4. The synergistic formulation of claim 1, wherein the encapsulating agent is skim milk powder.
5. The synergistic formulation of claim 1, wherein the moisture absorbent is precipitated silica.
6. The synergistic formulation of claim 1, wherein the carbohydrate source is dextrose.
7. The synergistic formulation of claim 1, wherein the PSB cell biomass has a moisture content of about 80%.
8. The synergistic formulation of claim 1, wherein the formulation comprises:
about 100 g of the PSB cell biomass;
about 10 g of the encapsulating agent;
about 100 g of the moisture absorbent; and
about 790 g of the carbohydrate source.
09. The synergistic formulation of claim 1, wherein the formulation is stable for at least 12 months at room temperature.
10. The synergistic formulation of claim 1, wherein the formulation improves nutrient availability, nutrient uptake ability, soil health, and microbiota.
11. The synergistic formulation of claim 1, wherein the formulation reduces chemical fertilizer usage.
12. The synergistic formulation of claim 1, wherein the formulation has improved consistent performance and faster action compared to chemical fertilizers alone.
13. The synergistic formulation of claim 1, wherein the formulation has more efficacy across various climatic and geographic zones.