DRY BIOLOGICAL COMPOSITIONS AND METHODS THEREOF

Abstract

This description generally relates to dry and stable biological compositions, high in colony-forming units, and methods of preparation and use thereof.

Description

[0002] Microbial insecticides, herbicides, fungicides, and growth promoters that incorporate viruses, bacteria, yeasts, and beneficial fungi targeting specific insect or plant species are of interest in agriculture due to their low impact on non-target species and the environment. Maintaining the viability of these products, however, is generally a challenge during storage and formulation processing. Currently, microbial pesticide products can be prepared in liquid or dry formulations. Liquid formulations typically include suspensions of these microorganisms in water, oils, or emulsions to maintain viability and efficacy. These liquid formulations, however, require low-temperature storage and transportation, which is often inconvenient and expensive.Dry formulations, on the other hand, generally involve formulating microorganisms into wettable powders, granules, pills, coated forms, or crystals to facilitate storage and transport. However, in order to be formulated in a dry form for easier handling, these microorganisms suffer cell death and stability problems due to the drying heat generated during the formulation process.

[0003] U.S. Patent No. 8,409,822 (Trevino et al.) describes and claims compositions for providing microorganisms in a dry mode comprising precipitated silica granules having a porous structure and microorganisms loaded through the pores of the precipitated silica granules, wherein the composition operates to permit the propagation of the microorganisms within the pores of the precipitated silica granules. Also, U.S. Patent No. 9,296,989 (Trevino et al.Trevino et al. describes and claims compositions for providing live cells in a dry mode comprising an inert carrier substrate having pores, live cells loaded within the pores of the inert carrier substrate, and a surface layer disposed on an external surface of the inert carrier substrate loaded with live cells, wherein the surface layer is permeable to molecules that aid cell growth in the live cells so that the composition can operate to allow increased propagation of live cells within the inert carrier substrate compared to other compositions lacking the surface layer. Although the compositions of Trevino et al. are described as being in "dry mode," they are not, in fact, dry since the liquids containing the live microorganisms are described as substantially loaded within the pores of precipitated silica granules. The silica in Trevino et al.It acts as an absorbent and becomes loaded with 25-75% live microorganisms. At this loading level, free-flowing loaded silica is defined as touch dry. Such compositions are relatively limited in their usefulness because the concentration of organisms and the water content in the silica are not utilized, and they will likely result in a loss of activity since the organisms can still respire.

[0004] Various protectants have been used, such as sulfoxides, alcohols, monosaccharides, polysaccharides, QCACnn / l 7P7 / B / Y amino acids, peptides, glycoproteins, and other additive agents to protect microorganisms against dehydration damage. U.S. Patent No. 5,360,607 (Eyal et al.) describes and claims improved, stable, dry, pill-shaped biopesticide compositions comprising an inert carrier capable of supporting fungal growth and promoting conidial sporulation, and an entomogenic fungal biomass prepared by submerged fermentation of isolated fungi, Paecilomyces fumosoroeus. This method, however, uses alginate to encapsulate natural solid globules, which are subject to variations, particularly moisture content (e.g., water activity (Aw) level), upon which microorganisms depend for survival and respiration.

[0005] There remains an unmet need in the state of the art to prepare microorganisms in a dry form, stable at high concentration. BRIEF DESCRIPTION OF THE INVENTION

[0006] The inventors of the present invention have surprisingly discovered that microorganisms such as mold spores and bacteria can be formulated and dried at a particular temperature on the surface of various substrates to provide dry biological compositions with improved viability and a higher concentration of colony-forming units (CFUs) than those of the prior art. To achieve the desired CFU level of these dry biological compositions (Concentrated Dry Biological Compositions), the present invention defines several interrelated parameters to create an optimal environment for the microorganisms to settle without sacrificing viability. First, a substrate is selected from a group of porous particles, for example, precipitated particles with a BET surface area between 10 and 400 m² / g. Second, the microorganisms are dried on this substrate to a target total water concentration of approximately 0.0.1% by weight and approximately 15% by weight. Achieving the target total water concentration in combination with the specific substrate parameter creates a defined water activity (Aw), since water activity depends on both the total amount of water and the relative availability of water controlled by the specific substrate. The ability to achieve an ideal surface water activity allows for the stabilization of the biological material in a desirable dormant state, characterized by a low change in colony-forming units (CFU) over time.

[0007] Therefore, in the first aspect, the invention provides a dry biological composition (Composition I) comprising, in one particular embodiment, essentially consisting of, and in another particular embodiment, consisting of, (i) a substrate and (ii) microorganisms loaded onto the surface of the substrate, wherein the composition has a total moisture content of approximately 0.01% by weight to approximately 15% by weight. It has been surprisingly discovered that microorganisms can be sustained on a certain substrate in a dormant state driven by the level of activity of the resulting surface water, at high concentration and with good viability. Preferably, in the first aspect, the invention provides Composition I as follows: 1.1 Composition I, wherein the composition has a total moisture content of approximately 0.01% by weight to approximately 8% by weight; 1.2 Composition I or 1.1, wherein the composition has a total moisture content of approximately 3% by weight up to approximately 8% by weight, preferably approximately 5% by weight up to approximately QCACnn / l 7Π7 / E / Y 8% by weight, preferably still, selected from 3% by weight, 5% by weight and 7% by weight; 1.3 Composition I or 1.1 or 1.2, wherein the composition has a water activity (Aw) value between approximately 0.01 and approximately 0.6, preferably between approximately 0.2 and approximately 0.6, and even more preferably between approximately 0.3 and approximately 0.5; 1.4 Composition I or any of 1.1-1.3, wherein the composition has more than approximately 107 CFU / g, preferably greater than or equal to approximately 108 CFU / g, preferably still greater than or equal to approximately 109 CFU / g, preferably still greater than or equal to approximately 1010 CFU / g, preferably still greater than or equal to approximately 1011 CFU / g, preferably still greater than or equal to approximately 1012 CFU / g; 1.5 Composition I or any of 1.1-1.4, wherein the substrate is selected from the group consisting of silica (for example, precipitated silica, in a particular form, hydrophilic silica, for example, SIPERNAT® 22 silica), diatomaceous earth, silica gel, silicates (for example, aluminosilicates such as ZEOLEX® 301 or clays) and water-insoluble natural fiber material such as cellulose; 1.6 Composition I or any of 1.1-1.5, where the substrate is silica; 1.7 Composition I or any of 1.1-1.6, where the substrate is precipitated silica; 1.8 Composition I or any of 1.1-1.7, where the substrate is hydrophilic silica, for example, SIPERNAT® 22 silica; 1.9 Composition I or any of 1.1-1.5, wherein the substrate is water-insoluble natural fiber material such as cellulose; 1.10 Composition I or any of 1.1-1.5, where the substrate is diatomaceous earth; 1.11 Composition I or any of 1.1-1.5, wherein the substrate is silica gel; 1.12 Composition I or any of 1.1-1.5, where the substrate is silicates (for example, aluminosilicates such as ZEOLEX® 301 or clays); 1.13 Composition I or any of 1.1-1.13, wherein the particle size (d50) of the substrate is approximately 5-200 micrometers, preferably, approximately 8-160 micrometers, preferably still, approximately 9-150 micrometers, preferably still, approximately 50-150 micrometers, preferably still, approximately 50-130 micrometers, preferably still, selected from a group consisting of approximately 50 micrometers, approximately 85 micrometers and approximately 120 micrometers; 1.14 Composition I or any of 1.1-1.14, wherein the BET surface area of ​​the substrate is approximately 2-400 m2 / g, preferably, approximately 5-400 m2 / g, preferably still, approximately 10-400 m2 / g, preferably still, approximately 30-400 m2 / g, preferably still, approximately 30-300 m2 / g, preferably still, approximately 40-200 m2 / g, preferably still, approximately 180 m2 / g; 1.15 Composition I or any of 1.1-1.14, wherein the BET surface area of ​​the substrate is approximately 2 m2 / g, preferably approximately 5 m2 / g; 1.16 Composition I or any of 1.1-1.14, wherein the BET surface area of ​​the substrate is approximately 180 m2 / g; QCACnn / l 7Π7 / Β / Y 1.17 Composition I or any of 1.1-1.16, wherein the pore volume of the substrate is approximately 0.01-1.20 cc / g, preferably approximately 0.05-1.20 cc / g, preferably still approximately 0.10-1.0 cc / g, preferably still approximately 0.20-0.95 cc / g; 1.18 Composition I or any of 1.1-1.17, wherein the composition comprises a precipitated silica having a BET surface area of ​​approximately 50-200 m2 / g, preferably approximately 180 m2 / g and the composition has a total moisture content of approximately 5% by weight to approximately 8% by weight; 1.19 Composition I or any of 1.1-1.18, wherein the composition comprises a precipitated silica having a BET surface area of ​​approximately 50-200 m2 / g, preferably approximately 180 m2 / g, a particle size of approximately 5-200 micrometers, preferably still approximately 120 micrometers, the composition having a total moisture content of approximately 5% by weight up to approximately 8% by weight; 1.20 Composition I or any of 1.1-1.19, wherein the final concentration of microorganism is between approximately 4 and approximately 40% by weight, preferably approximately 4 and approximately 20% by weight of the total composition; 1.21 Composition I or any of 1.1-1.20, wherein, the microorganisms are selected from the group consisting of Bacillus subtilis QST713, Pastearía usgae; Bassian beauty, Coniothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Brongniartii beauty, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp., Lecanicillium longisporum, Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var. acridum, Nomuraea ríleyi Sporothrix insectosorum; Cydia pomonella GV; Phytophthora palmivora, Lagenidium giganteum, Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, Bacillus spp. and Lactobacllus spp. or any combination of the same, preferably, are selected from group consisting of Bacillus thuríngiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea and any combination of the same; 1.22 Composition I or any of 1.1-1.21, wherein the microorganisms are Clonostachys rosea, or in another form, Pseudomonas fluorescens, 1.23 Composition I or any of 1.1-1.22 further comprises one or more excipients, in a particular embodiment, one or more agrochemically acceptable excipients; 1.24 Composition 1.22 in tablet form, fluid concentrated form, for example, for seed treatment or in the form of an oil dispersion; 1.25 Composition I or any of 1.1-1.24, wherein the composition does not require an exogenous protectant such as alginate encapsulation; 1.26 Composition I or any of 1.1-1.25, wherein the composition further comprises a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, other polysaccharides such as maltodextrin, guar gum (for example, hydroxypropyl guar gum) and polyethylene glycol; 1.27 Composition I or any of 1.1-1.26, wherein the composition further comprises a second substrate QCRcnn / i znz / R / γ as an outer layer; 1.28 Composition 1.27, wherein the second substrate is selected from a precipitated silica such as SIPERNAT® 50 S silica, for example, or a fuming silica such as AEROSIL® 200, AEROSIL® R 972 or AEROSIL® R 812S silica; 1.29 Composition I or any of 1.1-1.28, wherein the number of colony-forming units per gram of the composition (CFU / g) remains above approximately 107 CFU / g after storage at room temperature for 120 days; 1.30 Composition I or any of 1.1-1.29, wherein the number of colony-forming units per gram of the composition (CFU / g) remains above approximately 107 CFU / g after storage at 40°C for 40 days; 1.31 Composition I or any of 1.1-1.30, wherein the number of colony-forming units per gram of the composition (CFU / g) remains above approximately 107 CFU / g after storage at a relative humidity of 65% or less for 40 days; 1.32 Composition I or any of 1.1-1.31, wherein the compacted density of the composition is greater than 150% of the compacted density of the pure substrate material; 1.33 Composition I or any of 1.1-1.32, wherein the microorganisms are larger than the pore diameter of the substrate or the microorganisms are loaded onto the surface of the substrates; 1.34 Composition I or any of 1.1-1.24 or 1.27-1.33, wherein the composition further comprises (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic or other polysaccharides such as maltodextrin, guar gum (for example, hydroxypropyl guar gum), polyethylene glycol and, polyglycerol or (II) non-reducing polysaccharides such as trehalose or sucrose or (iii) skimmed milk or dimethyl sulfoxide; 1.35 Composition I or any of 1.1-1.24 or 1.27-1.33, wherein the composition further comprises non-reducing polysaccharides such as trehalose or sucrose; 1.36 Composition I or any of 1.1-1.24 or 1.27-1.33, wherein the composition further comprises a polymer such as polyglycerol, particularly a hyperbranched polyglycerol polymer; 1.37 Composition I or any of 1.1-1.36, wherein the secondary substrate is a finely divided hydrophobic or hydrophilic particle, wherein that particle is treated on the surface, for example, with silanes or silicone oil to modify the wettability or tendency of the samples to absorb water; 1.38 Composition I or any of 1.1-1.37, wherein the secondary substrate is a silica or clay, wherein that silica or clay is treated on the surface, for example, with silanes or silicone oil to modify the wettability or tendency of the samples to absorb water; 1.39 Composition I or any of 1.1-1.38, wherein the second substrate has a high BET surface area, for example, 50 to 750 m2 / g, in a particular modality, 50-380 m2 / g; 1.40 Composition I or any of 1.1-1.39, wherein the second substrate is a hydrophobic silica; 1.41 Composition I or any of 1.1-1.40, wherein the second substrate is a precipitated silica; 1.42 Composition I or any of 1.1-1.41, wherein the second substrate is a precipitated silica with a high BET surface area, for example, 50 to 750 m2 / g, in a particular modality, 50-380 m2 / g; QCACnn / l 7Π7 / Β / Y 1.43 Composition I or any of 1.1-1.42, wherein the second substrate is SIPERNAT® 50 or ZEOFREE® silica, in a particular form; SIPERNAT® 50 silica; 1.44 Composition I or any of 1.1-1.40, wherein the second substrate is a fuming silica; 1.45 Composition I or any of 1.1-1.44, wherein the second substrate is fuming silica; 1.46 Composition I or any of 1.1-1.44, wherein the second substrate is a fuming hydrophobic silica; 1.47 Composition I or any of 1.1-1.44, wherein the second substrate is a fuming hydrophobic silica having a BET surface area of ​​180 to 220 m2 / gya and a carbon content of 3.5 to 5% as AEROSIL® R202 silica; 1.48 Composition I or any of 1.1-1.6, 1.13, 1.17 or 1.20-1.47, wherein the primary substrate is a silica with a BET surface area of ​​400-600 m2 / g, preferably 500 m2 / g; 1.49 Composition 1.49, wherein silica has a pore volume greater than 1 cc / g, preferably 1.4 cc / g by the Barrett-Joyner-Halenda model or greater than 2 cc / g, preferably 2.2 by Mercury Pore Volume; 1.50 Composition 1.50, where the silica is SIPERNAT® 50 silica.

[0008] The second aspect of the invention provides a process for preparing a dried biological composition comprising, in one particular embodiment, essentially, and in another particular embodiment, a substrate and microorganisms loaded onto the substrate, wherein the composition has a moisture content of approximately 0.01% by weight to approximately 15% by weight, the process comprising, in one particular embodiment, essentially, and in another particular embodiment, the steps of (1) combining a mixture, solution, or suspension containing microorganisms with a substrate; and (2) drying the substrate-microorganism mixture to achieve a total moisture content of approximately 0.01% to approximately 15% by weight (Process I). Preferably, the invention provides Process I as follows: 2.1 Process I, wherein microorganisms are harvested from the surface of a seed by mechanically grinding or polishing the seed surface (step (a)), resulting in a fine fraction containing microorganisms, preferably fungal spores and some parts of the seed. Preferably, the yield of microorganisms in the fine fraction is greater than 10⁹ CFU / gram of the initially ground or polished seed. Still preferably, the process comprises sieving (step (b)) the resulting fine fraction to obtain a powder with a particle size distribution defined by subsequent process steps. Preferably, the powder is combined to prepare a mixture, solution, or suspension of microorganisms (step (c). 2.2 Process 2.1, wherein step (a) comprises grinding with a grinding stone to separate the seed from the fine fraction; 2.3 Process 2.1, wherein step (a) comprises grinding with a rotating shaft within an enveloping tube inside a slotted screen under pressure conditions followed by a sieve and filter to separate the seed from the fine fraction; 2.4 Process I or any of 2.1-2.3, wherein the sieving step of the fine fraction (b) comprises sieving with a sieve mesh size of 20 to 800 pm, preferably 100 pm to 300 pm; 2.5 Process I, where microorganisms are harvested from the surface of a seed by washing it with water and separating the seed and the liquid solution or suspension of microorganisms. Preferably, the seed is agitated in water. QCAcnn / i ζηζ / E / γ for 1 to 20 min. Still preferably, the solid-liquid separation is carried out in a pressure Nutsche filter; even more preferably, a mesh size of 1 to 3 mm is used in a pressure Nutsche filter. Still more preferably, the dewatering time in a pressure Nutsche filter is 20–200 seconds. Still more preferably, the filtration pressure in the Nutsche filter is 1 to 3 bar. Still more preferably, the microorganism solution or suspension is concentrated by separating the microorganisms from the liquid in a centrifuge field. Still more preferably, the concentration step comprises separation in a disc stack separator; even more preferably, the concentration step is repeated with dilution of the concentrate with water and a second subsequent concentration in a centrifuge field to separate the soluble parts of the microorganisms. 2.6 Process I or any of 2.1-2.5, wherein, wherein step (2) dries the substrate-microorganism mixture to a total moisture content of approximately 0.01% by weight to approximately 15% by weight, preferably, approximately 0.01% by weight to approximately 8% by weight, preferably still, approximately 3% by weight to approximately 8% by weight, preferably still, approximately 5% by weight to approximately 8% by weight, preferably still selected from 3% by weight, 5% by weight and 7% by weight; 2.7 Process I or any of 2.1-2.6, wherein the drying step (2) comprises fluidized bed drying of the substrate-microorganism mixture; 2.8 Process I or any of 2.1-2.6, wherein the drying step (2) comprises spray drying the substrate-microorganism mixture; 2.9 Process I or any of 2.1-2.6, wherein the drying step (2) comprises contact drying the substrate-microorganism mixture; 2.10 Process I or any of 2.1-2.6, wherein the drying step (2) comprises freeze-drying the substrate-microorganism mixture 2.11 Process I or any of 2.1-2.10, wherein the drying air temperature is less than or equal to approximately 130°C, preferably less than or equal to approximately 90°C, preferably still less than or equal to approximately 80°C, preferably still less than or equal to approximately 50°C, preferably still approximately 30°-50°C, preferably still approximately 40°-50°C, preferably still approximately 40°-45°C, preferably still approximately 43°C; 2.12 Process I or any of 2.1-2.11, wherein the powder bed is maintained at less than or equal to approximately 35°C, preferably less than or equal to approximately 30°C, preferably still between approximately 25°C and 35°C; 2.13 Process I or any of 2.1-2.7, where the dew rate is approximately 2 mL / g of substrate; 2.14 Process I or any of 2.1-2.13, wherein the resulting composition has a water activity (Aw) value between approximately 0.01 and approximately 0.6, preferably between approximately 0.2 and approximately 0.6, and even more preferably between approximately 0.3 and approximately 0.5; 2.15 Process I or any of 2.1-2.14, wherein the resulting composition has colony-forming units of microorganisms per gram of composition (CFU / g), for example, greater than 107 CFU / g, preferably greater than or equal to approximately 108 colony-forming units per gram (CFU / g), preferably greater QCRcnn / ι znz / R / γ which is or equal to approximately 109CFU / g, preferably even greater than or equal to approximately 1010CFU / g, preferably even greater than or equal to approximately 1011CFU / g, preferably even greater than or equal to approximately 1012CFU / g; 2.16 Process I or any of 2.1-2.15, wherein the substrate is selected from the group consisting of silica (for example, precipitated silica, in a particular modality, hydrophilic silica, for example, SIPERNAT® 22 silica), diatomaceous earth, silica gel, silicates (for example, aluminosilicates such as ZEOLEX® 301 or clays) and water-insoluble natural fiber material such as cellulose; 2.17 Process I or any of 2.1-2.16, where the substrate is silica; 2.18 Process I or any of 2.1-2.17, where the substrate is precipitated silica; 2.19 Process I or any of 2.1-2.18, wherein the substrate is a hydrophilic silica, for example, SIPERNAT® 22 silica; 2.20 Process I or any of 2.1-2.16, wherein the substrate is water-insoluble natural fiber material such as cellulose; 2.21 Process I or any of 2.1-2.16, where the substrate is diatomaceous earth; 2.22 Process I or any of 2.1-2.16, where the substrate is silica gel; 2.23 Process I or any of 2.1-2.16, where the substrate is silicates (for example, aluminosilicates such as ZEOLEX® 301 or clays); 2.24 Process I or any of 2.1-2.23, wherein the particle size (d50) of the substrate is approximately 5-200 micrometers, preferably, approximately 8-160 micrometers, preferably still, approximately 9-150 micrometers, preferably still, approximately 50-150 micrometers, preferably still, approximately 50-130 micrometers, preferably still, selected from a group consisting of approximately 50 micrometers, approximately 85 micrometers and approximately 120 micrometers; 2.25 Process I or any of 2.1-2.24, wherein the BET surface area of ​​the substrate is approximately 2400 m2 / g, preferably, approximately 5-400 m2 / g, preferably still, approximately 10-400 m2 / g, preferably still, approximately 30-400 m2 / g, preferably still, approximately 30-300 m2 / g, preferably still, approximately 40-200 m2 / g, preferably still, approximately 180 m2 / g; 2.26 Process I or any of 2.1-2.25, wherein the BET surface area of ​​the substrate is approximately 2 m2 / g, preferably approximately 5 m2 / g; 2.27 Process I or any of 2.1-2.25, where the BET surface area of ​​the substrate is approximately 180 m2 / g; 2.28 Process I or any of 2.1-2.27, wherein the substrate pore volume is approximately 0.01-1.20 cc / g, preferably approximately 0.05-1.20 cc / g, preferably still approximately 0.10-1.0 cc / g, preferably still approximately 0.20-0.95 cc / g; 2.29 Process I or any of 2.1-2.28, wherein the composition comprises a precipitated silica having a BET surface area of ​​approximately 50-200 m2 / g, preferably approximately 180 m2 / g and the composition has a total moisture content of approximately 5% by weight up to approximately 8% by weight QCAcnn / i znz / R / Y weight; 2.30 Process I or any of 2.1-2.29, wherein the composition comprises a precipitated silica having a BET surface area of ​​approximately 50-200 m2 / g, preferably approximately 180 m2 / g, a particle size of approximately 5-200 micrometers, preferably still approximately 120 micrometers, the composition having a total moisture content of approximately 5% by weight up to approximately 8% by weight; 2.31 Process I or any of 2.1-2.30, wherein step (1) comprises loading between approximately 4 and approximately 40% by weight, preferably approximately 4 and approximately 20% by weight of the total composition; 2.32 Process I or any of 2.1-2.31, wherein step (1) further comprises adding polymers selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic and other polysaccharides such as maltodextrin, guar gum (for example, hydroxypropyl guar gum) and polyethylene glycol; 2.33 Process I or any of 2.1-2.32, wherein step (1) further comprises a second substrate as an outer layer; 2.34 Process 2.33, wherein the second substrate is selected from a precipitated silica such as SIPERNAT® 50 S silica, for example, or a fuming silica such as AEROSIL® 200 silica, AEROSIL® R 972 silica, or AEROSIL® R 812S; 2.35 Process I or any of 2.1-2.34, wherein the microorganisms are selected from the group consisting of Bacillus subtilis QST713, Pasteuria usgae; Beauveria bassiana, Coníothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Beauveria brongniartii, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp., Lecanicillium longisporum, Lecanicillium muscarium, Lecaniciliium sp., Metarhizium anisopliae, Metarhizium anisopliae var. acridum, Nomuraea relay Sporothrix insectosorum; Cydia pomonella GV; Phytophthora palmivora, Lagenidium giganteum, Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, Bacillus spp. and Lactobacillus spp. or any combination of the same; 2.36 Process I or any of 2.1-2.34, wherein the microorganisms are selected from the group consisting of Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea',; 2.37 Process I or any of 2.1-2.34, where the microorganisms are Clonostachys rosea', 2.38 Process I or any of 2.1-2.34, wherein the resulting composition does not require an exogenous protectant such as alginate encapsulation; 2.39 Process I or any of 2.1-2.38, wherein the number of colony-forming units per gram of the composition (CFU / g) remains above 107 CFU / g after storage at room temperature for 120 days; 2.40 Process I or any of 2.1-2.39, wherein the number of colony-forming units per gram of the composition (CFU / g) remains above 107 CFU / g after storage at 40°C for 40 days; 2.41 Process I or any of 2.1-2.40, wherein the number of colony-forming units per gram of the composition (CFU / g) remains above 107CFU / g after storage at a relative humidity of 65% or less for 40 days; QCRCnn / l 7Π7 / Β / YILI 2.42 Process I or any of 2.1-2.41, wherein the compacted density of the composition is greater than 150% of the compacted density of the pure substrate material; 2.43 Process I or any of 2.1-2.31 or 2.33-2.37 or 2.39-2.42, wherein the composition further comprises (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic or other polysaccharides such as maltodextrin, guar gum (for example, hydroxypropyl guar gum), polyethylene glycol and, polyglycerol or (ii) non-reducing polysaccharides such as trehalose or sucrose or (iii) skimmed milk or dimethyl sulfoxide; 2.44 Process I or any of 2.1-2.31 or 2.33-2.37 or 2.39-2.42, wherein the composition further comprises non-reducing polysaccharides such as trehalose or sucrose; 2.45 Process I or any of 2.1-2.31 or 2.33-2.37 or 2.39-2.42, wherein the composition further comprises a polymer such as a polyglycerol, particularly a hyperbranched polyglycerol polymer; 2.46 Process I or any of 2.1-2.33 or 2.35-2.45, wherein the secondary substrate is a finely divided hydrophobic or hydrophilic particle, wherein that particle is treated on the surface, for example, with silanes or silicone oil to modify the wettability or tendency of the samples to absorb water; 2.47 Process I or any of 2.1-2.33 or 2.35-2.45, wherein the secondary substrate is a silica or clay, wherein that silica or clay is treated on the surface, for example, with silanes or silicon oil to modify the wettability or tendency of the samples to absorb water; 2.48 Process I or any of 2.1-2.33 or 2.35-2.45, wherein the secondary substrate having a high BET surface area, for example, 50 to 750 m2 / g, in a particular modality, 50-380 m2 / g; 2.49 Process I or any of 2.1-2.33 or 2.35-2.48, wherein the second substrate is a hydrophobic silica; 2.50 Process I or any of 2.1-2.33 or 2.35-2.49, wherein the second substrate is a precipitated silica; 2.51 Process I or any of 2.1-2.33 or 2.35-2.50, wherein the second substrate is a precipitated silica with a high BET surface area, for example, 50 to 750 m2 / g, in a particular modality, 50-380 m2 / g; 2.52 Process I or any of 2.1-2.33 or 2.35-2.50, wherein the second substrate is SIPERNAT® 50 silica or ZEOFREE®, in a particular modality; SIPERNAT® 50 silica; 2.53 Process I or any of 2.1-2.33 or 2.35-2.45, wherein the second substrate is fuming hydrophobic silica having a BET surface area of ​​180 to 220 m2 / gya and a carbon content of 3.5 to 5% as AEROSIL® R202 silica; 2.54 Process I or any of 2.1-2.33 or 2.35-2.45, where the second substrate is Aerosil® R202 silica; 2.55 Process I or any of 2.1-2.18, 2.24, 2.28, 2.31-2.54, wherein the BET surface area of ​​the primary substrate is 400-600 m2 / g, preferably 500 m2 / g; 2.56 Process 2.55, wherein the substrate has a pore volume greater than 1 cc / g, preferably 1.4 cc / g by the Barrett-Joyner-Halenda model or greater than 2 cc / g, preferably 2.2 by Mercury Pore Volume; 2.57 Process I or any of 2.33-2.56, wherein the second substrate is added to the microorganism suspension before the drying step (2); 2.58 Process I or any of 2.33-2.56, wherein the second substrate is added to the microorganism suspension during the drying step (2); QCAcnn / i ζηζ / E / γ 2.59 Process I or any of 2.33-2.56, wherein the second substrate is added to the microorganism suspension after the drying step (2); 2.60 Process I or any of 2.32-2.59, wherein the polymer or polysaccharide or non-reducing polysaccharide is added to the microorganism suspension prior to the drying step (2); 2.61 Process I or any of 2.32-2.59, wherein the polymer or polysaccharide or non-reducing polysaccharide is added to the microorganism suspension during the drying step (2); 2.62 Process I or any of 2.32-2.59, wherein the polymer or polysaccharide or non-reducing polysaccharide is added to the microorganism suspension after the drying step (2); 2.63 Process I or any of the foregoing, wherein microorganisms are harvested from the surface of a seed by mechanically grinding or polishing the substrate surface (step (a)), resulting in a fine fraction that includes microorganisms, preferably fungal spores, and some parts of the seed. Preferably, the yield of microorganisms in the fine fraction is greater than 10⁹ CFU / gram of the initially ground or polished seed. Still preferably, the process comprises sieving (step (b)) the resulting fine fraction to obtain a powder with a particle size distribution defined by subsequent process steps. Still preferably, the powder is combined to prepare a mixture, solution, or suspension of microorganisms (step (c)); 2.64 Process 2.63, wherein step (a) comprises grinding the seed with a grinding stone to separate it from the fine fraction; 2.65 Process 2.63, wherein step (a) comprises grinding with a rotating shaft within an enveloping tube inside a slotted screen under pressure conditions followed by a sieve and filter to separate the seed from the fine fraction; 2.66 Process 2.63, wherein the sieving time (b) of the fine fraction comprises sieving with a sieve mesh size of 20 to 800 pm, preferably from 100 pm to 300 pm; 2.67 Process I or any formula 2.1-2.63, wherein the microorganisms are harvested from the surface of a seed by washing them with water and separating the seed and the liquid solution or suspension of microorganisms. Preferably, the seed is agitated in water for 1 to 20 minutes. Still preferably, the solution-liquid separation is carried out in a pressure Nutsche filter. Preferably, a mesh size of 1 to 3 mm is used in a pressure Nutsche filter. Still preferably, the dewatering time in a pressure Nutsche filter is 20-200 seconds. Still preferably, the filtration pressure in the Nutsche filter is 1 to 3 bar. Still preferably, the microorganism solution or suspension is concentrated by separating the microorganisms from the liquid in a centrifuge field. Still preferably, the concentration step comprises separation in a disc stack separator.Still preferably, the concentration step is repeated with dilution of the concentrate with water and a subsequent second concentration in a centrifuge field to separate the soluble parts of the microorganisms; 2.68 Process I or any of the above, wherein the drying step (2) comprises drying the substrate-microorganism mixture in a fluidized bed; 2.69 Process I or any of the foregoing, wherein the drying step (2) comprises spray drying the QCACnn / l 7Π7 / Β / Y substrate-microorganism mixture; 2.70 Process I or any of the above, wherein the drying step (2) comprises contact drying the substrate-microorganism mixture; 2.71 Process I or any of the above, wherein the drying step (2) comprises freeze-drying the substrate-microorganism mixture; 2.72 Process I or any of the foregoing, wherein the drying air temperature is less than or equal to approximately 130°C, in a particular mode, less than or equal to approximately 90°C, preferably, less than or equal to approximately 80°C, preferably still, less than or equal to approximately 50°C, preferably still, approximately 30°-50°C, preferably still, approximately 40°-50°C, preferably still, approximately 40°-45°C, preferably still, approximately 43°C; 2.73 Process I or any of the foregoing, wherein the powder bed is maintained less than or equal to approximately 35°C, preferably from approximately 25°C to approximately 35°C; 2.74 Process I or any of the above, wherein the resulting composition has a water activity (Aw) value between approximately 0.01 and approximately 0.6, preferably between approximately 0.2 and approximately 0.6, and even more preferably between approximately 0.3 and approximately 0.5; 2.75 Process I or any of the foregoing, wherein the resulting composition has colony-forming units of microorganisms per gram of composition (CFU / g), for example, greater than approximately 107CFU / g, preferably greater than or equal to approximately 108 colony-forming units per gram (CFU / g), preferably greater than or equal to approximately 109CFU / g, preferably still greater than or equal to approximately 1010CFU / g, preferably still greater than or equal to approximately 1011CFU / g, preferably still greater than or equal to approximately 1012CFU / g; 2.76 Process I or any of the foregoing, wherein the drying air temperature is less than or equal to approximately 130°C, in a particular mode, less than or equal to approximately 90°C, preferably, less than or equal to approximately 80°C, preferably still, less than or equal to approximately 50°C, preferably still, approximately 30°-50°C, preferably still, approximately 40°-50°C, preferably still, approximately 40°-45°C, preferably still, approximately 43°C; 2.77 Process I or any of the foregoing, wherein the powder bed is maintained at less than or equal to approximately 35°C, preferably from approximately 25°C to approximately 35°C; 2.78 Process I or any of the above, wherein the resulting composition has a water activity (Aw) value between approximately 0.01 and approximately 0.6, preferably between approximately 0.2 and approximately 0.6, and even more preferably between approximately 0.3 and approximately 0.5; 2.79 Process I or any of the foregoing, wherein the resulting composition has colony-forming units of microorganisms per gram of composition (CFU / g), for example, greater than approximately 10⁷ CFU / g, preferably greater than or equal to approximately 10⁸ CFU / g, preferably greater than or equal to approximately 10⁹ CFU / g, preferably still greater than or equal to approximately 10¹⁰ CFU / g, preferably still greater than or equal to approximately 10¹¹ CFU / g, preferably QCRCnn / l 7Π7 / Β / Y even, greater than or equal to approximately 1012UFC / g!

[0009] In the third aspect, the invention provides a dry biological composition (Composition IIj) prepared by Process I or any of 2.1-2.79 of the present invention. In another embodiment of the third aspect, the invention provides a dry biological composition (Composition II-A) prepared by Process I or any of 2.1-2.42 of the present invention. In yet another embodiment of the third aspect, the invention provides a dry biological composition (Composition II-B) prepared by Process I or any of 2.43-2.79 of the present invention.

[0010] The compositions of the present invention are also useful for application to seeds to protect them against pests or to provide microorganisms with a biostimulatory function such as phosphorus release or nitrogen supply. Therefore, in the fourth aspect, the invention provides Composition I or any of 1.1-1.50 or Composition II or any of 2.1-2.79, further comprising, in one particular embodiment, essentially consisting of, and in another particular embodiment, consisting of, a seed to be treated (Composition III). In a further embodiment of the fourth aspect, the invention provides Composition I or any of 1.1-1.33 or Composition II-A, further comprising, in one particular embodiment, essentially consisting of, and in another particular embodiment, consisting of, a seed to be treated (Composition III-A). In another embodiment of the fourth aspect, the invention provides Composition I or any of 1.34-1.50 or Composition II-B, further comprises, in one particular form, consisting essentially of, and in another particular form, consisting of, a seed to be treated (Composition III-B). These compositions may optionally comprise a colorant.

[0011] In the fifth aspect, the invention provides a method for controlling insects, fungi, or nematodes on an area to be treated, optionally comprising reconstituting the concentrated dry biological composition of the invention (i.e., any of Composition I or any of 1.1-1.50) or Composition II or any of 2.1-2.79 or Composition III) and applying an effective quantity of the (optionally reconstituted) concentrated dry biological composition of the invention to the area to effect the treatment. In a further embodiment of the fifth aspect, the invention provides a method for controlling insects, fungi, or nematodes on an area to be treated, optionally comprising reconstituting the concentrated dry biological composition of the invention (i.e., any of Composition I or any of 1.1-1.50).33 or Composition II-A or Composition III-A) and applying an effective quantity of the concentrated (optionally reconstituted) dry biological composition of the invention to the area to be treated. In another embodiment of the fifth aspect, the invention provides a method for controlling insects, fungi, or nematodes on an area to be treated, optionally comprising reconstituting the concentrated dry biological composition of the invention (i.e., Composition I or any of 1.34-1.50), Composition II-B or any of 2.43-2.79 or Composition III-B) and applying an effective quantity of the concentrated (optionally reconstituted) dry biological composition of the invention to the area to be treated. In one embodiment, the area to be treated is a portion of a plant, including, without limitation, vegetative cuttings, roots, bulbs, tubers, stems, fruits, flowers, and / or leaves of plants, for example, corn, wheat, sorghum, soybeans, citrus and non-citrus fruits, nuts, and related plants.In another modality, the area to be treated is soil or seeds or mixtures thereof. BRIEF DESCRIPTION OF THE FIGURES

[0012] Figure 1 shows a graph of log UFC over time for Examples 12-23 stored at ocRcnn / i znz / R / v 40°C. The samples are labeled with additives.

[0013] Figure 2 shows the decimal reduction time, in units of weeks, for Examples 12-23 stored at 40°C. The error bars are the standard error of the regression.

[0014] Figure 3 shows the decline of log (CFU) with time for Examples 12-23 stored at high humidity.

[0015] Figure 4 shows the Decimal Reduction Time for each sample stored at high humidity. The error bars represent the standard error of the regression. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides a system for delivering microorganisms (e.g., microbial pesticides such as mold spores and other bacteria) in a dry, stable form and at high CFU levels compared to those of the prior art. It has been found that microorganisms can be dried onto certain substrates, for example, using the methods described herein, at a target total water concentration between approximately 0.01% by weight and approximately 15% by weight to create a suitable surface for the biological material to enter a dormant state. The exemplary substrate useful for the present invention includes, but is not limited to, silica, in a particular embodiment, precipitated silica; in another particular embodiment, hydrophilic silica; and in a specific embodiment, SIPERNAT® 22 silica.Another exemplary substrate also includes diatomaceous earth, silica gel, silicates (e.g., aluminosilicates such as ZEOLEX® 301 or clay), and water-insoluble natural fiber material such as cellulose. In another embodiment, the substrate is a silica with a BET surface area of ​​400–600 m² / g, preferably 500 m² / g. In yet another embodiment, the silica has a pore volume greater than 1 cc / g, preferably 1.4 cc / g according to the Barrett-Joyner-Halenda model, or greater than 2 cc / g, preferably 2.2 cc / g according to the Mercury Pore Volume, preferably SIPERNAT® 50 silica.

[0017] The typical particle size of the substrate of the compositions of the present invention may have a d50 of approximately 5-200 micrometers, preferably, approximately 8-160 micrometers, preferably, approximately 9-150 micrometers, preferably still, approximately 50-150 micrometers, preferably still, approximately 50-130 micrometers, preferably still, selected from a group consisting of approximately 50 micrometers, approximately 85 micrometers, and approximately 120 micrometers. The particle size of the silica may be measured by any method known to a person skilled in the art, for example, such as dry particle size analysis using laser light diffraction or Scanning Electron Microscopy (SEM) analysis.

[0018] The typical BET surface area of ​​the substrates of the composition of the present invention is approximately 2-400 m² / g, preferably approximately 5-400 m² / g, preferably approximately 10-400 m² / g, preferably still approximately 30-400 m² / g, preferably still approximately 30-300 m² / g, preferably still approximately 40-200 m² / g, preferably still approximately 180 m² / g. The BET surface area of ​​silica substrate is approximately 10-400 m² / g, preferably approximately 30-400 m² / g, preferably approximately 30-300 m² / g, preferably still approximately 40-200 m² / g, preferably still approximately 180 m² / g. The natural fiber substrate may have a lower BET surface area as QCACnn / l 7P7 / B / Y approximately 2 m² / g, preferably approximately 5 m² / g. In another embodiment, the BET surface area of ​​the substrates of the compositions and methods of the present invention is greater than 350 m² / g, preferably approximately 500 m² / g. Preferably, silica with a medium (greater than or equal to 350 m² / g) to high BET surface area (150 to 350 m² / g) is useful for the compositions and methods of the present invention. It is believed that such silica has better control of water activity and better preservation of CFUs. Therefore, in yet another embodiment, the substrate is a silica with a BET surface area of ​​400-600 m² / g, preferably 500 m² / g.Where the compositions or methods of the invention comprise a substrate with a high BET surface area such as SIPERNAT® 50 S silica, the compositions and methods preferably further comprise (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin, guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) non-reducing polysaccharides such as trehalose or sucrose, or (iii) skimmed milk or dimethyl sulfoxide. Silica of a low BET surface area (e.g., 50 to 150 m² / g) is also useful for the compositions of the invention.

[0019] The typical pore volume of the substrates of the compositions of the present invention is approximately 0.01-1.20 cc / g, preferably approximately 0.05-1.20 cc / g, preferably still approximately 0.10-1.0 cc / g, preferably still approximately 0.20-0.95 cc / g. Substrates such as silica having a pore volume of approximately 0.05-1.20 cc / g, preferably approximately 0.10-1.0 cc / g, preferably still 0.20-0.95 cc / g are useful for the present invention. Substrates such as cellulose having a lower pore volume, such as 0.01-1.2 cc / g, are also useful for the invention. These pore volume values ​​are measured on the basis of the Barrett-Joyner-Halenda model. In another embodiment, the substrate of the present invention is a silica with a pore volume greater than 1 cc / g, preferably 1.4 cc / g by the Barrett-Joyner-Halenda model or greater than 2 cc / g, preferably 2.2 by Mercury Pore Volume.

[0020] The substrates described herein provide a suitable surface for the microorganisms to be deposited and efficiently dried in the dryer to achieve a target total water content described herein, avoiding prolonged exposure to heat that decreases organism survival and viability after storage. In particular, the microorganisms are dried on the substrate surface, for example, using the methods described herein to a target total water content of between approximately 0.01% by weight and 15% by weight, in a particular modality, between approximately 0.01% by weight and approximately 8% by weight, preferably, approximately 5% by weight and approximately 8% by weight, preferably still, approximately 5% by weight and approximately 8% by weight, preferably still selected from approximately 3% by weight, approximately 5% by weight, and approximately 7% by weight.The water content level measures the amount of water present in a particular product and can be measured by methods known in the state of the art, for example, by measuring the amount of water (% by weight) lost per gram of product at approximately 100°C for a period of time at constant weight (i.e., loss after drying).

[0021] The selection of the substrate of the invention, together with the target moisture content level provided herein, creates an optimum defined water activity (Aw) level between approximately 0.01 and approximately 0.6, preferably between approximately 0.2 and approximately 0.6, and even more preferably between approximately 0.2 and approximately 0.5, and even more preferably between approximately 0.3 and approximately 0.5, where the microorganisms are believed to be inactivated but still live, thus providing a dry and stable system for supporting these microorganisms without sacrificing viability. Water activity is defined as the ratio of the partial pressure of water vapor in a product to the standard-state partial pressure of vapor of pure water. Water activity (Aw) measures the equilibrium amount of water available for the hydration of a particular material (i.e., water availability).Certain substances with the same moisture content can have different water activity levels. Water activity (Aw) can be measured using methods known in the prior art, such as resistive electrolytic hygrometers (REHs), capacitance hygrometers, and dew point hygrometers.

[0022] The present invention allows microorganisms to concentrate on the substrate to a high colony-forming unit concentration. In one particular embodiment, the microorganisms concentrate on the substrate (particularly silica) in the form of a crust. Therefore, the compositions of the present invention have more than approximately 10⁷ CFU / g, preferably greater than or equal to approximately 10⁸ CFU / g, preferably even greater than or equal to approximately 10⁹ CFU / g, preferably even greater than or equal to approximately 10¹⁰ CFU / g, preferably even greater than or equal to approximately 10¹¹ CFU / g, preferably even greater than or equal to approximately 10¹² CFU / g.The compositions of the present invention are particularly stable. In one specific embodiment, the number of colony-forming units per gram of the composition (CFU / g) remains above approximately 10⁷ CFU / g after storage at room temperature for 120 days. In another embodiment, it remains above approximately 10⁷ CFU / g after storage at 40°C for 40 days. In yet another embodiment, it remains above approximately 10⁷ CFU / g after storage at a relative humidity of 65% or lower for 40 days. In one particular embodiment, the compositions of the invention have a CFU loss of less than 5 log, preferably less than 3 log, more preferably less than 2 log, and more preferably less than 1 log, over ten weeks at room temperature, for example, 25°C.In another particular embodiment, the Composition of the Invention has a loss of CFU less than 5 log, preferably less than 3 log, more preferably less than 2 log, more preferably less than 1 log during ten weeks at room temperature (e.g. 25°C) and high humidity, e.g., at a relative humidity of 70%.

[0023] The microorganism useful for the invention includes natural or recombinant microorganisms that act as predators of or interfere with the life cycle of other undesirable microorganisms, or provide a beneficial effect to the area to be treated, or can produce a biologically active substance that is effective as a pesticide. Exemplary microorganisms useful for the invention include those that can be used in the agricultural field, which include, but are not limited to, Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, and similar microorganisms or any combination thereof.Other microorganisms useful for the invention also include, but are not limited to bacteria such as Bacillus subtilis QST713 and Pasteuria usgae; fungi such as Beauberia bassiana, Coníothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Beauberia brongniartíi, Hirsutella thompsoníí, Isaria fumosorosea, Isaria sp„ Lecanicillium longisporum,. QCACnn / l 7Π7 / Ε / Υ Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var. acridum, Nomuraea rileyi, and Sporothrix insectosorum; viruses such as Cydia pomonella GV; and oomycetes such as Phytophthora palmivora, Lagenidium giganteum, Bacillus spp., and Lactobacillus spp., or any combination thereof. Additional examples of fungi and subspecies useful for the invention can be found in Faria et al., Biological Control 43 (2007) 237–256, the contents of which are therefore incorporated by reference in their entirety. The present list is not intended to be exhaustive and may include other microorganisms useful in the agricultural field, as well as in other fields such as the food, medical, pharmaceutical, detergent, and energy sectors.

[0024] The substrate-microorganism mixture of the present invention does not require any exogenous protectant such as alginate encapsulation, but may optionally be treated with polymers or other materials such as fuming silica (e.g., Aerosil®) or a combination of polymers and fuming silica to provide additional moisture protection and insulation from high-temperature storage. Accordingly, in one embodiment, the composition further comprises (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin, guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol; or (ii) non-reducing polysaccharides such as trehalose or sucrose; or (iii) skimmed milk or dimethyl sulfoxide.In another embodiment, the composition of the invention further comprises a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, and other polysaccharides such as maltodextrin, guar gum (e.g., hydroxypropyl guar gum), and polyethylene glycol. In yet another embodiment, the polymer is polyglycerol, e.g., a hyperbranched polyglycerol polymer. In yet another embodiment, the composition further comprises non-reducing polysaccharides such as trehalose or sucrose. In yet another embodiment, the composition further comprises the combination of polymers and a secondary substrate as best described below. The amount of the polymers can be approximately 0.1–3% by weight in one particular embodiment, approximately 0.1–1% by weight in another particular embodiment, or approximately 1–1.5% by weight of the microorganism suspension.It should be noted that the polymer or polysaccharide or non-reducing polysaccharide herein may be added before, during or after the drying step (2).

[0025] The substrate-microorganism mixture of the present invention can also be treated with a second substrate, such as an inorganic material, to provide additional protection against moisture during storage. In one embodiment, the second substrate is selected from precipitated silica, such as SIPERNAT® 50 S silica, for example, at less than 3% as an outer layer. In another embodiment, the composition of the invention further comprises the addition of a second substrate, such as fuming silica, such as AEROSIL® 200 silica, AEROSIL® R 972 silica, or AEROSIL® R 812S silica, for example, at less than 2% as an outer layer. In yet another embodiment, the second substrate is a hydrophobic fuming silica having a BET surface area of ​​180 to 220 m² / g and a carbon content of 3.5 to 5%, such as AEROSIL® R202 silica. The amount of the second substrate can be approximately 0.1-3% by weight, in a particular modality, approximately 0.1-1% by weight, in a particular embodiment, approximately 0.1% by weight of the total composition. In a particular embodiment, the Compositions of the Invention comprise microorganisms and a substrate such as silica having a BET surface area greater than or equal to 350, for example, a BET surface area of ​​400-600 m² / g, preferably 500 m² / g, wherein such substrates are coated with one or more of (i) polymers selected from the group consisting of polyvinyl alcohol, xanthan gum, etc. QCACnn / l 7Π7 / Β / Υ gum arabic or polysaccharides such as maltodextrin, guar gum (e.g. hydroxypropyl guar gum), polyethylene glycol and, polyglycerol or (i) non-reducing polysaccharides such as trehalose or sucrose or (iii) skimmed milk or dimethyl sulfoxide. In another embodiment, the Compositions of the Invention comprise one or more microorganisms and a substrate, the microorganism-substrate further comprising a secondary substrate (as described below as a fuming hydrophobic silica having a BET surface area of ​​180 to 220 m2 / g and a carbon content of 3.5 to 5% as AEROSIL® R202 silica) and optionally one or more of (i) polymers selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or polysaccharides such as maltodextrin, guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) non-reducing polysaccharides such as trehalose or sucrose, or (iii) skimmed milk or dimethyl sulfoxide.In another embodiment, the Compositions of the Invention comprise a microorganism, a primary silica substrate, a secondary silica substrate, and one or more polymers from the group consisting of (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin, guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (ii) non-reducing polysaccharides such as trehalose or sucrose, or (iii) skimmed milk or dimethyl sulfoxide.In another embodiment, the Compositions of the Invention comprise microorganisms, a hydrophilic silica substrate (for example, a precipitated hydrophilic silica such as SIPERNAT® 22 silica), a secondary silica substrate (for example, a fuming hydrophilic or hydrophobic silica such as AEROSIL® R202 or 200 silica), and optionally one or more of (i) polymers selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or polysaccharides such as maltodextrin, guar gum (for example, hydroxypropyl guar gum), polyethylene glycol, and polyglycerol; or (ii) non-reducing polysaccharides such as trehalose or sucrose; or (iii) skimmed milk or dimethyl sulfoxide. In a particular embodiment, the secondary substrate is added after the drying step (2).

[0026] The compositions of the present invention can be applied directly to a treatment area such as a plant, seed, or pest, or they can be formulated into a biological formulation, for example, for application to that treatment area. Traditionally, aqueous formulations containing microbes or other biologically active materials are difficult to stabilize within the shelf life of a typical concentrated suspension formulation. The compositions discussed in this invention are intended to lower the overall formulation barrier. The present invention teaches approaches for creating a biological composition with a higher level of activity (CFU) and stability (low change in CFU over time). A more stable composition lowers the formulation hurdles required to generate a useful product suitable for use in an agricultural field application.The advantages of the compositions of the invention allow them to be formulated in both liquid and dry agrochemical formulations. Examples of such formulations include, but are not limited to, WP (Wettable Powder), WG (Water Dispersible Granules), SC (Suspension Concentrate), OD (Oil Dispersion), and FS (Seed Treatment). Therefore, in another aspect, the invention provides a biological formulation comprising the composition of the invention, for example, Composition I or any of 1.1-1.33 or 1.34-1.50, and one or more excipients. Since the microorganism of the invention may be useful for agricultural applications, agrochemically acceptable excipients or adjuvants are anticipated as wetting agents. Furthermore, combinations with other active agrochemical ingredients may be used. ocRcnn / i ζηζ / E / γ

[0027] Water-based formulations of the SC form may include one or more dispersants, polymers, binders, surfactants, coloring agents, and antifreeze compounds. The selection of specific formulation aids is within the knowledge of a person skilled in the art.

[0028] Dry formulations include powders (DP), seed coating powders (DS), granules (GR), microgranules (MG), water-dispersible granules (WG), wettable powders (WP), and may include one or more binding, dispersing, and wetting agents. The selection of specific formulation aids is also within the knowledge of a person skilled in the art.

[0029] The compositions of the invention are particularly useful in tablet form of formulation types ST (water-soluble tablets) and TB (tablet). Accordingly, in one particular embodiment, the invention provides a biological tablet comprising the composition of the invention, for example, Composition I or any of 1.1-1.33 or any of 1.34-1.50, and one or more excipients. The excipients useful for the tablet formulation of the invention may comprise one or more useful lubricants, binders, disintegrants, and fillers. Useful lubricants include, but are not limited to, talc, magnesium stearate, calcium stearate, stearic acid, boric acid, polyethylene glycol, and sodium stearyl fumarate. Useful binders include, but are not limited to, microcrystalline cellulose, cellulose acetate, carrageenan, dextrin, glucose, ethyl cellulose, and polyvinylpyrrolidone.Useful fillers include, but are not limited to, corn starch, potato starch, sodium starch glycolate, amylose, primogel, crospovidone, and croscarmellose sodium. Useful disintegrants include, but are not limited to, calcium silicates. Sample tablets can be made with 2 to 30% microorganism powder containing 10⁹ CFU per gram of organisms. Two-gram tablets are compressed to 20 kN. The tablets can be rapidly disintegrated using 7.5% FM1000.

[0030] The oily dispersion formulation of the invention comprises the composition of the present invention, for example, Composition I or any of 1.1-1.33 or any of 1.34-1.50, dispersed in a non-aqueous or water-insoluble liquid such as mineral, paraffinic, or vegetable oil and may contain one or more dispersants, emulsifying polymers, binders, or surfactants. The selection of specific formulation aids is within the knowledge of a person skilled in the art.

[0031] Agricultural oils useful for the formulation of the invention include paraffin oils such as octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane and mixtures thereof of those oils mixed with higher boiling homologues such as hepta-, octa-, nona-decane, eicosane, heneicosane, docosane, tricosane, tetracosane, pentacosane and, branched-chain isomers thereof; vegetable oils such as olive oil, kapok oil, castor oil, papaya oil, camellia oil, palm oil, sesame oil, corn oil, rice bran oil, peanut oil, cottonseed oil, soybean oil, rapeseed oil, linseed oil, tung oil, sunflower oil, safflower oil or transesterified products thereof, such as rapeseed oil methyl ester and rapeseed oil ethyl ester; animal oil, such as whale oil, cod liver oil or mink oil;other oils such as butanol, π-octanol, / -octanol, dodecanol, cyclopentanol, cyclohexanol, cyclooctanol, ethylene glycol, propylene glycol or benzyl alcohol, caproic acid, capric acid, caprylic acid, pelargonic acid, succinic acid, glutaric acid, benzoic acid, toluic acid, salicylic acid and italic acid, acetate; QCACnn / l 7Π7 / Β / Y of benzyl, ethyl ester of caproic acid, ethyl ester of pelargonic acid, methyl or ethyl ester of benzoic acid, methyl of salicylic acid, methyl, propyl or butyl ester of salicyl, diesters of italic acid with dimethyl ester, dibutyl ester, diisooctyl ester of saturated aliphatic italic acid or any combination thereof.

[0032] The invention also anticipates the composition of the invention for seed treatment or seed dressing. Accordingly, in one embodiment, the compositions of the invention provide a concentrated flowable form, for example, for seed treatment (FS form), which can be prepared by mixing Composition I or any of 1.1-1.33 or any of 1.34-1.50 with one or more dispersants, film-forming polymers, binders, surfactants, and coloring agents, and adding the mixture to a seed. Ingredients to aid the formulation in adhering to the seed, strengthen the coating, and reduce dustiness may also be included. The selection of specific formulation aids is also within the knowledge of a person skilled in the prior art.

[0033] In another aspect, the invention also provides processes for preparing a dried biological composition comprising, in one particular embodiment, essentially consisting of, and in another particular embodiment, consisting of the steps of (1) combining a mixture, solution, or suspension of microorganisms with a substrate; and (2) drying the substrate-microorganism mixture to achieve a total moisture content of approximately 0.01 to approximately 15% by weight, preferably approximately 3% to approximately 8% by weight, and preferably still approximately 5% to approximately 8% by weight, and preferably still selected from 3%, 5%, and 7% by weight. In a further embodiment, step (2) of the process of the present invention dries the composition to a resulting water activity (Aw) value of between approximately 0.01 and approximately 0.6, preferably between approximately 0.2 and approximately 0.6.6, preferably still, between approximately 0.3 and approximately 0.5.

[0034] The microorganisms to be used in the compositions of the present invention can be obtained by various means. In one embodiment, the microorganisms can be harvested from the surface of a seed by washing the seed with water, for example, a 1:1 water-to-seed ratio. In another embodiment, the microorganisms can be harvested from the surface of a seed by mechanically grinding or polishing the seed surface (step (a)), resulting in a fine fraction containing microorganisms, in a particular embodiment, containing fungal spores and some parts of the seed. In a particular embodiment, the yield of microorganisms in the fine fraction is greater than 10⁹ CFU / gram of the initially ground or polished seed. In a particular embodiment, the process comprises sieving (step (b)) the resulting fine fraction to obtain a powder with a particle size distribution defined by subsequent process steps.In one particular embodiment, the powder is combined to prepare a mixture, solution, or suspension of microorganisms (step c). In one particular embodiment, step (a) comprises grinding with a grinding stone to separate the seed from the fine fraction. In another particular embodiment, step (a) comprises grinding with a rotating shaft within a coiled tube inside a slotted screen under pressure, followed by sieving and filtering to separate the seed from the fine fraction. Sieving the fine fraction (step (b) may comprise sieving with a sieve mesh size of 20 to 800 µm, in one particular embodiment from 100 µm to 300 µm. QCRCnn / l 7f\7IW

[0035] In another embodiment, microorganisms can be harvested from the surface of a seed by washing it with water and separating the seed from the liquid solution or suspension of microorganisms. In one particular embodiment, the seed is agitated in water for 1 to 20 minutes. In another particular embodiment, solid-liquid separation is carried out in a pressure Nutsche filter; in another particular embodiment, a mesh size of 1 to 3 mm is used in a pressure Nutsche filter. In another particular embodiment, the dewatering time in a pressure Nutsche filter is 20–200 seconds. In one particular embodiment, the filtration pressure in the Nutsche filter is 1 to 3 bar. In another particular embodiment, the microorganism solution or suspension is concentrated by separating the microorganisms from the liquid in a centrifuge field. In one particular embodiment, the concentration step comprises separation in a disc stack separator.In one particular modality, the concentration step is repeated with dilution of the concentrate with water and a subsequent second concentration in a centrifuge field to separate the soluble parts of the microorganisms.

[0036] The substrate useful for step (1) of the process of the present invention is selected from a group consisting of silica (e.g., precipitated silica, in a particular embodiment, hydrophilic silica, e.g., SIPERNAT® 22 silica), diatomaceous earth, silica gel, silicates (e.g., aluminosilicates such as ZEOLEX® 301 or clays) and water-insoluble natural fiber material such as cellulose.In one embodiment, the substrate of step (1) of the process of the invention is silica; in a further embodiment, precipitated silica, for example, wherein the particle size (d50) of the substrate is approximately 5-200 micrometers, preferably, approximately 8-160 micrometers, preferably still, approximately 9-150 micrometers, preferably still, approximately 50-150 micrometers, preferably still, approximately 50-130 micrometers, preferably still, selected from a group consisting of approximately 50 micrometers, approximately 85 micrometers, and approximately 120 micrometers. In yet another embodiment, the substrate of step (1) of the process of the present invention is precipitated hydrophilic silica.In one further embodiment, the silica of step (1) of the process of the present invention has (i) a BET surface area of ​​approximately 2-600 m2 / g, in one further embodiment, 500 m2 / g, in another further embodiment, 2-400 m2 / g, preferably, approximately 5-400 m2 / g, preferably still, approximately 10-400 m2 / g, preferably still, approximately 30-400 m2 / g, preferably still, approximately 30-300 m2 / g, preferably still, approximately 40-200 m2 / g, preferably still, approximately 180 m2 / g; and / or (i) pore volume of approximately 0.01-1.20 cc / g, preferably approximately 0.05-1.20 cc / g, preferably still approximately 0.10-1.0 cc / g, preferably still approximately 0.20-0.95 cc / g by the Barrett-Joyner-Halenda model; and / or (ii) pore volume greater than 1 cc / g, preferably 1.4 cc / g by the Barrett-Joyner-Halenda model or greater than 2 cc / g, preferably 2.2 by Mercury Pore Volume.In one further modality, the substrate of step (1) is selected from SIPERNAT® 22 or SIPERNAT® 50 S silica.

[0037] The Process of the present invention described herein may further comprise adding after step (1), but in one embodiment, before step (2), in another embodiment, during step (2), and in yet another embodiment, after step (2), (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic, or other polysaccharides such as maltodextrin, guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol, and polyglycerol, or (i) non-reducing polysaccharides such as trehalose or sucrose, or (ii) skimmed milk or dimethyl QCRcnn / ι znz / R / γ sulfoxide, in a particular embodiment polyvinyl alcohol or polyglycerol (e.g., hyperbranched polyglycerol); and / or (i) a secondary substrate as an outer layer. In one particular embodiment, the secondary substrate is selected from a precipitated silica such as SIPERNAT® 50 S silica or a fuming silica such as AEROSIL® 200, AEROSIL® R 972, AEROSIL® R 812S, or AEROSIL® 202, preferably AEROSIL® 200 or R202, more preferably AEROSIL® R202 silica. The polymers described herein may be added without the secondary substrate. In another embodiment, the polymer described herein may be added with the secondary substrate and may be added before or after the secondary substrate. The polymers and / or the secondary substrate can be added before the drying step (2) or during the drying step (2) or after the drying step (2).

[0038] The drying step (2) of the process of the invention can be achieved by fluidized bed drying, spray drying, contact drying, or freeze drying. Fluidized bed drying can be achieved by allowing the inlet air temperature to be less than or equal to approximately 90°C, preferably less than or equal to approximately 80°C, preferably less than or equal to approximately 50°C, preferably still less than or equal to approximately 30°–50°C, preferably still less than or equal to approximately 40°–50°C, preferably still less than or equal to approximately 40°–45°C, preferably still less than or equal to approximately 43°C.In one particular embodiment, the drying step (2) of the process of the invention can be achieved by preheating the spray dryer with a very low fan speed using warm air at the inlet air temperature of less than or equal to approximately 50°C, preferably to approximately 30-50°C, preferably still to approximately 40-50°C, preferably still to approximately 40-45°C, and spraying the microorganism mixture into the chamber onto the substrate. Preferably, the pumping rate at laboratory scale is 1 mL / min, preferably still to 2 mL / min of substrate. Optionally, the drying step (2) also includes drying under reduced pressure (e.g., at 0.1 bar).

[0039] Spray drying can be achieved by allowing the inlet air temperature to be less than or equal to approximately 130°C, preferably less than or equal to 110°C, preferably even less than or equal to 100°C, preferably even less than or equal to approximately 90°C, preferably even less than or equal to approximately 80°C, preferably even less than or equal to approximately 50°C, preferably even at approximately 30°-50°C. Spray drying can be done with a gas flow.

[0040] Preferably, the drying step (2) of the process of the invention comprises maintaining the powder bed temperature less than or equal to approximately 35°C, preferably less than or equal to approximately 30°C, and preferably still between approximately 25°C and 35°C.

[0041] Drying time is believed to be proportional to the surface area of ​​the silica, and water activity control is inversely proportional to the surface area of ​​the silica. Therefore, in one embodiment, silica with a medium (such as 150 to 350 m² / g) to high (greater than 350 m² / g, such as 400–600 m² / g, e.g., 500 m² / g) BET surface area results in better water activity control and better preservation of the effluents. In another embodiment, the use of silica with a high BET surface area (greater than 350 m² / g, such as 400–600 m² / g, e.g., 500 m² / g) with wetting agents, polymers, or polysaccharides also results in good water activity control and preservation of the effluents for a long period of time. Preferably, silicas with a high BET surface area are dried under a short time and high temperature, for example, using spray drying at 100°C for a short period of time (for QCACnn / l 7Π7 / Β / Y example, residence time of 2-80 seconds).

[0042] The mixture, solution, or suspension of microorganisms from step (1) of the process of the invention can be fermented in a stirred batch fermenter by adding sugars and other nutrients in a batch reactor that is aerated or maintained in an anaerobic state to allow the organisms to multiply and reach an optimum state for harvesting, depending on the nature of the organism. Alternatively, the organisms can be grown on solid media such as cellulosic material, seeds, and other solid materials suspended in a stirred reactor. Furthermore, microorganisms grown on solid media in a dry, but humidified, environment are washed off the seeds when optimal.

[0043] For the purposes of this application, AEROSIL® 200 silica refers to a hydrophilic silica having a BET surface area of ​​200 m2 / g. AEROSIL® R 202, R 972, R 812 refers to hydrophobic fuming silica.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art. In case of conflict, this document, including the definitions, shall prevail. Preferred methods and materials are described below, although similar or equivalent methods and materials may be used in the practice or testing of the present invention. All publications, patent applications, patents, or other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples described herein are for illustrative purposes only and are not intended to be limiting.

[0045] The terms “comprises,” “includes,” “having,” “has,” “may,” “contains,” and variants thereof, as used herein, are intended to be open transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “one,” and “the” include references to the plural unless the context clearly dictates otherwise. This description also encompasses other modalities such as “comprising,” “consisting of,” and “essentially consisting of,” the modalities or elements presented herein, whether explicitly stated or not.

[0046] The conjunctive term “or” includes any and all combinations of one or more of the listed elements associated by the conjunctive term. For example, the phrase “an apparatus comprises A or B” may refer to an apparatus that includes A where B is not present, an apparatus that includes B where A is not present, or an apparatus where both A and B are present. The phrases “at least one of A, B,... and N” or “at least one of A, B,... N or combinations thereof” are defined in the broadest sense to mean one or more elements selected from the group consisting of A, B, ... and N, that is, any combination of one or more of the elements A, B, ... or N, including any element alone or in combination with one or more of the other elements, which may also include, in combination, additional unlisted elements.

[0047] The modifier “approximately” used in relation to a quantity is inclusive of the stated value and has the meaning indicated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The modifier “approximately” should also be regarded as describing the interval defined by the absolute values ​​of the two endpoints. For example, the expression “from approximately 2 to approximately 4” also describes an interval “from 2 to 4”. The term “approximately” may refer to QCAcnn / i ζηζ / E / γ plus or minus 10% of the stated number. For example, “approximately 10%” may indicate a range of 9% to 11% and “approximately 1” may mean from 0.9 to 1.1. Other meanings of “approximately” may be evident from the context, such as rounding, for example, “approximately 1” may also mean from 0.5 to 1.4. Examples

[0048] The foregoing may be better understood with reference to the following examples, which are presented for illustrative purposes and are not intended to limit the scope of the Invention.

[0049] Examples 1-10. Composition of the Invention Fluid Bed Drying: Approximately 50 grams of a drained suspension of a 1:1 water rinse of seeds containing Chlonostachys rosea is sprayed onto approximately 25 grams of substrate in a fluid bed dryer. The pump speed, atomizing air pressure, and fan speed are adjusted accordingly (e.g., to a pump speed of 1 ml / min and an atomizing air pressure of 0.1 bar) to allow the substrate spores to dry to an inlet temperature of approximately 45°C and a powder bed temperature of less than 28°C. The samples are heated until the desired moisture content is reached. The samples are analyzed using the following methods.

[0051] Dry Particle Size Test. The particle size measurement of the substrate is carried out on the HORIBA LA-950 laser diffraction dry particle size distribution analyzer through the angle of the diffracted laser light.

[0051] Total Moisture Content Measurements. The moisture content of the dried microorganism substrate powder is measured using a Sartorius moisture balance. A 0.1 g mass of the sample powder is weighed and held on an aluminum plate. Three replicates are performed while the sample is heated to a constant weight of 105°C, usually for 2 minutes.

[0052] Water Activity. The water activity (Aw) of test samples is measured by placing the sample in a water activity measuring device, which consists of a mirror above the test sample in a closed sample chamber. When the relative humidity reaches equilibrium, the mirror is cooled until condensation forms on the mirror due to the dew point. The temperature can then be calculated as the water activity level.

[0053] Scanning Electron Microscopy (SEM) Image. A Hitachi TM 3000 electron microscope is used to image the substrate-microorganisms of the invention to show the morphology and composition of the product particles. The images show that the spore cells are attached to the silica particles.

[0054] Mercury Pore Volume and Pore Diameter Test. The introduced mercury (Hg) pore volume is measured using a mercury porosimeter with a Micromeritics AutoPore IV 9520 apparatus. Pore diameters can be calculated using the Washburn equation with a contact angle, Theta (Θ), of 130° and a surface tension, gamma, of 485 dynes / cm. Mercury is forced into the particle voids as a function of pressure, and the volume of mercury introduced per gram of sample is calculated at each pressure setting. The pore volume expressed herein represents the cumulative volume of mercury introduced at pressures from 171 to 18,000 psia (12.0225 to 1265.5252 kg / cm²). The mercury introduced at these pressures corresponds to a diameter of QCACnn / l 7P7 / B / Y pore size ranges from 1000 to 10 nm. Volume increments (cm³ / g) at each pressure setting are plotted against the corresponding pore diameter for each pressure increment. The peak in the introduced volume versus the pore radius or diameter curve corresponds to the mode in the pore size distribution and identifies the most common pore size in the sample. Specifically, the sample size is adjusted to achieve a main volume of 25–75% in a powder penetrometer with a 5 mL bulb and a main volume of approximately 1.1 mL. Samples are evaluated at a pressure of 50 pHg and held for 5 minutes. Mercury fills the pores from 1.5 to 60,000 psia (0.1054604 to 4218.417478 kg / cm2) with an equilibrium time of 10 seconds at each of approximately 103 data collection points.

[0055] BET Surface Area and Pore Volume. The BET surface areas of the substrates (e.g., silica or silicate particles) were determined using a Micromeritics TriStar 3020 instrument by the nitrogen adsorption BET method of Brunaur et al., J. Am. Chem. Soc., 60, 309 (1938), which is well-known in the field of particulate materials, such as silica and silicate materials. Nitrogen adsorption-desorption isotherms were collected at 77 K. Powdered samples of 50–100 mg were degassed at 105°C for 2 hours prior to measurement. The Barrett-Joyner-Halenda (BJH) models were used to calculate the pore volume and BET surface area. Total pore volume calculations are taken from the total amount of nitrogen adsorbed at a partial pressure (P / Po) of 0.99.

[0056] CFU Test. The concentration of microorganisms is determined by plate counting using serial dilution techniques. The microorganism-substrate powder is stirred in sterile water with Triton surfactant present to mobilize the microorganisms. The resulting suspension of microorganisms is sequentially diluted several times, each time by a factor of 10. Each dilution sample is placed on sterile agar plates and incubated. After several days, the organisms present can be observed as spots on the agar. When the dilution is sufficient to reduce the number on the plate to a countable quantity, the number of colonies is counted and multiplied by the dilution factor to determine the population relative to the original population.

[0057] Using the analytical methods described or similarly described above, the physical properties of the different substrates are measured and summarized below in Table 1. QCACnn / l 7Π7 / Β / Y Table 1 Substrates Substrate Type BET Surface Area m2 / g BJH Pore Volume cc / g Hg Pore Volume 10-1000 nm cc / g Particle Size micrometers silica SIPERNAT® 50e silica 500 1.40 2.24 50 silica ZEOFREE®5161A silica 160 0.60 2.37 85 silica SIPERNAT® 22 silica 180 0.92 1.98 120 silica SIPERNAT® 22 silica 180 0.92 1.98 120 silica SIPERNAT® 22 silica 180 0.92 1.98 120 silica ZEOLEX® 201 silicate 75 silicate 0.20 1.45 14 silica ZEODENT®116 silica 55 0.24 1.20 9 ZEODENT®103 silica silica 38 0.15 0.52 8

[0058] Using the methods described or similarly described above, the total moisture content and water activity (Aw) level of the test samples after fluidized bed drying are measured and summarized in Table 2; the BET surface area, drying time, final moisture content, water activity (Aw) and initial CFU of the test samples are reported in Table 3; the effect of water activity (Aw) versus the effect of moisture content on CFU / g after 5 months at 25°C is summarized in Table 4. Table 2 QCRCnn / l 7Π7 / E / Y Example Substrate Total Moisture Content (%) Water Activity (Aw) Water Activity Temperature (°C) Drying Method 1 SIPERNAT®50 55 0.941 20.8 Fluidized bed drying 2 SIPERNAT®22 3.1 0.288 20.8 Fluidized bed drying 3 SIPERNAT®22 2.7 0.327 20.8 Fluidized bed drying 4 SIPERNAT®22 3.96 0.348 20.8 Fluidized bed drying 5 SIPERNAT®22 5.14 0.385 20.7 Fluidized bed drying 6 SIPERNAT®22 9.04 0.688 20.8 Fluidized bed drying 7 ZEOFREE5161A 7 0.545 20.7 Fluidized bed drying Table 3 Example: Substrate Pore Volume BJH (cc / g) Pore Volume Hg 101000 nm (cc / g) Total Moisture Content (%) Drying Time (min) Water Activity (Aw) Inlet Air Temperature (°C) Initial CFU / g Drying Method 1 SIPERNA T®50 (silicon dioxide) 1.40 2.24 55 60 0.94 45 1x106 Fluidized Bed Drying 2 SIPERNAT @22 (silicon dioxide) 0.92 1.98 3.1 40 0.288 45 1.9x109 Spray drying 7 ZEOFREE ®5161A (silicon dioxide) 0.60 2.37 7.00 35 0.55 45 1x107 Fluidized bed drying 8 ZEOLEX® 201 (sodium aluminosilicate) 0.20 1.45 6.8 33 0.581 45 2.9x107 Fluidized bed drying 9 ZEODENT 116 (silicon dioxide) 0.24 1.20 8.64 28 0.280 45 2.8x107 Fluidized bed drying 10 ZEODENT 103 (silicon dioxide) 0.15 0.52 8.67 17 0.384 45 1.9x108 Fluid bed drying QCAcnn / i znz / R / Y Table 4 Example Substrate Total Moisture Content (% by weight) Water Activity (Aw) Inlet Air Temperature (°C) CFU / g after storage for 5 months at 25°C 1 SIPERNAT® 50 55 0.941 45 ~101 2 SIPERNAT® 22 3.1 0.288 45 1x109 5 SIPERNAT® 22 5.14 0.385 45 7.2 x105 6 SIPERNAT® 22 9.04 0.688 45 ~101 7 ZEOFREE 5161A 7 0.545 45 1.5x106

[0059] As can be seen from the tables above, the substrate with a high BET surface area and larger pore volume, such as SIPERNAT® 50, surprisingly does not dry as quickly in a fluidized bed dryer, resulting in the organisms being exposed to excessive amounts of time in the dryer. Substrates with lower BET surface area and pore volume dried more quickly in a fluidized bed dryer, allowing for less stress on the organisms and higher CFU / g after drying, which can result in potential cost savings per CFU / g due to lower drying costs. Table 4 also shows that the higher the total moisture content, the greater the drop in CFU / g during storage. Silica must be kept low during storage to maintain high CFU / g.The present invention shows, therefore, that the selection of substrates with optimum BET surface area and pore volume results in a low total moisture content with fast drying time, which creates less stress on the microorganisms and thus produces high initial CFU / g and also allows higher CFU / g values ​​after 5 months.

[0060] Example 11. Composition of the Invention Spray Drying: The bacterial biomass of Pseudomonas fluorescens is harvested from the culture overnight in a flask with centrifugal shaking at 8000 g for 10 minutes. The cell mass is resuspended in sodium chloride solution (0.9% w / w) and added to a suspension of the substrate Sipernat® 50 silica and gum arabic. The resulting suspension contains approximately 8% silica, 7% gum arabic, 3% dry biomass, and 81% water. The suspension is then spray dried in a Büchi B-290 laboratory spray dryer at a gas inlet temperature of 78°C. The spraying is carried out using a two-fluid nozzle at an atomization pressure of approximately 1.35 bar. The drying airflow rate is 38 m³ / h. The spray rate is approximately 5 mL / min. The established parameters result in an outlet temperature of 53°C and a residual moisture content of 6.3% in the product.The UFC count in the resulting powder is 3.4x107ufc / g.

[0061] Aqueous Harvesting of Fungal Spores: 15 g of initial spores with fungal pores on the surface are washed with water, the mass of water being 3-10 times the mass of the spores. The resulting suspensions are filtered through a 3 mm mesh after mixing with a stirrer (disc shaker) for 20 min. The suspension is filtered using a laboratory pressure jet of 380 ml. The operating conditions are at room temperature and 1 bar (abs). The dewatering time is 120 seconds. The filtrate is analyzed via spore count analysis. Further concentration of the filtrate is achieved by separation in a laboratory centrifuge at 2100 g for 5 min to reduce the water content before use in fluidized bed spraying.

[0062] Dry harvesting of fungal spores: 100 g of seeds with fungal spores on the surface are ground in a grinding machine with a rotating grinding stone for a residence time of 20 seconds. A fine fraction is generated due to the grinding of the seed surface and is collected separately from the seed residue, weighed, and the number of fungal spores (fs) in the sample is determined. The fs achieved in the fine fraction is 5 x 10⁹ fs per gram of seeds. The resulting fine fraction is sieved using a 300 µm mesh sieve. The resulting powder is mixed with water to obtain a suspension for subsequent spraying and dried in a fluidized bed.

[0063] Examples 12-23. The following examples were carried out to determine the effect of additives on improving the thermal and moisture stability of microorganisms.

[0064] Washing Procedure. The seeds are washed by mixing them with an equal mass of water until the water turns a light brown color. The spore suspension is strained from the seeds until half the initial volume of water is collected. More water is added if necessary to collect the final volume, which includes the added volume of the standard solutions. The additives are mixed directly into the suspension (AEROSIL® 200 silica and HPG) or into a concentrated standard solution (PVA) up to 2% gram to the final volume (mL).

[0065] Fluidized Bed Drying. The collected spore suspension is sprayed at approximately 4 g / min from above onto SIPERNAT® 22 silica, equal to the weight of the sprayed suspension. The fan speed is 8 Hz, and the inlet air temperature is set at 45°C for samples without additives in the suspension and 55°C for samples with additive(s). The initial powder temperature is 28°C. The powder is considered dry when the temperature rises rapidly from the initial temperature (28°C) after a few minutes, indicating dryness.

[0066] CFU Count. CFU, or colony-forming units, is the number of viable spores in one gram of product. Spore powder is mixed with Triton solution and, using the serial dilution method, placed on potato dextrose agar containing 0.1% streptomycin and incubated at room temperature for 5 days. CFU are determined by counting the plate containing 30–300 spores and multiplying by the dilution factor.

[0067] Subsequent Addition of AEROSIL® R 202 Silica. AEROSIL® R 202 silica is added to the final powder at 1% g / g of the selected samples. This is mixed in the Turbula, a low-energy mixer, for 5 minutes to uniformly coat the spore powder.

[0068] Thermal Stability. Spore powder with a sufficiently low water activity is stored in an oven at 40°C, and the CFU are counted at various time points to measure the decline in viable cell density in the powder.

[0069] Humidity Stability. The spore powder is stored in a humidity chamber (Associated Environmental Systems) at 70% relative humidity at 25°C in semi-porous Tubulin bags, which are permeable to water vapor but not to spores. CFU are measured at various time points to monitor progress.

[0070] Water Activity. Water activity is defined as the vapor pressure of water in a sealed sample. It is measured by the dew point on a cooled mirror in a sealed chamber as the mirror temperature drops. Water activity is measured in an AquaLab Model 3.

[0071] Decimal Reduction Time. Decimal reduction time is defined as the time to reduce the viable microbe population by 90%. It is calculated using the inverse slope of the survival curve, which is a graph of log CFU versus time.

[0072] Results. The samples used in the stability experiments were initially designed to have high CFU and low water activity. Samples that did not meet these requirements were discarded and prepared again. The water activity of each sample used can be seen in Table 6 below. The samples that met these two requirements were split and mixed half with AEROSIL® R 202 silica. The resulting powders were then stored in an oven at 40°C or in a humidity chamber at 25°C / 70% relative humidity. Table 5 summarizes the sample preparation below: QCRCnn / l 7Π7 / Β / Y Examples Additive(s) Description 12 - The spore suspension was sprayed onto SIPERNAT® 22 13 AEROSIL® 200 silica 2% AEROSIL® 200 silica was added to the spore suspension and sprayed onto SIPERNAT® 22 14 HPG 2% HPG was added to the spore suspension and sprayed onto SIPERNAT® 22 silica 15 PVA 2% PVA was added to the spore suspension and sprayed onto SIPERNAT® 22 silica 16 AEROSIL® R 202 silica Sample 12 was mixed with 1% AEROSIL® R 202 silica (for the thermal stability test) 17 AEROSIL® 200 and AEROSIL® R 202 silica Sample 13 mixed with 1% AEROSIL® R 202 silica (for the thermal stability test) 18 HPG silica and AEROSIL® R 202 Sample 14 mixed with 1% AEROSIL® R 202 silica (for the thermal stability test) 19 PVA silica and AEROSIL® R 202 Sample 15 mixed with 1% AEROSIL® R 202 silica (for the thermal stability test) 20 AEROSIL® R 202 silica Sample 12 mixed with 1% AEROSIL® R 202 silica (for the humidity stability test) 21AEROSIL® 200 silica and AEROSIL® R 202 Sample 13 mixed with 1% AEROSIL® R 202 silica (for moisture stability test) 22 HPG silica and AEROSIL® R 202 Sample 14 mixed with 1% AEROSIL® R 202 silica (for moisture stability test) 23 PVA silica and AEROSIL® R 202 Sample 15 mixed with 1% AEROSIL® R 202 silica (for moisture stability test) Table 6. Corresponding initial water samples and activities. Examples Additives Water Activity 12 - 0.121 13 2% silica AEROSIL® 200 0.165 14 2% HPG 0.25 15 2% PVA 0.15

[0073] Samples tested for thermal stability are stored in a monitored oven for a period of 10 weeks. CFUs are measured at various time points, as shown in Figure 1. The QCACnn / l 7P7 / E / Y decimal reduction time (D value) and can be observed in Figure 2. As can be seen, during the first six weeks, the values ​​did not show significant divergence. At the 10-week mark, however, the samples mixed with AEROSIL® R 202 silica were observed to be more stable than those without it. The samples that were not subsequently treated with AEROSIL® R 202 silica had a low UFO value at this time point, too low to be accurately counted. This shows that the addition of AEROSIL® R 202 silica improves the stability of the spore powder and extends the powder's shelf life. The combination of PVA and AEROSIL® R 202 silica is the most stable in the long term. PVA, however, has a lower initial UFO.Although some of this may be attributed to variations in processing, the spore suspension is also more dilute because PVA is difficult to dissolve and is therefore added to the spore suspension as a concentrated stock solution.

[0074] Although most samples show comparable trends, the control performs the worst in the thermal stability test. The sample with no additives also starts with the lowest CFU. The sample with AEROSIL® R 202 silica only also starts with a low CFU; however, it performs much better throughout the stability test. These CFU trends can be observed in Figure 3.

[0075] As shown in Figure 4, the samples containing HPG performed best. The sample with AEROSIL® R 202 silica alone also had a similar decimal reduction time. The samples with additives mixed into the spore solution (AEROSIL® 200 silica, HPG, PVA) showed no further improvement when subsequently mixed with AEROSIL® R 202 silica. Although AEROSIL® R 202 silica improved moisture stability compared to the control, it did not further improve moisture stability when used in addition to other additives. It is believed that the samples with additives were better able to keep moisture away from the spores under humid conditions.

[0076] The subsequent addition of AEROSIL® R 202 silica shows a clear improvement in thermal stability compared to samples without it. Similarly, AEROSIL® R 202 silica also has improved moisture stability, but it does not further improve moisture stability when used in conjunction with other additives. Therefore, the addition of AEROSIL® R 202 silica is the most effective way to improve long-term thermal and moisture stability.

[0077] Examples 24-25 were prepared as described below: Example 24 QCRCnn / l 7Π7 / Β / Y Ingredients % by Weight of Dispersion % by Weight of Product Sipernat® Silica 50 8 32 Gum Arabic 7 28 Trehalose 5 20 Pseudomonas fluorescens 3 20 Sodium Chloride Solution (0.9% w / w) 77 0 Example 25 Ingredients % by Weight of Dispersion % by Weight of Product Sipernat® Silica 50 4 17 Gum Arabic 4 15 Trehalose 9 34 Pseudomonas fluorescens 8 34 Sodium Chloride Solution (0.9% w / w) 75 0 ocRcnn / i ζηζ / E / γ

[0078] The biomass of Pseudomonas fluorescens is fermented in minimal medium and harvested using a disc centrifuge to obtain a concentrated cell suspension. A physiological saline solution is prepared and mixed with trehalose, gum arabic, and Sipernat® 50 silica. The harvested cell suspension is mixed with the trehalose / gum arabic / Sipernat® silica suspension. The components of the suspension in Example 24, after mixing, account for up to 8% silica, 7% gum arabic, 3% dry biomass, 77% sodium chloride solution, and 5% trehalose. The components of the suspension in Example 25 account for up to 4% silica, 4% gum arabic, 8% dry biomass, 75% sodium chloride solution, and 9% trehalose. The suspensions of Example 24 and Example 25 are spray dried separately in a Niro Minor spray dryer using a two-fluid nozzle at an atomization pressure of 2.3 bar.The gas inlet temperature is 100°C and the mass flow rate of the slurry is 0.9 kg / h for Example 24, and the gas inlet temperature is 110°C and the mass flow rate of the slurry is 1.6 kg / h for Example 25. This results in an outlet temperature of 50°C for both Examples 24 and 25. The drying gas flow rate is 45 m³ / h for both Examples 24 and 25. The product moisture content for Examples 24 and 25 is 7% by weight and the water activity is 0.3. The fumed energy content (FEC) of the final product for Example 24 is 2 x 10¹⁰ FEC / g, and for Example 25 it is 3.6 x 10¹⁰ FEC / g.

Claims

1. A dry biological composition (1) comprises a substrate and microorganisms loaded (2) onto the surface of the substrate, wherein the composition has a total moisture content of approximately 0.01% by weight to approximately 15% by weight, preferably, approximately 0.01% by weight to approximately 8% by weight, preferably, approximately 3% by weight to approximately 8% by weight, preferably, approximately 5% by weight to approximately 8% by weight, preferably still, selected from 3% by weight, 5% by weight and 7% by weight; 2. The composition according to claim 1, wherein the composition has a water activity (Aw) value between approximately 0.01 and approximately 0.6, preferably between approximately 0.2 and approximately 0.6, and even more preferably between approximately 0.3 and approximately 0.

5.

3. The composition according to claim 1 or 2, wherein the composition has more than approximately 107 CFU / g, preferably greater than or equal to approximately 108 CFU / g, preferably even greater than or equal to approximately 109 CFU / g, preferably even greater than or equal to approximately 1010 CFU / g, preferably even greater than or equal to approximately 1011 CFU / g, preferably even greater than or equal to approximately 1012 CFU / g.

4. The composition according to any of claims 1-3, wherein the substrate is selected from the group consisting of silica (e.g., precipitated silica, in a particular embodiment, hydrophilic silica, e.g., SIPERNAT® 22 silica), diatomaceous earth, silica gel, silicates (e.g., aluminosilicates such as ZEOLEX® 201 or clays) and water-insoluble natural fiber material such as cellulose.

5. The composition according to any of claims 1-4, wherein the substrate is silica.

6. The composition according to any of claims 1-5, wherein the substrate is precipitated silica.

7. The composition according to any of claims 1-6, wherein the substrate is hydrophilic silica.

8. The composition according to any of claims 1-7, wherein the particle size (d50) of the substrate is approximately 5-200 micrometers, preferably, approximately 8-160 micrometers, preferably still, approximately 9-150 micrometers, preferably still, approximately 50-150 micrometers, preferably also, approximately 50-130 micrometers, preferably selected from a group consisting of approximately 50 micrometers, approximately 85 micrometers, and approximately 120 micrometers.

9. The composition according to any of claims 1-8, wherein the BET surface area of ​​the substrate is approximately 2-600 m2 / g, preferably 2-400 m2 / g, preferably approximately 5-400 m2 / g, preferably still approximately 10-400 m2 / g, preferably still approximately 30-400 m2 / g, preferably also approximately 30-300 m2 / g, preferably still approximately 40-200 m2 / g, preferably still approximately 180 m2 / g.

10. The composition according to any of claims 1-9, wherein the BET surface area of ​​the substrate is approximately 2 m2 / g, preferably approximately 5 m2 / g.

11. The composition according to any of claims 1-9, wherein the BET surface area of ​​the QCAcnn / i ζηζ / E / γ substrate is approximately 180 m2 / g.

12. The composition according to any of claims 1-11, wherein the pore volume of the substrate is approximately 0.01-1.20 cc / g, preferably approximately 0.05-1.20 cc / g, preferably still approximately 0.10-1.0 cc / g, preferably still approximately 0.20-0.95 cc / g based on the Barrett-Joyner-Halenda model or the BET surface area of ​​the substrate is approximately 400-600 m² / g, preferably 500 m² / g and the pore volume is greater than 1 cc / g, preferably 1.4 cc / g by the Barrett-Joyner-Halenda model or greater than 2 cc / g, preferably 2.2 cc / g by the Mercury Pore Volume.

13. The composition according to any of claims 1-12, wherein the final concentration of microorganisms is between approximately 4 and approximately 40% by weight, preferably approximately 4 and approximately 20% by weight of the total composition.

14. The composition according to any one of claims 1-13, wherein the microorganisms are selected from the group consisting of Bacillus subtilis QST713, Pastearía usgae; Bassian beauty, Coniothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Brongniartii beauty, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp., Lecanicillium longisporum, Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var. acridum, Nomuraea rileyi Sporothrix insectosorum; Cydia pomonella GV; Phytophthora palmivora, Lagenidium giganteum, Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, Bacillus spp. and Lactobacillus spp. or any combination of the same, preferably, are selected from group consisting of Bacillus thuríngiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea and any combination of the same.

15. The composition according to any of claims 1-14, wherein the microorganisms are Clonostachys rosea or Pseudomonas fluorescens.

16. The composition according to any of claims 1-15 in tablet form, fluid concentrated form, for example, for seed treatment or in oil dispersion form further comprises one or more excipients, preferably one or more agrochemically acceptable excipients.

17. The composition according to any of claims 1-16, wherein the composition does not require an exogenous protectant such as alginate encapsulation.

18. The composition according to any of claims 1-17, wherein the composition further comprises (i) a polymer selected from the group consisting of polyvinyl alcohol, xanthan gum, gum arabic or other polysaccharides such as maltodextrin, guar gum (e.g., hydroxypropyl guar gum), polyethylene glycol and, polyglycerol or (ii) non-reducing polysaccharides such as trehalose or sucrose or (iii) skimmed milk or dimethyl sulfoxide.

19. The composition according to any of claims 1-18, wherein the composition further comprises a second substrate as an outer layer, preferably the second substrate being selected from (i) a precipitated silica, preferably hydrophobic precipitated silica, preferably having a high BET surface area, preferably 50-750 m² / g, preferably 50-380 m² / g, preferably SIPERNAT® 50 S or ZEOFREE® silica or (ii) a fuming silica, preferably fuming silica, preferably selected from the group consisting of AEROSIL® 200, AEROSIL® R202, AEROSIL® R 972 and AEROSIL® R 812S silica. QCRcnn / i znz / R / γ 20. The composition according to any of claims 1-19, wherein the number of colony-forming units per gram of the composition (CFU / g) remains above approximately 107 CFU / g after storage at (a) room temperature for 120 days; (b) 40°C for 40 days; (c) a relative humidity of 65% or less for 40 days.

21. The composition according to any of claims 1-20, wherein the compacted density of the composition is greater than 150% of the compacted density of the pure substrate material.

22. A process for preparing a dry biological composition comprises (1) combining a mixture of microorganisms, solution or suspension with a substrate; and (2) drying the substrate-microorganism mixture to achieve a total moisture content of approximately 0.01 to approximately 15% by weight, preferably, approximately 0.01% by weight to approximately 8% by weight, preferably still, approximately 3% by weight to approximately 8% by weight, preferably still, approximately 5% by weight to approximately 8% by weight, preferably still selected from 3% by weight, 5% by weight and 7% by weight.

23. The process of claim 22, wherein the microorganisms are harvested from the surface of a seed by (a) mechanically grinding or polishing the substrate surface resulting in a fine fraction that includes microorganisms and some parts of the seed; and optionally (b) sieving the resulting fine fraction to obtain a powder having a particle size distribution defined by subsequent process steps.

24. The process of claim 23, wherein step (a) comprises grinding the seed with a grinding stone to separate the seed from the fine fraction.

25. The process of claim 23, wherein step (a) comprises grinding with a rotating shaft within an enclosed tube inside a slotted screen under pressure, followed by a sieve and filter to separate the seed from the fine fraction.

26. The process of claim 23, wherein the sieving time (b) of the fine fraction comprises sieving with a sieve mesh size of 20 to 800 pm, preferably from 100 pm to 300 pm.

27. The process of claim 22 wherein the microorganisms are harvested from the surface of a seed by washing them with water and separating the seed and the liquid solution or suspension of microorganisms, preferably the seed is agitated in water for 1 to 20 min, preferably further the solution-liquid separation is carried out in a pressure Nutsche filter, preferably further a mesh size of 1 to 3 mm is used in a pressure Nutsche filter, preferably further the dewatering time in a pressure Nutsche filter is 20-200 seconds, preferably further the filtration pressure in the Nutsche filter is 1 to 3 bar, preferably further the microorganism solution or suspension is concentrated by separating the microorganisms from the liquid in a centrifuge field, preferably further the concentration step comprises separating in a disc stack separator, preferablyThe concentration step is repeated with dilution of the concentrate with water and a subsequent second concentration in a centrifuge field to separate the soluble parts of the microorganisms.

28. The process of any of claims 22-27, wherein the drying step (2) comprises fluid bed drying of the substrate-microorganism mixture.

29. The process of any of claims 22-27-, wherein the drying step (2) comprises drying the substrate-microorganism mixture by QCAcnn / i ζηζ / E / γ spray.

30. The process of claim 22-27, wherein the drying step (2) comprises contact drying the substrate-microorganism mixture.

31. The process of claim 22-27, wherein the drying step (2) comprises freeze-drying the substrate-microorganism mixture.

32. The process of any of claims 22-31, wherein the drying air temperature is less than or equal to approximately 130°C, in a particular embodiment, less than or equal to approximately 90°C, preferably, less than or equal to approximately 80°C, preferably still, less than or equal to approximately 50°C, preferably still, approximately 30°-50°C, preferably still, approximately 40°-50°C, preferably still, approximately 40°-45°C, preferably still, approximately 43°C.

33. The process of any of claims 22-32, wherein the powder bed is maintained less than or equal to approximately 35°C, preferably from approximately 25°C to approximately 35°C.

34. The process of any of claims 22-33, wherein the resulting composition has a water activity (Aw) value between approximately 0.01 and approximately 0.6, preferably between approximately 0.2 and approximately 0.6, and even more preferably between approximately 0.3 and approximately 0.

5.

35. The process of any of claims 22-34, wherein the resulting composition has colony-forming units of microorganisms per gram of composition (CFU / g), for example, greater than approximately 107 CFU / g, preferably greater than or equal to approximately 108 colony-forming units per gram (CFU / g), preferably greater than or equal to approximately 109 CFU / g, preferably still greater than or equal to approximately 1010 CFU / g, preferably still greater than or equal to approximately 1011 CFU / g, preferably still greater than or equal to approximately 1012 CFU / g.

36. The process of any of claims 22-35, wherein the substrate is selected from the group consisting of silica (e.g., precipitated silica, in a particular embodiment, hydrophilic silica, e.g., SIPERNAT® 22 silica), diatomaceous earth, silica gel, silicates (e.g., aluminosilicates such as ZEOLEX® 301 or clays) and water-insoluble natural fiber material such as cellulose.

37. The process of any of claims 22-36, wherein the substrate is silica, preferably precipitated silica, preferably a hydrophilic silica.

38. The process of any of claims 22-37, wherein the substrate is selected from those having a BET surface area of ​​approximately 400-600 m2 / g, preferably 500 m2 / g and a pore volume greater than 1 cc / g, preferably 1.4 cc / g by the Barrett-Joyner-Halenda model or greater than 2 cc / g, preferably 2.2 by Mercury Pore Volume.

39. The process of any of claims 22-37, wherein the substrate is selected from those having: (i) a particle size (d50) of approximately 5-200 micrometers, preferably approximately 8-160 micrometers, preferably still, approximately 9-150 micrometers, preferably still, approximately 50-150 micrometers, preferably still, approximately 50-130 micrometers, preferably still, selected from a group consisting of approximately 50 micrometers, approximately 85 micrometers, and approximately 120 micrometers; (i) a BET surface area of ​​approximately 2-400 m2 / g, preferably, approximately 5-400 m2 / g, preferably still, approximately 10-400 m2 / g, preferably still, approximately 30-400 m2 / g, preferably still, approximately 30-300 m2 / g, preferably still, approximately 40-200 m2 / g, preferably still, approximately 180 m2 / g; (iii) a substrate pore volume of approximately 0.01-1.20 cc / g, preferably, approximately 0.05-1.20 cc / g, preferably still, approximately 0.10-1.0 cc / g, preferably still, approximately 0.20-0.95 cc / g; (iv) or any combination thereof.

40. The process of any of claims 22-39, wherein step (1) comprises loading from approximately 4 to approximately 40% by weight, preferably approximately 4 to approximately 20% by weight of the total composition.

41. The process of any of claims 22-40, wherein microorganisms are selected from the group consisting of Bacillus subtilis QST713, Pasteuria usgae; Beauveria bassiana, Coniothyrium minitans, Chondrostereum purpureum, Paecilomyces lilacinus, Aschersonia aleyrodis, Beauveria brongniartii, Hirsutella thompsonii, Isaria fumosorosea, Isaria sp., Lecanicillium longisporum, Lecanicillium muscarium, Lecanicillium sp., Metarhizium anisopliae, Metarhizium anisopliae var. acridum, Nomuraea rileyi Sporothrix insectorum; Cydia pomonella GV; Phytophthora palmivora, Lagenidium giganteum, Bacillus thuringiensis, Pseudomonas fluorescens, Bradyrhizobium, Mycorrhiza, Clonostachys rosea, Bacillus spp. and Lactobacillus spp. or any combination thereof.

42. The process of any of claims 22-41, wherein the resulting composition does not require an exogenous protectant such as alginate encapsulation.

43. A dry biological composition prepared by the process of any of claims 2242.

44. The dry biological composition according to any of claims 1-21 or 43 further comprises a seed that will be treated.

45. A method for controlling insects, fungi, or nematodes over an area to be treated, optionally comprising reconstituting the dry biological composition of any of claims 1-19 or 43, and applying an effective quantity of the optionally reconstituted composition to the area to effect the treatment.

46. ​​The method of claim 45, wherein the area to be treated is a portion of a plant, including without limitation, vegetative cutting, root, bulb, tuber, stem, fruit, flower and / or leaf of a plant, for example, corn, wheat, sorghum, soybeans, citrus and non-citrus fruits, nuts and the like.