Process for obtaining a solid composition for microbiological immobilisation, solid composition, uses and method for applying same

Abstract

The present invention relates to a process for obtaining a solid composition of microbiological immobiliser that provides a solid composition with better activation and dispersion of microorganisms, which does not require a controlled environment to guarantee its conservation and stability during transport, storage and handling. The solid composition, uses and application methods thereof are also described. The present invention belongs to the technological sector of biological components for the agro-industrial sector and bioremediation.

Description

[0001] DESCRIPTIVE REPORT

[0002] PROCESS FOR OBTAINING A SOLID COMPOSITION OF MICROBIOLOGICAL IMMOBILIZED, SOLID COMPOSITION, USES AND METHOD FOR APPLYING THE SAME

[0003] Technical sector

[0004]

[0001] The present invention belongs to the technological sector of biological inputs, more specifically it refers to a process for obtaining, composition, use and method for applying a solid composition of microbiological immobilized that allows better activation and dispersion of microorganisms and that does not require a controlled environment to guarantee the conservation and stability of the bioinput during transport, storage and handling of the product.

[0005] State of the art

[0006]

[0002] There is an increasing demand for ways to clean and preserve the environment, water, and soil, as well as to increase agricultural production by reducing or even eliminating the use of agrochemicals. Since the beginning of time, microorganisms have played a role in these applications; however, their natural concentrations have been declining due to anthropogenic factors. Studies have shown that increasing the concentrations of these microorganisms in their environments, and according to their specific mechanisms of action, can replace the use of chemicals and help restore the environment's balance, combating soil and plant diseases, inoculating seeds, and recovering contaminated water. Thus, microbial bioinputs have been used in agricultural ecosystems to stimulate natural processes, helping plant metabolism adapt to a wide range of stress situations, whether caused by pests, diseases, or abiotic factors.

[0007]

[0003] To this end, enabling the production of a bioinput containing immobilized microbiological strains, so that their preservation outside the culture medium is possible, maintaining their morphological, physiological and genetic characteristics, providing cellular stability and, furthermore, protection of the strains against environmental and physical-chemical adversities, such as, for example, application under solar radiation and resistance to agricultural pesticides for associated use, is of great commercial interest.

[0008]

[0004] However, the provision of liquid or powdered bioinputs requires a controlled environment to ensure the conservation and stability of the cells during transport, storage and handling. In addition, the reactivation and replication of microorganisms transported in the liquid and / or powder medium requires a biofactory-type cultivation structure, time (on average 24-72h) and a specialized team, with equipment for the supply of nutrients and maintenance of cultivation conditions. Also, in biofactories, problems may occur in the processing of this inoculum, such as product with inadequate concentrations of microorganisms, contamination, with the growth of opportunistic microorganisms, even pathogenic to humans, animals and plants, resulting in products with low or no efficiency.

[0009]

[0005] In terms of dilution and application of the bioinput in the field, the bioinput in powder form may be difficult to disperse in the water of the application tank (agricultural machine), with the particles remaining suspended on the surface of the liquid without a homogeneous dispersion, which causes concentration failures during application and, also, the volume of product to be applied.

[0010]

[0006] Microbial metabolism has a wide diversity of energy strategies. Thus, microorganisms can use macro and micronutrients from organic and inorganic chemical compounds to carry out their metabolic processes of growth and reproduction.

[0011]

[0007] The direct application of biological products in the sowing / planting furrow promotes good efficiency and productivity of the bioinput, since it avoids contact with other inputs, such as insecticides, fungicides, fertilizers, other biofertilizers and micronutrients, which can be applied in seed treatment beforehand, in addition to delivering the active bioinput to the soil, reducing the time of exposure to sunlight, which could cause the mortality of microorganisms.

[0012]

[0008] Similarly, there is the option of applying biological products via foliar application, which aims to combat aerial diseases, such as leaf spots and rust. However, care must be taken with exposure to sunlight, which can cause mortality of microorganisms present in the inoculum.

[0013]

[0009] Patent document BR 102019 013058-0 describes a process for formulating a polymeric matrix composed of commercial potassium alginate and cornstarch, with bacterial cell encapsulation properties, for use in bioremediation. In the document, the bacterial cells are first cultivated in a liquid culture medium with subsequent cell aggregation in the polymeric matrix composed of potassium alginate and cornstarch, with the polymer and starch acting as carriers. This aggregation is achieved through the drip technique and polymerization in calcium chloride. Therefore, the large-scale production of the microbiological immobilized material becomes laborious. Furthermore, in the document, the microorganisms are present in a liquid medium, which requires storage in a controlled environment and also does not present a substance with the potential to provide rapid disintegration of the polymeric matrix and release of the inoculum.

[0014]

[0010] US patent document 20230320991 describes a process for producing probiotics encapsulated in calcium-alginate hydrogel. The encapsulated probiotics are grown in a culture medium and lyophilized, that is, transformed into a powder to increase the body's resistance to bile acids. Therefore, the probiotic in the document requires storage in a controlled environment until use.

[0015]

[0011] The use of microbiological bioinputs optimizes the physiological and biochemical processes of crops, resulting in increased soil fertility and nutrition, sustainable crop productivity, and improved worker and ecosystem health. However, it is necessary to select preservative agents and appropriate carriers to promote the immobilization of microorganisms to ensure medium- and long-term cell conservation, preservation outside the culture medium, maintaining their morphological, physiological, and genetic characteristics, and protecting the strains from environmental and chemical adversities, such as application of the bioinput under solar radiation and resistance to agricultural pesticides. Therefore, research is being developed to provide appropriate production processes and substances to promote the nutrition, protection, conservation, and effective delivery of microbial cells, culminating in the quality of the bioinputs.

[0016] Novelties and purpose of the invention

[0017]

[0012] In the present invention, the process for obtaining a solid composition of microbiological immobilized material includes a step of pre-mixing the microorganisms with a polyol, preferably glycerin. This mixture aims to coat the microbial cells in order to hydrate and protect the inoculum for subsequent mixing with the other ingredients of the composition. Furthermore, the composition allows for effective reactivation of the inoculum provided by the nutrients present in the formulation (carbon, oxygen, sulfur, phosphorus and sugars), and dispersion in the field, carried out through the reaction of the effervescent agent with water.

[0018]

[0013] In a first object, a process is revealed for obtaining a solid composition of microbiological immobilized material comprising the steps of: i) forming a premixture of microorganisms and at least one polyol at room temperature and pH of 6 to 8; ii) adding an effervescent agent to the premixture (i), mixing until a homogenate is obtained; iii) forming an aqueous solution comprising calcium salt; iv) mixing the homogenate of (ii) and the aqueous solution of (iii), forming a solid mass; v) molding or extruding the solid mass of (iv); vi) refrigerating at a temperature of 0 to 10 °C; vii) drying.

[0019]

[0014] In a second object, a solid composition of microbiological immobilized material is presented, comprising 20% ​​to 50% by weight of microorganisms; from 10% to 20% by weight of a polyol; 5% to 20% of effervescent agent; from 5% to 20% by weight of calcium salt.

[0020]

[0015] In a third object, the use of a solid composition of microbiological immobilized obtained by the process according to the first object is presented, for fertilization, treatment and control of plant and seed diseases.

[0021]

[0016] In a fourth object, the use of a solid composition of microbiological immobilized material obtained by the process according to the first object is presented, for bioremediation of soils, waters and effluent treatment.

[0022]

[0017] In a fifth object, a method is presented for applying a solid composition of microbiological immobilized material obtained according to the first object, comprising diluting said composition in a solvent and applying it via a seeding furrow executed in the soil or via foliar application.

[0023]

[0018] In a sixth object, a method is presented for applying a solid composition of microbiological immobilized material obtained according to the first object, comprising the direct addition of the composition to water and effluents.

[0019] The invention has as its objective a process for obtaining, solid composition, use of the composition and method for applying a solid composition of microbiological immobilized material that does not require a controlled environment to ensure the conservation of microorganisms during transport and handling of the product. Furthermore, the composition allows for effective reactivation, replication of the inoculum and dispersion in the field. In the invention, the integrity and stability of microbial cells, even outside a controlled environment, is achieved by mixing the microbial inoculum with glycerin and subsequent homogenization with the other components of the formulation.The reactivation and replication of microorganisms is provided by the nutrients present in the formulation (carbon, oxygen, sulfur, phosphorus and sugars).

[0024] Advantages and technical effects of the invention

[0025]

[0020] It was found that, surprisingly, the step of pre-mixing the microorganisms with polyol ensured the stability and protection of the microbial cells in such a way that, even outside a controlled environment, the activity and viability of the cells were maintained.

[0026]

[0021] The process for obtaining, composing, using the composition and method for applying the microbiological immobilized composition, objects of the invention, result in the following advantages and achieve the following technical effects on the incorporators of the state of the art:

[0027] • The lyophilized product contains a high concentration of microorganisms (2.1x10 11UFC / g) is mixed with polyol and, subsequently, the other constituents of the formulation are added, and can then go through the extrusion or molding stage to present a weight and structure defined for each application;

[0028] • The high concentration of microorganisms immobilized in the composition allows for greater application performance, as well as compact packaging, resulting in ease of transportation;

[0029] • The effervescence reaction, provided by sodium bicarbonate, allows the accelerated disintegration and dispersion of the composition with uniform spreading of the active ingredient in solution;

[0030] • The solid composition does not need to be activated in biofactories for application, its application method involves diluting the solid composition in solvent, according to the hectares to be treated, directly in the planter or sprayer tank; • The ingredients of the composition itself also have the function of serving as nutrients for microorganisms, when activated.

[0031] • The process of obtaining the composition, comprising the pre-mixing of microorganisms with polyol, promotes protection of the microbial cells against the liquid solutions to be inserted into the mixture and substances that form the solid composition; stabilization and conservation of the inoculum during storage, preventing the microbiological immobilized material from undergoing cellular changes (degradation of cells with loss of viability and reproduction capacity) during transportation and storage, until effective application in the field;

[0032] • The selection of microbial species to be used in the composition may vary according to the final use of the product and the synergistic action of the microorganisms;

[0033] • With the protection and stabilization of the inoculum provided by the polyol, the invention does not require a controlled environment to ensure its conservation during transport, storage and handling, thus guaranteeing the preservation of the cells for later reactivation and ease of application in the field;

[0034] • Product with a shelf life of at least 12 months, not requiring storage in a refrigerated environment;

[0035] • In the composition containing an ultraviolet (UV) radiation filter substance, refraction, dispersion and absorption of ultraviolet rays occur, resulting in the protection of microbial cells against the action of UV rays when the composition is applied via foliar application.

[0036] List of attached drawings

[0037]

[0022] In order for the present invention to be fully understood and put into practice by any technician in this technological sector, it is described in a clear, precise and sufficient manner, based on the attached Figure, listed below, which illustrates the steps of the process for obtaining the composition of the microbiological immobilized:

[0038] - Figure 1 - Flowchart demonstrating steps of the process for obtaining the solid composition comprising, after premixing microorganisms and polyol, addition of effervescent agent and aqueous solution with calcium salt. - Figure 2 - Flowchart demonstrating steps of the process for obtaining the solid composition comprising, after premixing microorganisms and polyol, addition of effervescent agent, aqueous solution with calcium salt and, additionally, binding agent.

[0039] Detailed description of the invention

[0040]

[0023] For the bioinput to be effective in delivering the inoculum, adequate immobilization of the microorganisms is necessary in order to guarantee the conservation and viability of the microbial cells. This is achieved by the present invention.

[0041]

[0024] In a first object, a process is disclosed for obtaining a solid composition of microbiological immobilized material comprising the steps of: i) forming a premixture of microorganisms and at least one polyol at room temperature and pH of 6 to 8; ii) adding an effervescent agent to the premixture (i), mixing until a homogenate is obtained; iii) forming an aqueous solution comprising calcium salt; iv) mixing the homogenate of (ii) and the aqueous solution of (iii), forming a solid mass; v) molding or extruding the solid mass of (iv); vi) refrigerating at a temperature of 0 to 10 °C; vii) drying.

[0042]

[0025] In one embodiment, the drying step (vii) occurs under vacuum with silica.

[0043]

[0026] In one embodiment, the aqueous solution of step (iii) comprises a binding agent.

[0044]

[0027] In one embodiment, the aqueous solution of step (iii) comprises a UV radiation filter.

[0045]

[0028] In one embodiment, the molding or extrusion step (v) provides a sphere or cork shape.

[0046]

[0029] In a second object, a solid composition of microbiological immobilized material is provided comprising 20% ​​to 50% by weight of microorganisms; from 10% to 20% by weight of a polyol; 5% to 20% by weight of effervescent agent; from 5% to 20% by weight of calcium salt.

[0047]

[0030] In one embodiment, the composition comprises microorganisms in a concentration of 25% to 35%, preferably 30% to 50% by weight, by weight of the composition.

[0048]

[0031] In one embodiment, the polyol is selected from: glycerol, erythritol, mannitol, sorbitol, and xylitol, and disaccharides lactitol, maltitol, and isomalt, or mixtures thereof; preferably the polyol is glycerol.

[0049]

[0032] In one embodiment, the composition further comprises a binding agent selected from: alginate, xanthan gum, gum arabic, or mixtures thereof. Preferably the gum is alginate. The binding agent may be in concentrations of 10% to 35% by weight, preferably 20% to 30% by weight, by weight of the composition.

[0050]

[0033] In one embodiment, the composition further comprises an ultraviolet radiation filter selected from: titanium dioxide, zinc oxide. The ultraviolet radiation filter may be in concentrations of 5% to 15% by weight, by weight of the composition.

[0051]

[0034] In one embodiment, the microorganism is selected from bacteria of the genus Bacillus sp; or fungi of the genus Trichoderma sp. The bacteria may be selected from: Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mucilaginosus, or a mixture thereof.

[0052]

[0035] In one embodiment, the solid composition is a molded or extruded, preferably having a sphere or cork shape.

[0053]

[0036] In a third object, the use of the solid composition of microbiological immobilized for fertilization, treatment and control of plant and seed diseases is presented.

[0054]

[0037] In a fourth object, the use of the solid composition of microbiological immobilized material for bioremediation of soils, water and effluent treatment is revealed.

[0055]

[0038] In a fifth object, a method is presented for applying the solid immobilized composition comprising diluting said composition in a solvent and applying it via a seeding furrow executed in the soil or via foliar application.

[0056]

[0039] In a sixth object, a method is presented for applying the solid composition of immobilized assets comprising the direct addition of said composition to water and effluents.

[0057]

[0040] For the purposes of this application, unless otherwise specified, all concentrations represented in “%” are % (w / w) concentrations based on the weight of the final total composition. Process for obtaining the solid composition

[0058]

[0041] The step of pre-mixing microorganisms and polyol is crucial for obtaining a solid composition according to the present invention.

[0059]

[0042] Figure 1 shows a flowchart showing the pre-mixing step of polyol and microorganisms, followed by mixing with other ingredients, e.g., effervescent agent, aqueous solution with calcium salt. After the mixing step, the solid mass is extruded or molded, cooled and dried.

[0060]

[0043] Figure 2 shows a flowchart showing the pre-mixing step of polyol and microorganisms, followed by mixing with other ingredients, e.g., effervescent agent, aqueous solution with calcium salt, and highlighting the binding agent. After the mixing step, the solid mass is extruded or molded, cooled, and dried.

[0061] Microorganisms

[0062]

[0044] In the context of the present invention, the microorganisms are lyophilized microorganisms. Any microorganisms commonly used in the agro-industrial and bioremediation sector can be included.

[0063]

[0045] Non-limiting examples of suitable microorganisms are bacteria of the genus Bacillus sp., for example Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus mucilaginosus or a mixture thereof; and fungi of the genus Trichoderma sp.

[0064] Polyols

[0065]

[0046] Polyols, or sugar alcohols, are carbohydrates obtained by replacing an aldehyde group with a hydroxyl group. Glycerin, or glycerol, (C3HS(OH)3) is a polyol and is a colorless, odorless, viscous liquid, widely used as a vehicle due to its solubility in water and ethanol. Glycerin is used in the preservation of cryopreserved cells, as it protects them against freezing damage, mainly by reducing the formation of intracellular ice crystals and differences in osmotic pressure. Thus, glycerin, in small amounts, keeps the constituents in solution, presenting a suitable culture medium, preventing the water molecule from approaching the microbial cell.

[0066]

[0047] The glycerol used in the process of obtaining the composition may come from the reuse of used soybean oil. Calcium salts

[0067]

[0048] The addition of crosslinking agents produces networks or reticles through the formation of crosslinks between the polymer chains. Calcium chloride (CaCl2), as well as calcium carbonate (CaCOs) and calcium sulfate (CaSO4), also called gypsum, can be used in the crosslinking process. Calcium chloride has high solubility in aqueous solutions and a rapid gelation reaction, while calcium carbonate (CaCOs) and calcium sulfate (CaSO4), especially calcium sulfate, promote a more gradual internal gelation of the alginate, where calcium is released in a controlled manner simultaneously throughout the system, which creates a more homogeneous and less dense network compared to the gelation achieved by the rapid diffusion process.

[0068] Effervescent agent

[0069]

[0049] The use of effervescent agents such as acidic substances and carbonates or hydrogen carbonates (sodium bicarbonate, NaHCOs) aims at the rapid and complete solubilization of the formulation components in the liquid medium. The effervescent agents react rapidly in the presence of water with the release of carbon dioxide (CO2) and dissolution of the binder.

[0070] Binding agents

[0071]

[0050] Binding agents are substances that promote the cohesion of solid particles, forming agglomerates. In the present invention, the preferred binding agent is alginate, even more preferably hydrolyzed alginate.

[0072]

[0051] Alginate is a water-soluble, polyanionic linear polysaccharide containing blocks of [3-D-mannuromic and α-L-guluromic acids joined by (1,4) bonds, obtained from brown algae and some soil bacteria, with pore sizes ranging from 5-200 mm, thus having good molecular diffusion inside and outside the gel and good biocompatibility, and can be used as a colloidal stabilizer, thickening and gelling agent, with extrusion properties, in addition to participating in intermolecular crosslinking with Ca 2+ forming hydrogels.

[0073]

[0052] Definitions of:

[0074]

[0053] Ambient temperature (AT): a range of 20 to 25 °C measured at atmospheric pressure should be understood.

[0075]

[0054] Microbiological immobilized: confinement and limitation of the mobility of viable microbial cells to a specific region defined in space, using synthetic or natural polymers, in order to protect the microorganism against physical, chemical and environmental stresses.

[0076]

[0055] Bioinput or biological input: are products, processes or technologies of biological origin for use in production, storage or processing in agricultural, livestock, forestry and aquatic systems. In agricultural systems, bioinputs act for the fertility, health and nutrition of plants and soil.

[0077]

[0056] Inoculum: suspension of microorganisms.

[0078]

[0057] Solvent: in the context of the method of applying the solid composition, it should be understood as any solvent that is capable of solubilizing said composition to enable its application, for example water or ethyl alcohol, or mixtures thereof.

[0079]

[0058] Examples

[0080]

[0059] Example 1 - Composition of microbiological immobilized with binding agent.

[0081] Table 1. Example of composition of microbiological immobilized material with binding agent. lUPAC: International Union of Pure and Applied Chemistry.

[0082] (---) does not apply.

[0083] Commercial alginate from the manufacturer Dentsply Sirona: (Potassium Alginate, Calcium Sulfate, Sodium Tetrapyrophosphate, Potassium Fluotitanate, Polyethylene Glycol, Magnesium Oxide, Diatomite, Aroma, Chlorhexidine, Anhydrous Alcohol and Phenophthalein).

[0084]

[0060] The process of obtaining the microbiological immobilized composition with binding agent follows the following steps:

[0085] 1. form a premix of microorganisms with glycerin at room temperature and pH 7;

[0086] 2. add sodium bicarbonate to the premix, mixing until smooth;

[0087] 3. form an aqueous solution with 40 to 100 ml of distilled water and calcium sulfate until dissolved;

[0088] 4. form a paste by adding alginate to 100 to 160 ml of water;

[0089] 5. mix the premix with the aqueous solution and the aqueous paste comprising alginate for about 10 minutes;

[0090] 6. shape or extrude the solid mass into a sphere or cork shape;

[0091] 7. Refrigerate at a temperature of 0°C to 10°C;

[0092] 8. Remove the solid mass from the mold and place it under vacuum in silica gel until completely dry (8 days), pH 7 of the dry solid mass.

[0093] 9. Remove the solid mass of silica, weigh it and pack it in an aluminum material to avoid contact with moisture and radiation.

[0094]

[0061] Example 2 - Composition of microbiological immobilized material without binding agent.

[0095] Table 2. Example of microbiological immobilized composition without binding agent. lUPAC: International Union of Pure and Applied Chemistry, International Union of

[0096] Pure and Applied Chemistry.

[0097] (---) does not apply.

[0098] Commercial alginate from the manufacturer Dentsply Sirona: (Potassium Alginate, Calcium Sulfate, Sodium Tetrapyrophosphate, Potassium Fluotitanate, Polyethylene

[0099] Glycol, Magnesium Oxide, Diatomite, Aroma, Chlorhexidine, Anhydrous Alcohol and Phenophthalein).

[0100]

[0062] The process of obtaining the microbiological immobilized composition without the binding agent follows the steps:

[0101] 1. form a premix of microorganisms with glycerin at room temperature and pH 7;

[0102] 2. Add sodium bicarbonate to the premix, mixing until a homogenized mixture is obtained; 3. Form an aqueous solution with 40 to 100 ml of distilled water and calcium sulfate until dissolved;

[0103] 4. mix the premix with the aqueous solution for about 10 minutes; 5. shape or extrude the solid mass into a sphere or cork shape;

[0104] 6. refrigerate the solid mass to a temperature of 0°C to 10°C;

[0105] 7. remove the solid mass from the mold and place it under vacuum in silica gel until completely dry (8 days), pH 7 of the dry solid mass.

[0106] 8. Remove the solid mass of silica, weigh it and pack it in an aluminum material to avoid contact with moisture and radiation.

[0107]

[0063] Example 3 - Composition of microbiological immobilized material with ultraviolet ray filter^ Table 3. Example of composition of microbiological immobilized material with ultraviolet ray filter.

[0108] IUPAC: International Union of Pure and Applied Chemistry. International Union of

[0109] Pure and Applied Chemistry. (---) not applicable. Commercial alginate from the manufacturer Dentsply Sirona: (Potassium Alginate, Calcium Sulfate, Sodium Tetraphosphate, Potassium Fluotitanate, Polyethylene Glycol, Magnesium Oxide, Diatomite, Aroma, Chlorhexidine, Anhydrous Alcohol and Phenophthalein).

[0110]

[0064] The process of obtaining the microbiological immobilized composition with ultraviolet ray filter follows the same steps as in Example 2.

[0111]

[0065] Example 4 - Cell viability test

[0112]

[0066] To determine the viability of microbial cells (inoculum) and their reactivation and replication when dissolved in water, viability tests were carried out using the microorganism counting method, for which the following steps were carried out:

[0113] - Weigh one gram of the solid composition of microbiological immobilized material;

[0114] - Add 10 mL of sterile deionized water followed by homogenization for one minute in a vortex;

[0115] - Dilute the suspension in sterile saline solution and plate in nutrient agar culture medium;

[0116] - Incubate at 28°C for 24 hours for subsequent counting of colony forming units (CFU) using a colony counter. The entire procedure was performed in triplicate.

[0117] Table 4. Count of Bacillus sp. present in the microbiological immobilized sample.

[0118]

[0067] Thus, surprisingly, it was found that even after dilution in water, the composition of microorganisms according to Example 1 maintained the levels of microorganisms initially added in the composition and even with increased levels of microorganisms, suggesting that the immobilized mixture and grouping of microorganisms in the solid composition promotes a suitable nutritional environment for rapid proliferation of microorganisms.

Claims

CLAIMS 1. “PROCESS FOR OBTAINING A SOLID COMPOSITION OF MICROBIOLOGICAL IMMOBILIZED”, characterized by comprising the steps of: i) forming a premixture of microorganisms and at least one polyol at room temperature and pH of 6 to 8; ii) adding an effervescent agent to the premixture (i), mixing until a homogenate is obtained; iii) forming an aqueous solution comprising calcium salt; iv) mixing the homogenate of (ii) and the aqueous solution of (iii), forming a solid mass; v) molding or extruding the solid mass of (iv); vi) refrigerating at a temperature of 0 to 10 °C; vii) drying.

2. “PROCESS FOR OBTAINING A COMPOSITION”, according to claim 1, characterized in that the drying step (vii) occurs under vacuum with silica.

3. “PROCESS FOR OBTAINING A COMPOSITION”, according to any one of the previous claims, characterized in that the aqueous solution of step (iii) comprises a binding agent.

4. “SOLID COMPOSITION OF MICROBIOLOGICAL IMMOBILIZATION”, obtained by the process defined in claim 1, characterized by comprising from 20% to 50% by weight of microorganisms; from 10% to 20% by weight of a polyol; 5% to 20% of effervescent agent; from 5% to 20% by weight of calcium salt.

5. “SOLID COMPOSITION”, according to claim 4, characterized in that the polyol is selected from: glycerol, erythritol, mannitol, sorbitol and xylitol and disaccharides lactitol, maltitol and isomalt, or mixtures thereof; preferably the polyol is glycerol.

6. “SOLID COMPOSITION”, according to claim 4 or 5, characterized by comprising a binding agent in a concentration of 10% to 35% by weight; in which the binding agent is selected from: alginate, xanthan gum, gum arabic, or mixtures thereof; preferably the binding agent is alginate.

7. “SOLID COMPOSITION” according to any one of claims 4 to 6, characterized by comprising an ultraviolet radiation filter selected from: titanium dioxide, zinc oxide; preferably zinc oxide.

8. “SOLID COMPOSITION”, according to any one of claims 4 to 7, characterized in that the microorganisms are selected from bacteria of the genus Bacillus sp; or fungi of the genus Trichoderma sp.

9. “SOLID COMPOSITION” according to any one of claims 4 to 8, characterized in that it is molded or extruded, preferably having the shape of spheres or corks.

10. “USE OF A SOLID COMPOSITION OF MICROBIOLOGICAL IMMOBILIZATION” obtained by the process defined in any one of claims 1 to 3, characterized by being for fertilization, treatment and control of plant and seed diseases.

11. “USE OF A SOLID COMPOSITION OF MICROBIOLOGICAL IMMOBILIZED” obtained by the process defined in any one of claims 1 to 3, characterized by being for bioremediation of soil, water and effluent treatment.

12. “METHOD FOR APPLYING A SOLID COMPOSITION OF MICROBIOLOGICAL IMMOBILIZED obtained by the process defined in any one of claims 1 to 3, characterized by comprising diluting said composition in a solvent and applying via a seeding furrow executed in the soil or via foliar application.

13. “METHOD FOR APPLYING A SOLID COMPOSITION OF MICROBIOLOGICAL IMMOBILIZED obtained by the process defined in any one of claims 1 to 3, characterized by comprising the direct addition of said composition to water and effluents.

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